PROTEIN COMPOSITION AND METHODS FOR ANALYSING MICROBIOTA

Abstract

A method of isotope-labelling a microbiota sample. It involves providing a first microbiota sample that was obtained from a given source; exposing the first microbiota sample to an isotope enriched medium; and culturing the exposed first microbiota sample in the isotope enriched medium to obtain an isotope-labelled microbiota sample, wherein the isotope labelled metaproteome of the isotope-labelled microbiota sample is taxon specific for taxa present in the first microbiota sample when initially obtained from the given source.

Description

TECHNICAL FIELD

The present application relates generally to compositions and methods for metaproteomics and more specifically compositions and methods for microbiota protein composition analysis.

BACKGROUND

The human body harbors trillions of microbes which together comprise the human microbiome. Accumulating evidence has associated changes in microbiota composition with many diseases including inflammatory bowel diseases (IBD), obesity, diabetes, cancer, heart disease, urolithiasis, allergies etc. [2]. The microbiome is a highly complex and extremely sensitive system and it has been demonstrated that the microbiome composition is susceptible to alterations due to exposure to various compounds including but not limited to therapeutics, excipients, additives, preservatives, chemicals, stress, exercise, foods and beverages, which can impact both maintenance of health as well as development of disease. In addition to the overall microbiome, organ and region specific microbiomes have been described, in which subsets of these microbes form populations, such as the intestines, skin, vagina, oral cavity, kidney, bladder, eyes, lungs and breasts. Furthermore, it has been shown that the changes induced by many of these environmental, chemical and alimentary compounds have the potential to induce positive changes to these regional microbiomes which improve the composition and diversity of the microbiome, while others induce negative changes consistent with a diseased or unhealthy microbiome. For example, it has been demonstrated that emulsifiers, a ubiquitous component of processed food, causes negative changes in the intestinal microbiome composition indicative of inflammation and disease, while prebiotics have been shown to increase the levels of beneficial microbes, increasing the health of the intestinal microbiome. Exactly how these compositional changes affect overall function, however, remains an outstanding and important question.

One of the largest populations of microbes resides in the gastrointestinal tract and constitutes the intestinal microbiota [1]. The importance of intestinal microbiota for human health was illustrated with the use of fecal microbiota transplantation (FMT) for treating recurrent Clostridium difficile infections [3]. Furthermore, it has been shown that the microbiome continues to undergo changes throughout the course of a disease, which was recently demonstrated in IBD in which changes in the intestinal microbiome composition were tightly correlated with disease severity in Crohn's disease. Clearly, the microbiota appears to be involved in the development, progression and resolution of multiple diseases and in some instances, can be modulated to impact disease outcome, making the microbiota of global interest in both scientific and public health communities.

Beyond the human microbiome many additional microbial communities, or microbiomes, have been identified and described. One of the most prominent is the soil microbiome, which is a complex microbial community that can differ by region, climate and cultivation. The composition of the soil microbiome can be assessed to determine biodiversity and the subsequent function of these microbes in the generation and breakdown of nutrients holds wide-ranging implications for agriculture. Biofilms are another well-studied microbiome, which can be of varying complexity, occur in numerous environmental and industrial settings, and can contribute to contamination or toxicity. Understanding the composition and function of the microbes within biofilms is important for understanding biofilm prevention and dissolution. Finally, the microbiome of animals, both in agricultural and lab settings are important to the advancement of food production and research respectively. The microbiome of agricultural animals can be effected by antibiotics as well as feed and these changes have the potential to impact, breeding, animal health and subsequently food production. Additionally, it has recently been recognized that the microbiome of laboratory research animals can have profound effects on their response to therapeutics. A deeper understanding of the microbiome composition and function of these animals could possibly lead to an improved understanding of drug metabolism and the microbiome effect of therapeutic response.

Next-generation sequencing (NGS), such as metagenomics and metatranscriptomics, is well suited for examining the microbiota composition and predicting potential functions, however, it does not provide direct evidence on whether the genes are translated into proteins or not. This information, therefore, does not provide data on changes in the function of the microbiota, which are needed to understand how the alterations in composition impact function and if it is impacted in a physiologically meaningful way. Instead, metaproteomics can provide invaluable information on the functional activities of the microbiome by directly profiling protein expression levels, which are indicative of function [4, 5]. In stark contrast to metagenomics, however, metaproteomics approaches have only been applied to a limited number of studies on the microbiota. This is due, at least in part, to challenges related to both identification and quantification of the microbial peptides/proteins. Peptide/protein identification algorithms have recently been significantly improved by the use of iterative searching of large microbial protein databases [6]. In contrast, accurate methods for peptide/protein quantification are still lacking. Most current metaproteomic approaches are based on label free quantification (LFQ) which suffers from significant variability during the separate sample processing and mass spectrometry runs, making data extremely difficult to compare across experiments or datasets. Stable isotope labeling by amino acids in cell culture (SILAC) and stable isotope labeling of mammals (SILAM) are currently the most widely used approach for quantitative proteomics and provides lower variability [7]. In SILAC/SILAM, proteins in one test or reference sample are metabolically labeled with isotopically heavy amino acids, enabling for quantitative comparison between different samples. However, the application of this approach in bacteria, particularly complex bacterial populations such as the microbiome, has been limited. One of the challenges of applying these metabolic labelling approaches to the microbiota is the inherently high species diversity, which is not present in mammalian cells. Furthermore, the diverse microbiota populations frequently include both aerobic and many anaerobic species, which are extremely difficult to culture in order to achieve sufficient labeling. These complex populations also almost inevitably result in a diverse metabolic capacity to biosynthesize amino acids, which hampers the full incorporation of heavy-labelled amino acids into microbial proteins. Instead, complete metabolic labeling of nitrogen or carbon has only been applied to single bacteria [8], and environmental microbial communities, such as acid mine drainage biofilms [9]. However, its application to characterize the microbiota proteome is lacking.

There is therefore a need for better metaproteomics approach for the analysis of the microbiota.

SUMMARY

A first broad aspect is a fast and cost-effective strategy for metabolic labeling of the whole human microbiota, termed stable isotopically labelled microbiota (SILAMi). It will be understood that by whole human microbiota, it is meant that SILAMi may be used to provide metabolic labeling of any microbiota sample taken from the human microbiota. By samples from the human microbiota, it is meant such samples as, but not limited to samples form the intestinal microbiota, samples from the vaginal microbiota, samples from the oral microbiota, samples from the cutis microbiota, samples from the vaginal microbiota, samples from the bladder microbiota, samples from the kidney microbiota, samples from the lung microbiota, samples from the eye microbiota, samples from the breast microbiota, samples from the penile microbiota, a microbiota mucosal sample, etc. A skilled person will also readily understand that SILAMi can also be applied to a microbiota originating from an animal sample, wherein that animal is, for instance, a mammal, a bird, a reptile, etc.

Applicant has discovered that it is possible to use an isotope-labelling standard for a given microbiota sample having a large microbe population. Prior to the Applicant's discovery, it was believed that the diverse microbiota population could not be labelled simultaneously and that the microbiota would change too rapidly during incorporation of the labelling to achieve a reliable standard that is representative of the original microbiota sampled. However, applicant has discovered that the isotope-labelled standard achieved following the isotope-incorporation process is in fact representative of a significant population of the microbiota of the original sample and the proteins representative thereof. This isotope labelling of the microbiota is entitled SILAMi.

Therefore, SILAMi is a method for achieving the successful labelling of a large microbe population (a microbiota), such as one found in a human or animal subject. Difficulties in obtaining such a standard lie for instance in the fragility and low abundance of some of the microbe species found in the desired microbiota sample, for instance sensitive to changes in environment or exposure to oxygen (e.g. as some of these microbes grow in anaerobic conditions). Moreover, not all of these microbes have the same cell cycle or life cycle, and some take longer to replicate and incorporate the isotope. However, the more time that is taken to grow and incorporate the isotopes in culture, the greater the risk that certain of the species found in the microbiota sample will die off or disproportionately proliferate, not providing a faithful depiction of the microbiota found in the sample as obtained. SILAMi has overcome these prior problems and successfully provides an isotope-labelled standard for a given microbiota from a microbiota sample, where the sample may be taken from, for instance, a human, an animal, soil, plant, water or any other source with a significant population of microbes.

Moreover, Applicant has discovered that using an isotope-labelled standard, such as the one achieved using SILAMi, allows for the study of a large population of microbes in a given microbiota sample. The standard allows for the determination of the functionality and the composition, including changes in the determination of the functionality and composition of the microbiota sample. Furthermore, the standard provides a means of reducing variability when performing a metaproteomic analysis of the microbiota sample. This may be achieved, for instance, by adding a known amount of the standard to the microbiota sample (with a known ratio and measuring the heavy to light ratios for the sample, while comparing the ratios to theoretical known results).

In some examples, the analysis of the microbiota of a subject, such as a human or animal subject, may provide an indication of a disease, illness or other condition afflicting the subject, where these conditions have a measured effect on the microbiota of the subject found at different locations on the subject (e.g. the subject's organs). A comparison between the isotope-labelled standard and the microbiota sample may provide indication, for instance, as to the effectiveness of a treatment, the nature, including diagnosis and therapeutic response to of the disease or illness, the potential for weight gain or loss of the subject, etc.

Another broad aspect is a human microbiota labelled-proteins standard having labelled proteins representative of a metaproteome from a human microbiota as described herein. In some examples, said labelled proteins have at least about 50%, preferable 90%, and more preferably at least about 95% average heavy isotopic enrichment rate.

In a further embodiment there is provided a human intestinal microbiota labelled-proteins standard that may have labelled proteins representative of a metaproteome from an intestinal microbiota. In some embodiments, said labelled proteins may have at least about 90% and preferably at least about 95% average heavy isotopic enrichment rate.

In yet another embodiment the microbiota labelled-proteins standard may be taxon-specific for at least about 90% and preferably at least 95% of the microbes present in the microbiota sample. Preferably the labelled proteins are taxon-specific for 100% of Kingdoms present in the microbiota sample. Preferably the labelled proteins are also taxon-specific for 95% and preferably at least 100% of Phyla present in the microbiota sample. Preferably the labelled proteins are also taxon-specific for at least about 90% and preferably at least 95% of Genera present in the microbiota sample. Preferably the labelled proteins are also taxon-specific for at least about 90% and preferably at least 95% of species present in the microbiota sample.

In yet another embodiment the microbiota labelled-proteins standard may be taxon-specific for at least about 90% and preferably at least 95% of the microbes present in the intestinal microbiota. The labelled proteins may be taxon-specific for 100% of Kingdoms present in the intestinal microbiota. The labelled proteins may also be taxon-specific for 95% and preferably at least 100% of Phyla present in the intestinal microbiota. The labelled proteins may also be taxon-specific for at least about 90% and preferably at least 95% of Genera present in the intestinal microbiota. The labelled proteins may also be taxon-specific for at least about 70% and preferably at least 95% of species present in the intestinal microbiota.

In yet another embodiment the microbiota labelled-proteins standard may be taxon-specific for at least about 90% and preferably at least 95% of the microbes present in the microbiota sample from including but not limited to vaginal, oral, skin, bladder, kidney, lung, eye and breast. The labelled proteins may be taxon-specific for 100% of Kingdoms present in the microbiota. The labelled proteins may also be taxon-specific for 95% and preferably at least 100% of Phyla present in the microbiota. The labelled proteins may be also taxon-specific for at least about 90% and preferably at least 95% of Genera present in the microbiota. The labelled proteins may also be taxon-specific for at least about 90% and preferably at least 95% of the species present in the microbiota.

For an exemplary intestinal microbiota samples, the Domains may be Bacteria, Eukaryota and Archaea, the Phyla are, but not limited to, Bacteroidetes, Proteobacteria, Verrucomicrobia, Fusobacteria, Synergistetes, Thaumarchaeota, Fimicutes, Actinobacteria, Ascomycota, Basidiomycota, Euryarchaeota, Apicomplexa, Arthropoda, Chordata, Nematoda, Streptophyta, the Genera are those listed in table 1, the Species are those listed in table 2.

TABLE 1
An exemplary set of genera that can be found
in an exemplary intestinal microbiota sample.
Abiotrophia	Edwardsiella	Nitrososphaera	Tyzzerella
Acidaminococcus	Eggerthella	Odoribacter	Veillonella
Acidovorax	Enterobacter	Oribacterium	Vibrio
Acinetobacter	Enterococcus	Oscillibacter	Weissella
Actinobacillus	Erwinia	Oxalobacter	Xanthomonas
Actinomyces	Erysipelatoclostridium	Paenibacillus	Yokenella
Adlercreutzia	Escherichia	Parabacteroides
Aerococcus	Eubacterium	Paraprevotella
Aeromonas	Facklamia	Parasutterella
Akkermansia	Faecalibacterium	Parvimonas
Alcanivorax	Faecalitalea	Pediococcus
Alistipes	Ferrimonas	Peptoclostridium
Alteromonas	Filobasidiella	Peptostreptococcus
Anaerobaculum	Finegoldia	Phascolarctobacterium
Anaerococcus	Flavonifractor	Photobacterium
Anaerofustis	Fusarium	Piscirickettsia
Anaerostipes	Fusobacterium	Plasmodium
Anaerotruncus	Gemella	Porphyromonas
Arcobacter	Gordonibacter	Prevotella
Aspergillus	Granulicatella	Propionibacterium
Atopobium	Haemophilus	Proteus
Bacillus	Hafnia	Providencia
Bacteroides	Hahella	Pseudoflavonifractor
Barnesiella	Helicobacter	Pseudomonas
Bifidobacterium	Holdemanella	Pseudoxanthomonas
Bilophila	Holdemania	Ralstonia
Blautia	Hungatella	Rhodotorula
Burkholderia	Intestinibacter	Roseburia
Butyrivibrio	Klebsiella	Rothia
Campylobacter	Lachnoanaerobaculum	Ruminiclostridium
Candida	Lachnoclostridium	Ruminococcus
Carnobacterium	Lactobacillus	Salmonella
Catenibacterium	Leptotrichia	Selenomonas
Cellvibrio	Leuconostoc	Serratia
Citrobacter	Listeria	Shewanella
Clostridium	Marinomonas	Slackia
Collinsella	Marvinbryantia	Staphylococcus
Coprobacillus	Megamonas	Streptococcus
Coprococcus	Methanobrevibacter	Subdoligranulum
Corynebacterium	Methanosphaera	Succinatimonas
Debaryomyces	Methylobacterium	Sutterella
Desulfitobacterium	Meyerozyma	Synergistes
Desulfovibrio	Mitsuokella	Tannerella
Dialister	Mogibacterium	Terrisporobacter
Dorea	Moraxella	Thermoplasma
Dysgonomonas	Neisseria	Turicibacter

TABLE 2
An exemplary set of species that can be found
in an exemplary intestinal microbiota sample.
Abiotrophia defectiva            Bacteroides intestinalis
Acidaminococcus intestini        Bacteroides oleiciplenus
Acidovorax avenae                Bacteroides ovatus
Acinetobacter junii              Bacteroides pectinophilus
Actinobacillus suis              Bacteroides plebeius
Actinomyces georgiae             Bacteroides stercoris
Actinomyces massiliensis         Bacteroides thetaiotaomicron
Actinomyces odontolyticus        Bacteroides uniformis
Adlercreutzia equolifaciens      Bacteroides vulgatus
Aerococcus viridans              Bacteroides xylanisolvens
Aeromonas hydrophila             Barnesiella intestinihominis
Aeromonas veronii                Bifidobacterium adolescentis
Akkermansia muciniphila          Bifidobacterium angulatum
Alcanivorax dieselolei           Bifidobacterium bifidum
Alistipes finegoldii             Bifidobacterium breve
Alistipes indistinctus           Bifidobacterium catenulatum
Alistipes putredinis             Bifidobacterium dentium
Alistipes shahii                 Bifidobacterium gallicum
Alteromonas macleodii            Bifidobacterium longum
Anaerobaculum hydrogeniformans   Bifidobacterium pseudocatenulatum
Anaerococcus hydrogenalis        Bilophila wadsworthia
Anaerofustis stercorihominis     Blautia hansenii
Anaerostipes caccae              Blautia hydrogenotrophica
Anaerostipes hadrus              Blautia obeum
Anaerotruncus colihominis        Butyrivibrio crossotus
Arcobacter butzleri              Butyrivibrio fibrisolvens
Aspergillus fumigatus            Campylobacter concisus
Atopobium minutum                Campylobacter upsaliensis
Atopobium parvulum               Candida albicans
Atopobium rimae                  Candidatus Nitrososphaera gargensis
Bacillus cereus                  Catenibacterium mitsuokai
Bacillus smithii                 Cellvibrio japonicas
Bacteroides caccae               Citrobacter freundii
Bacteroides cellulosilyticus     Citrobacter youngae
Bacteroides clarus               Clostridium asparagiforme
Bacteroides coprocola            Clostridium bolteae
Bacteroides coprophilus          Clostridium butyricum
Bacteroides dorei                Clostridium citroniae
Bacteroides eggerthii            Clostridium clostridioforme
Bacteroides finegoldii           Clostridium hiranonis
Bacteroides fluxus               Clostridium hylemonae
Bacteroides fragilis             Bacteroides intestinalis
Clostridium innocuum             Eubacterium siraeum
Clostridium leptum               Eubacterium ventriosum
Clostridium methylpentosum       Facklamia ignava
Clostridium perfringens          Faecalibacterium prausnitzii
Clostridium saccharolyticum      Faecalitalea cylindroides
Clostridium scindens             Ferrimonas balearica
Clostridium spiroforme           Finegoldia magna
Clostridium symbiosum            Flavonifractor plautii
Collinsella aerofaciens          Fusarium graminearum
Collinsella intestinalis         Fusobacterium gonidiaformans
Collinsella stercoris            Fusobacterium mortiferum
Collinsella tanakaei             Fusobacterium necrophorum
Coprococcus catus                Fusobacterium nucleatum
Coprococcus comes                Fusobacterium periodonticum
Coprococcus eutactus             Fusobacterium ulcerans
Corynebacterium ammoniagenes     Fusobacterium varium
Corynebacterium durum            Gemella sanguinis
Cryptococcus gattii              Gordonibacter pamelaeae
Debaryomyces hansenii            Granulicatella adiacens
Desulfitobacterium hafniense     Hafnia alvei
Desulfovibrio desulfuricans      Hahella chejuensis
Desulfovibrio piger              Helicobacter bilis
Dialister invisus                Helicobacter Canadensis
Dialister succinatiphilus        Helicobacter cinaedi
Dorea formicigenerans            Helicobacter pullorum
Dorea longicatena                Helicobacter pylori
Dysgonomonas gadei               Helicobacter winghamensis
Dysgonomonas mossii              Holdemanella biformis
Edwardsiella tarda               Holdemania filiformis
Eggerthella lenta                Hungatella hathewayi
Enterobacter cancerogenus        Intestinibacter bartlettii
Enterobacter cloacae             Klebsiella pneumoniae
Enterococcus faecalis            Lachnoanaerobaculum saburreum
Enterococcus faecium             Lactobacillus acidophilus
Enterococcus haemoperoxidus      Lactobacillus amylolyticus
Enterococcus saccharolyticus     Lactobacillus antri
Erwinia amylovora                Lactobacillus brevis
Escherichia coli                 Lactobacillus delbrueckii
Eubacterium dolichum             Lactobacillus fermentum
Eubacterium hallii               Lactobacillus helveticus
Eubacterium rectale              Lactobacillus iners
Lactobacillus plantarum          Prevotella stercorea
Lactobacillus reuteri            Prevotella veroralis
Lactobacillus rhamnosus          Propionibacterium acnes
Lactobacillus ruminis            Proteus mirabilis
Lactobacillus ultunensis         Proteus penneri
Leptotrichia goodfellowii        Providencia alcalifaciens
Leuconostoc mesenteroides        Providencia rettgeri
Listeria grayi                   Providencia rustigianii
Listeria innocua                 Providencia stuartii
Marinomonas profundimaris        Pseudoflavonifractor capillosus
Marvinbryantia formatexigens     Pseudomonas aeruginosa
Megamonas funiformis             Pseudoxanthomonas spadix
Megamonas hypermegale            Ralstonia pickettii
Methanobrevibacter smithii       Rhodotorula glutinis
Methanosphaera stadtmanae        Roseburia intestinalis
Methylobacterium nodulans        Roseburia inulinivorans
Meyerozyma guilliermondii        Rothia aeria
Mitsuokella multacida            Rothia mucilaginosa
Mogibacterium timidum            Ruminococcus bromii
Moraxella catarrhalis            Ruminococcus champanellensis
Neisseria bacilliformis          Ruminococcus gnavus
Odoribacter laneus               Ruminococcus lactaris
Oribacterium sinus               Ruminococcus torques
Oxalobacter formigenes           Salmonella enterica
Paenibacillus lactis             Selenomonas sputigena
Parabacteroides distasonis       Serratia marcescens
Parabacteroides johnsonii        Shewanella putrefaciens
Parabacteroides merdae           Slackia exigua
Paraprevotella clara             Slackia piriformis
Paraprevotella xylaniphila       Staphylococcus aureus
Parasutterella excrementihominis Streptococcus equinus
Parvimonas micra                 Streptococcus thermophiles
Pediococcus acidilactici         Subdoligranulum variabile
Peptoclostridium difficile       Succinatimonas hippie
Peptostreptococcus anaerobius    Sutterella parvirubra
Phascolarctobacterium succinatutens Sutterella wadsworthensis
Photobacterium damelae           Terrisporobacter othiniensis
Piscirickettsia salmonis         Thermoplasma volcanium
Porphyromonas endodontalis       Turicibacter sanguinis
Prevotella copri                 Tyzzerella nexilis
Prevotella salivae               Veillonella dispar
Veillonella parvula
Weissella paramesenteroides
Yokenella regensburgei

In another aspect, the labelled-proteins standard may have isotope(s) labelled proteins and wherein the isotopes(s) can be stable or radioactive isotopes. The isotopes can be selected, for example, from ¹³C, ¹⁴C, ¹⁵N, ³²S, ³⁵S, ³²P and Deuterium, and combination thereof.

In a further aspect, there is provided a method for obtaining a microbiota labelled-proteins standard, as described above, comprising: obtaining a microbiota sample from an individual; exposing said sample to an isotope enriching growth medium (i.e. an enriched media, such as an isotope enriched media, also defined herein as an isotope enriched medium); and culturing said exposed sample for a period of time sufficient to obtain a predetermined level of enrichment.

In a further aspect, there is provided a method for obtaining a microbiota labelled-proteins standard, as described above, comprising: obtaining a microbiota sample from an individual including but not limited to intestinal, vaginal, oral, skin, bladder, kidney, lung, eye or breast microbiota; exposing said sample to an isotope enriching medium; and culturing said exposed sample for a period of time sufficient to obtain a predetermined level of enrichment.

In another aspect, there is provided a method for measuring an amount of one or more proteins in a microbiota sample comprising obtaining a protein extract from the microbiota sample and spiking the protein extract with the standard, as described above, and obtaining labelled/unlabeled protein ratios of the standard and the one or more bacteria (or, as the case may be, other forms of microbes) in the microbiota sample.

In another aspect, there is also provided a method for measuring an amount of one or more proteins in an intestinal microbiota sample comprising obtaining a protein extract from the microbiota sample and spiking the protein extract with the standard, as described above, and obtaining labelled/unlabeled protein ratios of the standard and the one or more bacteria in the intestinal microbiota sample.

In another aspect, there is also provided a method for measuring an amount of one or more proteins in a microbiota sample comprising obtaining a protein extract from the microbiota sample including but not limited to vaginal, oral, skin, bladder, kidney, lung, eye, or bladder microbiota and spiking the protein extract with the standard, as described above, and obtaining labelled/unlabeled protein ratios of the standard and the one or more bacteria in the microbiota sample.

The method for measuring an amount of one or more proteins in an microbiota sample may further comprise obtaining a label free quantification (LFQ) of the microbiota sample. The SILAMi and LFQ method can be combined to improve the accuracy of protein measurement in a sample. The method may also involve performing gas chromatography/mass spectrometry. In some embodiments, the method may involve performing mass spectrometry.

In some embodiments, the method for measuring an amount of one or more proteins in an intestinal microbiota sample may further comprise obtaining a label free quantification (LFQ) of the intestinal microbiota sample. The SILAMi and LFQ method can be combined to improve the accuracy of protein measurement in a sample.

The method for measuring an amount of one or more proteins in a microbiota sample including but not limited to vaginal, oral, skin, bladder, kidney, lung, eye, or bladder microbiota may further comprise obtaining a label free quantification (LFQ) of the microbiota sample. The SILAMi and LFQ method can be combined to improve the accuracy of protein measurement in a sample

In yet another embodiment there is provided a method for diagnosing a disease such as, but not limited to, an intestinal disease (IBD for example) comprising measuring an amount of one or more proteins in a microbiota sample (wherein the measuring is performed using the standard such as described with respect to the method for measuring an amount of one or more proteins in a microbiota sample as described herein) from a patient and wherein deviation from normal is indicative of disease. In an aspect of this method the measuring is performed at one or more time point and is compared to control samples optimally obtained at a predetermined time in the life of an individual or from an individual in a predetermined state of health.

A method for treating a patient with a disease is also provided that involves assessing said patient's microbiota as described above to diagnose the disease and treating the patient according to the diagnostic.

In yet another aspect, there is provided a method for determining treatment response in a patient with a disease comprising measuring one or more proteins in a microbiota sample from a patient and wherein derivation away from diseased and/or toward normal is indicative of favorable treatment response. In an aspect of this method the measuring is performed at a one or more time point and is compared to control samples optimally obtained at a predetermined time in the life of an individual or from an individual in a predetermined state of health or disease.

In yet another aspect, there is provided a method for determining remission in a patient with a disease comprising measuring one or more proteins in a microbiota sample from a patient and wherein normal levels are indicative of the absence of a previously present disease. In an aspect of this method the measuring is performed at a one or more time point and is compared to control and/or disease samples optimally obtained at a predetermined time in the life of an individual or from an individual in a predetermined state of health or disease.

In another aspect there is provided a method for screening xenobiotics effect on a human microbiota comprising exposing the microbiota to one or more xenobiotics and measuring an amount of one or more protein as described above.

In another aspect, there is provided a method for screening xenobiotics effect on a human microbiota, including but not limited to intestinal, vaginal, oral, skin, bladder, kidney, lung, eye and breast microbiome, comprising exposing the microbiota to one or more xenobiotics and measuring an amount of one or more protein as described above.

In another aspect, there is provided a method for screening xenobiotics effect on an intestinal human microbiota comprising exposing the microbiota to one or more xenobiotics, including but not limited to chemicals, toxins, environmental toxins, and poisons and measuring an amount of one or more protein as described above.

From the screening of xenobiotics effect a profile may be generated based on proteins measurements. The profile can be integrated into a method of diagnostic or prognostic.

In a further aspect, there is provided a method for screening the effect of therapeutics on a human microbiota, including but not limited to immunotherapies, antibiotics, checkpoint inhibitors, chemotherapies, antidepressants, antiepileptic, antiemetic, analgesics, antivirals, sedatives, antidiabetic, antipsychotics, and anticoagulants, comprising exposing the microbiota to one or more drugs and measuring the amount of one or more proteins as described above.

In a further aspect, there is provided a method for screening the effect of therapeutics on a human microbiota, using the RapidAIM and/or SILAMi technique disclosed herein, including but not limited to therapies or antibodies targeted to, PD-1/PDCD1/CD279; PD-L1/CD274; PD-L2/PDCD1LG2; CTLA-4/CD152; CD80/B7/B7-1; CD86; TIM-3/HAVCR2; Galectin-9/GAL9/LGALS9; TIGIT; CD155/PVR; LAG3; VISTA/C10orf54; B7-H3/CD276; B7-H4/VTCN1; BTLA/CD272; HVEM/TR2/TNFRSF14; A2AR; CD28; CD80/B7/B7-1; CD86; ICOS/CD278; CD275/ICOSLG/B7RP1; CD40L/CD154; CD40; CD137/4-1BB; CD137L; CD27; CD70/CD27L; OX40/CD134/TNFRSF4; OX40L/TNFSF4; GITR; GITRL; SIRPα; CD47 comprising exposing the microbiota to one or more drugs and measuring the amount of one or more proteins as described above.

In a further aspect, there is provided a method for screening the effect of foods on a human microbiota comprising exposing the microbiota to one or more foods and measuring the amount of one or more proteins as described above.

In a further aspect, there is provided a method for screening the effect of food ingredients on a human microbiota, including but not limited to food additives, amino acids, flavorings, dyes, emulsifiers, sweetners, hydrocolloids and preservatives, comprising exposing the microbiota to one or more ingredients and measuring the amount of one or more proteins as described above.

In a further aspect, there is provided a method for screening the effect of beverages on a human microbiota, including but not limited to soda, sports beverages, infant formula, milk, alcohol, juice, drinkable yogurt, and fermented teas, comprising exposing the microbiota to one or more beverages and measuring the amount of one or more proteins as described above.

In another aspect, there is provided a method for screening the effect of packaging components on a human microbiota, including but not limited to coatings and plastics, comprising exposing the microbiota to one or more packaging component and measuring the amount of one or more proteins as described above.

In another aspect, there is provided a method for screening the effect of cosmetics and cosmetic components including but not limited to excipients, natural and synthetic pigments, thickeners, and emulsifiers, on a human microbiota, comprising exposing the microbiota to one or more cosmetics or cosmetic components and measuring the amount of one or more proteins as described above.

In another aspect, there is provided a method for screening the effect of consumer products including but not limited to infant products, household cleaners, lotions, shampoos and perfumes on a human microbiota, comprising exposing the microbiota to one or more consumer products and measuring the amount of one or more proteins as described above.

In another aspect, there is provided a method for screening the effect of consumer health products including but not limited to supplements, vitamins, amino acids, and plant extracts on a human microbiota, comprising exposing the microbiota to one or more consumer products and measuring the amount of one or more proteins as described above

In another aspect, there is provided a fast and cost-effective method for metabolic labeling of a soil microbiota. The method for obtaining a soil microbiota labelled-proteins standard, as described above, involves obtaining a soil microbiota sample; exposing said sample to an isotope enriching medium; and culturing said exposed sample for a period of time sufficient to obtain a pre-determined level of enrichment.

Another aspect is a method for measuring an amount of one or more proteins in a soil microbiota sample comprising obtaining a protein extract from the microbiota sample and spiking the protein extract with the standard, as described above, and obtaining labelled/unlabeled protein ratios of the standard and the one or more bacteria in the microbiota sample.

In another aspect, there is provided a method for screening xenobiotics effect on a soil microbiota, involving exposing the microbiota to one or more xenobiotics including but not limited to pesticides, toxins, amino acids, and nitrates and then measuring an amount of one or more proteins.

In another aspect, there is provided a fast and cost-effective method for metabolic labeling of an animal microbiota, wherein the animal microbiota may originate from a microbiota sample from an animal, such as a cow, pig, chicken, llama, sheep, goat, rabbit, mouse, rat, etc.

In a further aspect, there is provided a method for obtaining an animal microbiota labelled-proteins standard, as described above, involving obtaining an animal microbiota sample such as a cow, pig, chicken, llama, sheep, goat, rabbit, mouse, rat, etc; exposing said sample to an isotope enriching medium; and culturing said exposed sample for a period of time sufficient to obtain a pre-determined level of enrichment.

In yet another aspect there is provided a method for diagnosing a disease including measuring an amount of one or more proteins in a microbiota sample from an animal, including but not limited to cows, pigs, chickens, llamas, sheep, goats, rabbits, mice and rats and wherein deviation from normal is indicative of disease. In an aspect of this method the measuring is performed at a one or more time point and is compared to control samples optionally obtained at a predetermined time in the life of an animal or from an animal in a pre-determined state of health.

In another aspect there is provided a method for screening xenobiotics effect on an animal microbiota, including but not limited to cows, pigs, chickens, llamas, sheep, goats, rabbits, mice and rats; comprising exposing the microbiota to one or more xenobiotics, including but not limited to feed, amino acids, supplements, pesticides, and toxins, and measuring an amount of one or more proteins.

In one aspect, there is provided a fast and cost-effective method for metabolic labeling of a biofilm microbiota. The method for screening xenobiotics effect on a biofilm microbiota; includes exposing the microbiota to one or more xenobiotics, including but not limited to chemicals, pesticides, toxins, and soaps, and measuring an amount of one or more proteins.

In another aspect, there is provided a fast and cost-effective strategy for metabolic labeling of a microbiota from an industrial manufacturing facility.

Another broad aspect is a method of labelling a microbiota sample that includes providing a microbiota sample that was obtained from a given source. The method involves exposing the microbiota sample to an enriched medium, and culturing the microbiota sample to obtain a microbiota sample with a labeled proteome. In some embodiments, the labelled microbiota sample may be taxon specific for taxa present in the first microbiota sample when initially obtained from the given source.

In some embodiments, the enriched medium may be an isotope enriched medium, wherein the proteome of the microbiota sample may be isotope-labelled. However, the label enriched medium may provide for labelling other than isotopes.

Another broad aspect is a microbiota labelled-proteins standard obtained by performing the method of obtaining a labelled microbiota sample as defined herein, wherein the microbiota labelled-proteins standard has labelled proteins representative of a proteome from a selected microbiota.

Another broad aspect is a method for labelling protein of a microbiota sample comprising providing a first microbiota sample that was obtained from a given source; exposing the first microbiota sample to an enriched medium; and culturing the exposed first microbiota sample in the enriched medium to obtain an labelled microbiota sample, wherein the labelled metaproteome of the labelled microbiota sample is taxon specific for taxa present in the first microbiota sample when initially obtained from the given source. In some embodiments, the method may further comprise characterizing said labelled microbiota sample by performing a metaproteomic analysis of said labelled microbiota sample. In some embodiments, said labelled microbiota sample may be taxon specific for a predetermined proportion of microbe populations present in the first microbiota sample when initially obtained from the given source. In some embodiments, the enriched medium may be an isotope enriched medium.

In some embodiments, the labelled microbiota sample is taxon specific for a predetermined proportion of microbe populations present in the first microbiota sample when initially obtained from the given source. By pre-determined proportion it is meant that some taxa of microbes are specifically sought to be labelled in the labelling of the labelled sample. For instance, a user may be searching for specific bacterial species that are associated with a given disease (e.g. atopobium parvulum in the case of certain intestinal disease). In this example, the pre-determined proportion would be or would include the bacterial species that are known for that disease. Moreover, certain microbial populations may be known to react positively or negatively when a patient is given a specific compound (e.g. a drug) or when a patient is responding to a given diagnostic treatment. In these examples, the pre-determined populations may be or may include those reactive microbial populations or taxa.

In some embodiments, the method may involve characterizing the labelled microbiota sample by performing a metaproteomic analysis of the -labelled microbiota sample. The culturing of the exposed first microbiota sample may be for a period to obtain an average level of enrichment of the labelled proteins representative of the metaproteome of at least 70% and to be taxon specific for a predetermined proportion of microbe populations present in the first microbiota sample when initially obtained from the given source.

The culturing of the exposed first microbiota sample may be for a period to obtain an average level of enrichment of the labelled proteins representative of the metaproteome of at least 90% and to be taxon specific for a predetermined proportion of microbe populations present in the first microbiota sample when initially obtained from the given source. The culturing of the exposed first microbiota sample may be for a period to obtain an average level of enrichment of the labelled proteins representative of the metaproteome of at least 95% and to be taxon specific for a predetermined proportion of microbe populations present in the first microbiota sample when initially obtained from the given source. The culturing the exposed first microbiota sample may be for a period to obtain a predetermined average level of enrichment of the labelled proteins representative of the metaproteome of the exposed first microbiota sample and to be taxon specific for at least 50% of the microbe populations present in the first microbiota sample when initially obtained from the given source. The culturing the exposed first microbiota sample may be for a period to obtain a predetermined average level of enrichment of the labelled proteins representative of the metaproteome of the exposed first microbiota sample and to be taxon specific for at least 90% of the microbe populations present in the first microbiota sample when initially obtained from the given source. The culturing the exposed first microbiota sample may be for a period to obtain a predetermined average level of enrichment of the labelled proteins representative of the metaproteome of the exposed first microbiota sample and to be taxon specific for 90% of Phyla present in the first microbiota sample when initially obtained from the given source. The culturing the exposed first microbiota sample may be for a period to obtain a predetermined average level of enrichment of the labelled proteins representative of the metaproteome of the exposed first microbiota sample and to be taxon specific for at least about 90% of Genera present in the first microbiota sample when initially obtained from the given source. The culturing the exposed first microbiota sample may be for a period to obtain a predetermined average level of enrichment of the labelled proteins representative of the metaproteome of the exposed first microbiota sample and to be taxon specific for at least about 90% of species present in the first microbiota sample when initially obtained from the given source.

In some embodiments, an isotope enriched medium to which the first microbiota sample is exposed may contain an isotope selected from 13C, 14C, 15N, 32S, 35S, 32P and Deuterium, and combination thereof. The isotope enriched medium to which the microbiota sample is exposed may contain as an isotope 15N.

In some embodiments, the first microbiota sample that is provided may be obtained from a human subject. In other embodiments, the first microbiota sample that is provided may be obtained from an animal subject.

the providing a first microbiota sample may be providing a type of microbiota sample, wherein the microbiota sample type may be an intestinal microbiota sample, a cutis microbiota sample, a vaginal microbiota sample, an oral microbiota sample, a lung microbiota sample, a mucosal microbiota sample, a bladder microbiota sample, a kidney microbiota sample, an eye microbiota sample, a penile microbiota sample, or a breast microbiota sample.

Another broad aspect may be a method of performing a compositional analysis of a second microbiota sample that involves using a labelled microbiota sample, obtained by performing a method such a sample as described herein, as a labelled standard to perform compositional analysis of a second microbiota sample, wherein the compositional analysis is enhanced as a result of the employment of the labelled-standard.

The compositional analysis may be performed on a second microbiota sample having a same microbiota sample type as that of the first microbiota sample. The method may include, prior to the employing the labelled-standard to perform compositional analysis of a second microbiota sample, providing the second microbiota sample that was obtained from the same source as the microbiota sample used to obtain the labelled standard. The using a labelled microbiota sample as a labelled standard to perform compositional analysis of a second microbiota sample may include performing metaproteomic analysis, and the metaproteomic analysis may be for measuring an amount of one or more protein in the second microbiota sample. The metaproteomic analysis may include obtaining a protein extract from the second microbiota sample, spiking the protein extract with the labelled standard; and obtaining labelled/unlabelled protein ratios of the labelled standard and the one or more protein in the second microbiota sample. The metaproteomic analysis may also involve obtaining a label free quantification (LFQ) of the second microbiota sample. The using a labelled microbiota sample as a labelled standard to perform compositional analysis of a second microbiota sample may involve performing metagenomic analysis. In some embodiments, the metagenomic analysis may involve 16S-based sequencing. The metagenomic analysis may involve shotgun sequencing.

The compositional analysis may be performed to achieve disease diagnosis in a target subject, assessing treatment response in a target subject, assessing remission in a subject receiving treatment, screening for xenobiotic effects on a microbiome of a target subject, screening for effects of a compound on a microbiome of a target subject, wherein the compound is one of a food, a drug, a chemical, a therapeutic agent, a toxin, a poison, a beverage, a food additive, a cosmetic, a cosmetic ingredient, packaging material, a pesticide, a herbicide, a consumer product, and/or screening a microbiome to identify the responsiveness of a subject to a therapy or treatment.

The compositional analysis may be performed to achieve the screening for xenobiotic effects on a microbiome of a target subject, and the second microbiota sample may be obtained from the target subject, and the compositional analysis may be performed subsequent to the target subject being exposed to one or more xenobiotics. The compositional analysis may be performed to achieve screening for effects of a compound on a microbiome of a target subject, wherein the second microbiota sample may be obtained from the target subject, and the compositional analysis may be performed subsequent to the target subject being exposed to one or more compounds. A profile may be generated based on the compositional analysis. The profile may be integrated into a method of diagnosis or prognosis. The compositional analysis may be performed to achieve the disease diagnosis in a target subject, wherein the using a labelled microbiota sample as a labelled standard to perform compositional analysis may also include measuring an amount of the one or more protein in the second microbiota sample and wherein deviation from normal is indicative of the disease. The metaproteomic analysis may be performed at one or more time points using a time-point microbiota sample taken at the one or more time points, and, following a metaproteomic analysis performed on the time-point microbiota sample, a measured one or more proteins from the time-point microbiota sample may be compared to a control sample. The control sample may be a standard control sample taken from a subject in a predetermined state of health, and/or a control sample obtained at a predetermined time in the life of the target subject. Another broad aspect is a method for treating a patient with a disease comprising assessing the patient's microbiota to diagnose the disease and treat the patient in accordance with the diagnostic.

Another broad aspect is a method of high throughput screening of multiple microbiota samples for metaproteomic analysis of the samples. The method entails culturing multiple microbiota samples wherein each sample of the multiple microbiota samples is cultured in a well of a multi-well receptacle. The method involves washing the cells of the multiple microbiota culture samples, re-suspending in lysis buffer with a protease inhibitor the microbiota culture samples, lysing the cells of the multiple microbiota culture samples, diluting the multiple microbiota culture samples, and digesting the proteins contained in the microbiota culture samples. The method adds performing simultaneous metaproteome identification and quantification of the multiple microbiota samples by using a microbial gene catalog of a given subject type and an iterative database search strategy.

In some embodiments, the microbial gene catalog of a given subject type is a microbial gene catalog of a human. In some embodiments, the microbial gene catalog of a given subject type may be a microbial gene catalog of an animal.

In some embodiments, prior to digesting the proteins contained in the microbiota culture samples, the method may involve spiking the microbiota culture samples with an isotope labelled standard corresponding to a given microbiota sample. In some embodiments, prior to the digesting, the method may involve reducing and alkylating of cysteines in the proteins contained in the microbiota culture samples. The spiking may involve adding sufficient isotope labelled-standard to reach a 1:1 protein mass ratio with the protein contained in the microbiota culture samples.

In some embodiments, the multi-well receptacle is a multi-well plate.

In some embodiments, the method may involve assessing the results of the metaproteome identification and quantification of the multiple microbiota samples to perform disease diagnosis in a target subject, assessing treatment response in a target subject, assessing remission in a subject receiving treatment, screening for xenobiotic effects on a microbiome of a target subject, screening for effects of a compound on a microbiome of a target subject, wherein the compound is one of a food, a drug, a chemical, a therapeutic agent, a toxin, a poison, a beverage, a food additive, a cosmetic, a cosmetic ingredient, packaging material, a pesticide, a herbicide, a consumer product, and/or screening a microbiome to identify the responsiveness of a subject to a therapy or treatment.

Another broad aspect is a method of high throughput screening of multiple microbiota samples for meta-omic analysis of the samples includes providing a plurality of microbiota samples in culture. The method also involves performing a pre-screening using a meta-omic technique to identify changes in microbiomes of the microbiota samples, selecting the microbiomes exhibiting predetermined changes, and analyzing the selected microbiomes to characterize the changes. The provided microbiota samples may be cultured in micro-well receptacles. The provided microbiota samples may be cultured in micro-well plates.

In some embodiments, the analyzing may involve using a microbial gene catalog of a given subject type and an iterative database search strategy. The analyzing may involve performing a metaproteomic analysis combined with a metagenomic analysis. The microbial gene catalog of a given subject type may be a microbial gene catalog of a human. The microbial gene catalog of a given subject type may be a microbial gene catalog of an animal.

In some embodiments, the method may involve, after the providing, spiking the plurality microbiota culture samples with an isotope labelled standard corresponding to a given microbiota sample. The spiking may involve adding sufficient isotope labelled-standard to reach a 1:1 protein mass ratio with the protein contained in the plurality of microbiota culture samples. The performing a pre-screening using a meta-omic technique may involve performing metaproteomics.

The method may involve assessing the results of the analysis of the selected microbiomes to perform disease diagnosis in a target subject; assessing treatment response in a target subject; assessing remission in a subject receiving treatment; screening for xenobiotic effects on a microbiome of a target subject; screening for effects of a compound on a microbiome of a target subject, wherein the compound is one of a food, a drug, a chemical, a therapeutic agent, a toxin, a poison, a beverage, a food additive, a cosmetic, a cosmetic ingredient, packaging material, a pesticide, a herbicide, a consumer product; and/or screening a microbiome to identify the responsiveness of a subject to a therapy or treatment.

Another aspect is a method for isotope-labelling protein of a microbiota sample comprising:

• • providing a first microbiota sample that was obtained from a given source;
  • exposing said first microbiota sample to an isotope enriched medium; andculturing said exposed first microbiota sample in said isotope enriched medium to obtain an isotope-labelled microbiota sample, wherein the isotope labelled metaproteome of said isotope-labelled microbiota sample is taxon specific for taxa present in said first microbiota sample when initially obtained from said given source.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood by way of the following detailed description of embodiments of the invention with reference to the appended drawings, in which:

FIG. 1 is a schematic diagram of the exemplary labelling method comprising isotopic ¹⁵N metabolic labeling of human microbiota for quantitative metaproteomics. (A) Brief workflow of the stable isotope labeling of microbiota (SILAMi), and the SILAMi-based quantitative metaproteomic approaches, which can be applied to any human microbiota sample. (B) ¹⁵N isotopic enrichment of identified intestinal microbial peptides. Mucosal-luminal interface aspirate samples, or stool samples yielding slurries from five different individuals were labelled separately with three technical replicates. The average ¹⁵N enrichment rates of all the identified peptides for each individual's microbiota were shown. It will be understood that even though a Mucosal-luminal interface aspirate samples or a stool sample (slurry) were used as a microbiota sample, any intestinal microbiota sample may be used to perform the following method. Moreover, a skilled person will understand that any microbiota sample used from a human or animal may be used when performing the following method.

FIG. 2 describes the quantitation accuracy of the SILAMi-based metaproteomics. (A) Density plot showing the calculated L/H ratios of quantified protein groups in samples with different L/H spike-in ratios (1:1, 1.25:1, 2:1, and 5:1). Scatter plot shows the correlation between the calculated L/H protein ratios (median) and spike-in ratios. Pearson's r-value was indicated; (B) Density plot showing the distribution of fold changes when compared to the sample with 1:1 spike-in ratio. Log 2-transformed L/H ratios or fold changes were used for generating the density plots with a band width of 0.2. Dashed lines indicate median values. The percentage of proteins within two-fold difference to median was shown in the brackets.

FIG. 3 illustrates examples of SILAMi-based quantitative metaproteomics for microbiota studies and the use of this technique for screening the effect of chemicals and/or compounds on microbiota protein expression overtime. (A) Principal component analysis score plot of FOS-mediated metaproteome changes. (B) Representative total ion currents (TICs) of quantified peptides of protein EF-Tu. Both heavy (red) and light (blue) are shown. (C) Heatmap of 246 microbial protein which significantly changed upon the supplementation of monosaccharides during in vitro cultivation. Both column and row clusterings were based on Euclidean distance. Blue square, N-acetyl glucosamine (GlcNAc); blue circle, glucose; yellow circle, galactose; green circle, mannose; red triangle, fucose; C, control. (D) Quantified proteins involved in bacterial fucose utilization pathway. Mean±SD was shown in the bar charts. DHAP, dihydroxyacetone phosphate; FucP, L-fucose:H+ symporter permease; FucM, L-fucose mutarotase; FucI, L-fucose isomerase; FucA, L-fuculose-1-phosphate aldolase; fucK, L-fuculokinase; FucO, L-1,2-propanediol oxidoreductase or lactaldehyde reductase. It will be understood that while microbiota samples were treated with monosaccharides, any compound could be used in this method to treat the microbiota samples and the subsequent effect on microbiome protein expression assessed.

FIG. 4 is a graph illustrating ¹⁴N and ¹⁵N peptide identification for each passage of the five human intestinal microbiota samples during metabolic labeling. The mean and standard error of the identified unique peptide sequences are shown.

FIG. 5 illustrates microbiota composition at the initial inoculum (Passage 0) and SILAMi at phylum and genus levels. Taxonomic analysis was performed using metaproteomics. For metaproteomic analysis, phyla (A) and genera (B) were considered present if they had ≥2 detected unique peptide sequences. Orange indicates the presence of the taxa in only Passage 0, while purple indicates the taxa are present at both Passage 0 and in SILAMi.

FIG. 6 represents a heat map of the 187 identified protein groups altered by fructooligosaccharide (FOS) treatment. Complete protein names are listed in Table 3; a few proteins of interest are indicated. The clustering of rows was generated based on Euclidean distance in Perseus. It will be understood that while this example demonstrates changes from treatment with FOS, a heatmap of changes could be generated from microbiome protein changes following treatment with any compound using the methods described herein.

FIG. 7 illustrates the influence of monosaccharides on the relative abundance of N-acetyl glucosamine-degrading related proteins in human microbiome samples. Log 2-transformed L/H ratios are shown and expressed as mean±SD. A two sample t-test was used to compare differences between the non-treated control group (n=3) and the treated sample (n=3). * P<0.05, **P<0.01, ***P<0.001.

FIG. 8 illustrates an exemplary workflow of RapidAIM. Rapid Analysis of Individual Microbiota (RapidAIM) through fast-pass metaproteomics for rapid screening and in-depth metaproteomics/metagenomics for mechanism interpretation. The workflow includes high-performance, easy-to-use software platforms for rapidly identifying positive hits and providing functional insights into microbiome changes.

FIG. 9 illustrates an exemplary 96-well-based culturing and metaproteomic experimental workflow for the RapidAIM assay, which can be used to assess any set of microbiome samples, including those from multiple individuals, in a multi-well format. Briefly, the microbiome samples cultured in 96 deepwells are washed with PBS and re-suspended in urea lysis buffer. Then the samples are sonicated with a multi-channel sonicator for cell lysis. The protein concentrations of control samples (in triplicates) are tested and all samples will be digested in a volume equivalent to 100 μg proteins in the control. 1:1 protein content of SILAMi reference can be spiked-in in this step (but spiking with the SILAMi reference is not required). After reduction and alkylation, the proteins are digested by trypsin and are desalted. This workflow allows for accurate and reproducible high throughput metaproteomic analysis in a multi-well. This provides a screening platform that is capable of assessing changes due to disease, drug treatment or any other manipulation or treatment of the microbiome samples while in culture. Furthermore, this allow for the simultaneous culture and assessment of the microbiome samples from multiple individuals in a multi-well format, allowing for high-throughput screening in a compact and time-efficient manner.

FIG. 10 illustrates exemplary results from a RapidAIM assay for samples treated with a high (4-BBH), medium (3-BBM) or low (2-BBL) dose of berberine compared to a sample that is normal control (1-CN). The taxonomic composition at the species level were quantified in each of the cultured microbiome sample on the MetaLab bioinformatics platform: FIG. 10A shows the results from the loadings plot of a principal component analysis (PCA) with all bacterial species. FIG. 10B shows that the abundances of the species originating from the Akkermansia genus (identified in FIG. 10B as Akkermansia species 1, Akkermansia species 2 and Akkermansia species 3) were significantly increased when treated with high concentration of Berberine. Akkermansia spp has been reported to be beneficial bacterial in the gut microbiome, which has been shown in other literatures to be increased by another antidiabetic drug, Metformin. It will be understood that while microbiota samples were treated with Berberine, any compound could be used in this screening platform to treat the microbiome samples and the subsequent effect on microbiome protein expression assessed with a system to perform functional and quantitative analysis of the samples.

DETAILED DESCRIPTION

SILAMi is a labelling technique that yields an isotope-labelled standard for a given microbiota sample. The original microbiota sample may have a large diversity of microbes. The microbe populations contained in the sample may range from prokaryotes (bacteria and archaea) to eukaryotes, where the eukaryotes may include fungi, protists.

In SILAMi, microbiota samples are inoculated into ¹⁵N-labeled bacterial growth media, cultured under anaerobic conditions and passaged every 24 hours. Once the ¹⁵N isotope is incorporated, the labelled microbiota can be used as an internal standard for the study of unlabelled samples. In the examples provided herein, a fresh intestinal microbiota sample was used. However, the skilled person will readily understand that other microbiota samples may be obtained and used in SILAMi without departing from the present teachings.

In the present application, by “compositional analysis” it is meant an analysis technique to determine the composition of a microbiota sample. Such analysis may involve, for example, metaproteomic analysis, metagenomic analysis or any other analytical technique employed to determine the composition (may it be the protein composition, the microbe composition), or a combination thereof, of the microbiota sample.

Moreover, by “microbe populations” it is meant the different taxa present in a microbiota (this includes, for example, the Domain, Kingdom, Phyla, Class, Order, Family Genera, Species found in the sample). In some examples, the microbe populations as herein defined may relate to the microdiversity of a microbiota sample, or to the diverse taxa found in the microbiota sample.

By “microbiota sample” it is meant a sample that contains a microbiota from a particular source. Even though the experiments described herein focus upon microbiota samples originating from a human (e.g. an intestine of a human as shown in FIG. 1A and following), it will be understood that these are but examples of the fact that an isotope-labelled standard may be achieved for such a diverse population as that found in a human subject. Therefore, it will be appreciated by a skilled person in the art that other microbiota samples may similarly be obtained and cultured to reach an isotope-labelled standard by employing the SILAMi technique as described herein. For example, it will be readily understood that such microbiota samples may originate from animals, soil or water, and may even involve certain plants with a diverse microbiota population residing therein or thereon.

By “isotopically metabolic labelling” it is meant the technique of incorporating isotopes into a given microbiota population as described herein.

Experiment 1: Effectiveness of the SILAMi Technique to Label a Diverse Microbe Population:

In an experiment to demonstrate the efficacy of the SILAMi microbiome labeling technique in a diverse mixed microbe populations, it was first examined whether intestinal metaproteomes could be efficiently labeled with ¹⁵N. The intestinal metaproteome was selected for this experiment due to its diverse microbiome—indicative that other diverse microbiomes may similarly be labelled to provide isotope-labelled standards and importance in intestinal disease.

Experiment Protocol:

Five intestinal microbiome samples were aspirated from colons. In some examples, as shown in FIG. 1A, the samples may be obtained from stool 101 of subjects. The microbiota samples were transported to an anaerobic workstation (37° C., 10% H2, 10% CO2, and 80% N2) for processing and culturing. The samples were individually cultured in 15N-labeled growth media for 5 days 102 (passages) and kept during this time in anaerobic conditions. An optimized enriched media that is used may be modified dependent upon the microbiota sample used. In some embodiments, a skilled person will understand the enriched media used may be adapted to be suitable for the particular microbiota sample. For instance, in the case of the present intestinal microbiota samples, a 15N bacteria growth medium was supplemented with 0.1% w/v sodium thioglycolate and a 0.5 g/L bile salts mixture to accommodate intestinal bacteria. It will be understood that other adjustments may be made in order to provide a suitable growth medium that may be used in combination with other and/or additional labelled isotopes for the microbiome found in the sample. Moreover, it will be understood that the growth medium may include other isotopes that are to be incorporated into the microbiota. Even though 15N was used for the present intestinal microbiota sample, other isotopes may be used depending upon the nature of the microbiota present within the sample, the point of origination of the sample itself, and the desired labelling (e.g. a sulfur isotope may be used if only cysteine is to be labelled).

Results:

After each passage 102, as shown in FIG. 1A, the microbiota sample was analyzed by mass spectrometry, as is known in the art, to determine the 15N enrichment level of the microbiota sample. These results are shown in FIG. 1B. As illustrated in FIG. 1B, for the five individuals tested, the 15N enrichment rate of the samples was approximately 95% after two passages. This illustrates that it is possible to achieve a high isotope enrichment rate for a microbiota sample with a diverse microbe population (e.g. a microbiota sample originating from the intestine). A skilled person will appreciate that even though an intestinal microbiota sample was used in the present experiment to achieve this high enrichment rate, such isotope enrichment may be achieved by using other microbiota populations with a diverse microbe population, other than the one found in the intestine of a human subject (e.g. lung microbiota, cutis microbiota). Moreover, based upon these results, such microbiota samples may also be obtained from animal subjects, wherein animal subjects similarly have diverse microbial populations.

Moreover, for certain microbiota samples exposed to air (and oxygen), it may not be necessary to maintain anaerobic conditions.

Furthermore, a skilled person will also understand that by using other isotope enriched growth media, where the isotope is one other that 15N, it is appreciated that labelling a microbiota sample with other isotopes can be performed while still yielding a high enrichment rate, based upon these results, as presented in FIG. 1B.

The metaproteomes were analyzed by mass spectrometry. It will be readily understood that gas chromatography-mass spectrometry may also be used. It will be readily understood that the intestinal microbiota sample was selected because of its diversity of microbiota to demonstrate SILAMi's ability to provide a standard for such a complex microbiota population. However, it will be apparent that any other microbiota population with a diverse microbiota may be similarly used without departing from the present teachings (e.g. mucosal, lung, cutis, etc.).

Moreover, the number of peptides identified with complete ¹⁵N labeling increased (up to 11,800 peptides/sample after three days labeling), while the unlabeled peptides were minimally identified (less than 100 peptides/sample; FIG. 4). Percent atomic enrichment calculation using Census [10] also showed that all five microbiota tested reached an average ¹⁵N enrichment of >95%, which is more than sufficient for ¹⁵N-based quantitative proteomics, within three passages/days (FIG. 1B). However, even though over 95% enrichment rate is explained herein as being sufficient for quantitative proteomics as illustrated in the example of FIG. 1B, showing a very high and optimal enrichment rate, it will be understood by a person skilled in the art that the enrichment rate for quantitative proteomics may be anywhere over 50% and still provide acceptable results. In some examples, an enrichment rate of over 90%, as shown for the majority of subjects after 1 passage in FIG. 1B, is sufficient for quantitative proteomics or any other compositional analysis. These data demonstrate that this technique is capable of efficiently labeling a complex and metabolically diverse population of microbes.

In order for this labeling approach to have broad applicability to a microbiome, the labeling is to be occurring across the various phyla and species represented within the microbiome samples. For instance, in some examples, in order to examine the representability of the ¹⁵N-labelled SILAMi, the SILAMi microbial composition was compared to the initial inoculum (Passage 0) using metaproteomics-based methods. This demonstrates if the SILAMi labeled proteins were representative of the initial population in the microbiota sample. Briefly, all the identified peptide sequences (i.e. ¹⁵N peptides in SILAMi and ¹⁴N peptides in Passage 0) were phylogenetically classified using Unipept, which assigns taxonomic information for peptides based on lowest common ancestor (LCA) algorithm, UniProt database and NCBI taxonomy [11]. As shown in FIG. 5, 16 of 18 microbial phyla (including those belong to Bacteria, Archaea, and Eukaryota kingdoms), and 138 of 142 genera that were detected in Passage 0 remained in the SILAMi reference (FIG. 5). This demonstrates that this labeling approach can efficiently label across all kingdoms as well as the majority of genera which are present in human microbiome samples. It will be appreciate that such an isotope-labelling standard may be used to perform accurate and reproducible metaproteomics.

In some examples, an isotope-labelled standard that has labelled 50% or more of the microbe population corresponding to an initial microbiota sample (an initial microbiota sample being the microbe population of the sample when initially obtained from the patient) may be used. However, it will be understood that the percentage of the microbe population of an initial microbiota sample that is to labelled to obtain an effective standard may vary depending upon the nature of the experiment (if only certain populations are desirable, such as the study of hydrogen sulfide producing bacteria in the study of inflammatory bowel disease).

Experiment 2: Accurate Ratio Measurement Using SILAMi Labeled Samples

It was next tested whether accurate ratio measurement could be obtained using the SILAMi-based quantitative metaproteomics. Briefly, the same amount of SILAMi proteomes were spiked into different amounts of the unlabelled human gut metaproteome samples at L/H ratios of 1:1, 1.25:1, 2:1, and 5:1, respectively (e.g. FIG. 1A, 103). The mixtures were then processed for 4 hr gradient MS analysis on an Orbitrap Elite. A total of 6,943 unique peptide sequences corresponding to 4,014 protein groups were quantified and the L/H ratios 104 were calculated using Census. The distributions of calculated L/H ratios for all the quantified protein groups are shown in FIG. 2A, which demonstrated that the median L/H ratios were in great agreement with the spike-in ratios (Pearson's r=0.99). The fold change (FC) of each protein group between the four samples was then calculated by the “ratio of the L/H ratios” (FIG. 2B), which showed that narrow FC distributions were obtained, with 81-92% of the protein groups having less than two-fold difference to the median. Moreover, the median protein FCs (1.23, 1.93 and 4.39 folds; FIG. 2B) were in great agreement with the theoretical FC values (1.25, 2 and 5 folds, respectively). This demonstrates that metabolically labeled SILAMi microbiome can be used efficiently as an internal standard to reduce interexperiment and/or intraexperiment variability and optimize quantification in metaproteomics analysis. For instance, the use of an isotope-labelled standard for a microbiota population as described herein may identify false positives, changes in protein levels due to or experimental error or provide an indication if certain increases or reductions in the presence of certain proteins is a result of the preparation itself, or may be in fact due to a change in the microbiota as found in the original microbiota sample.

In summary, SILAMi represents a fast (3 days or less), efficient and cost-effective approach for generating metabolically labelled proteomes of intestinal microbial community, and allows accurate metaproteomic analysis of multiple samples with highly flexible experimental designs and implementations. SILAMi allows for highly standardized and quantitative analysis of the metaproteome, which will facilitate the use of metaproteomics analysis in the characterization of microbiome composition and function.

Example 1: Use of SILAMi to Assess Changes in a Microbiome as a Result of Treatment with a Compound

As a proof-of-principle example demonstrating the application of SILAMi to assess changes in a microbiome treated with a compound overtime, the approach was applied for evaluating the effects of fructooligosaccharide (FOS), a known prebiotic, on the microbiota. Briefly, unlabelled intestinal microbiota were cultured in basal culture medium (BCM) with or without 10 mg/ml FOS for 13 and 36 hours. The proteomes extracted from each microbial culture were spiked with the labelled SILAMi reference and analyzed by mass spectrometry. Principal component analysis of the 2,280 quantified proteins showed that FOS markedly shifted the overall metaproteome along the first principal component (explains 37.5% of the total variance; FIG. 3A). 187 proteins were significantly changed, as shown in Table 3:

TABLE 3
187 identified protein groups altered by fructo-oligosacchride (FOS) treatment
Protein_ID	Protein_name	Taxa
YP_007784111.1	LSU ribosomal protein L12P	Ruminococcus sp. SR1/5
CBK99399.1	pyruvate:ferredoxin (flavodoxin)	Faecalibacterium prausnitzii L2-6
	oxidoreductase, homodimeric
EEU98313.1	pyruvate synthase	Faecalibacterium prausnitzii A2-165
EFQ07801.1	pyruvate synthase	Faecalibacterium cf. prausnitzii KLE1255
EFJ59195.1	DNA-binding protein H-NS	Escherichia coli MS 200-1
CBL23538.1	Phosphotransferase system, HPr-	Ruminococcus obeum A2-162
	related proteins
EET15673.1	rubredoxin	Bacteroides sp. 4_3_47FAA
EHJ38221.1	glutamate dehydrogenase, NAD-	Prevotella stercorea DSM 18206
	specific
EEJ50816.1	pyruvate, phosphate dikinase	Oribacterium sinus F0268
EDR45788.1	pyruvate, phosphate dikinase	Dorea formicigenerans ATCC 27755
EFE12305.1	rubredoxin	Clostridium sp. M62/1
EDM52622.1	rubredoxin	Eubacterium ventriosum ATCC 27560
EEJ51973.1	chaperonin GroL	Oribacterium sinus F0268
CBK95920.1	glutamate dehydrogenase (NADP)	Eubacterium siraeum 70/3
EDP26881.1	Glu/Leu/Phe/Val dehydrogenase,	Coprococcus eutactus ATCC 27759
	dimerization domain protein
EEC56529.1	Glu/Leu/Phe/Val dehydrogenase,	Bacteroides pectinophilus ATCC 43243
	dimerization domain protein
WP_008981262.1	glutamate dehydrogenase	Ruminococcaceae bacterium D16
EEO57674.1	ribosomal protein L7/L12	Bacteroides sp. 2_2_4
EET14416.1	ribosomal protein L7/L12	Bacteroides sp. 4_3_47FAA
EDO55515.1	ribosomal protein L7/L12	Bacteroides uniformis ATCC 8492
EKU90753.1	acyl carrier protein	Bacteroides oleiciplenus YIT 12058
EFV67501.1	glyceraldehyde 3-phosphate	Bacteroides sp. 3_1_40A
	dehydrogenase
EDO52619.1	glyceraldehyde-3-phosphate	Bacteroides uniformis ATCC 8492
	dehydrogenase, type I
EDS13393.1	glyceraldehyde-3-phosphate	Bacteroides stercoris ATCC 43183
	dehydrogenase, type I
CBL23659.1	Glutamate dehydrogenase/leucine	Ruminococcus obeum A2-162
	dehydrogenase
CBK78198.1	glyceraldehyde-3-phosphate	Clostridium cf. saccharolyticum K10
	dehydrogenase, type I
EJZ69496.1	glyceraldehyde-3-phosphate	Lachnoanaerobaculum sp. OBRC5-5
	dehydrogenase, type I
EDO59009.1	glyceraldehyde-3-phosphate	Clostridium sp. L2-50
	dehydrogenase, type I
YP_007775284.1	glyceraldehyde-3-phosphate	Eubacterium siraeum 70/3
	dehydrogenase, type I
EEG49252.1	hypothetical protein RUMHYD_01832	Blautia hydrogenotrophica DSM 10507
EDP25141.1	hypothetical protein COPEUT_02635	Coprococcus eutactus ATCC 27759
EFE14625.1	hypothetical protein CLOM621_05456	Clostridium sp. M62/1
WP_009005634.1	hypothetical protein	Clostridium sp. D5
EGG85123.1	hypothetical protein	Lachnospiraceae bacterium 6_1_63FAA
	HMPREF0992_00050
EEG49378.1	ketol-acid reductoisomerase	Blautia hydrogenotrophica DSM 10507
CBK94839.1	chaperonin GroL	Eubacterium rectale M104/1
EFC97322.1	chaperonin GroL	Clostridium hathewayi DSM 13479
EFU71919.1	chaperone GroEL	Campylobacter upsaliensis JV21
EDO56757.1	Glu/Leu/Phe/Val dehydrogenase,	Clostridium sp. L2-50
	dimerization domain protein
ACD05861.1	Glutamate dehydrogenase (NADP(+))	Akkermansia muciniphila ATCC BAA-835
EEZ62164.1	Glu/Leu/Phe/Val dehydrogenase,	Slackia exigua ATCC 700122
	dimerization domain protein
EGW52698.1	NADP-specific glutamate	Desulfovibrio sp. 6_1_46AFAA
	dehydrogenase
EHH00248.1	hypothetical protein	Paraprevotella clara YIT 11840
	HMPREF9441_01482
EJZ66498.1	hypothetical protein	Barnesiella intestinihominis YIT 11860
	HMPREF9448_00677
EMZ42216.1	hypothetical protein	Atopobium minutum 10063974
	HMPREF1091_01190
EFI05647.1	conserved hypothetical protein	Bacteroides sp. 1_1_14
WP_010168870.1	Rubrerythrin	Epulopiscium sp. ‘N.t. morphotype B’
YP_007784205.1	Rubrerythrin	Ruminococcus sp. SR1/5
EID25347.1	glyceraldehyde-3-phosphate	Streptococcus pseudopneumoniae ATCC
	dehydrogenase, type I	BAA-960
AGJ88568.1	ADP-L-glycero-D-mannoheptose-6-	Raoultella omithinolytica B6
	epimerase
EFJ91797.1	ADP-glyceromanno-heptose	Escherichia coli MS 45-1
	6-epimerase
EGB20520.1	ribosomal protein S8	Clostridium symbiosum WAL-14673
EEG56094.1	hypothetical protein	Clostridium asparagiforme DSM 15981
	CLOSTASPAR_01820
EHP50162.1	reverse rubrerythrin-1	Clostridium perfringens WAL-14572
EGX68663.1	triosephosphate isomerase	Dorea formicigenerans 4_6_53AFAA
EDM62185.1	triose-phosphate isomerase	Dorea longicatena DSM 13814
EHL68608.1	triosephosphate isomerase	Bacillus sp. 7_6_55CFAA_CT2
WP_009261992.1	triosephosphate isomerase	Lachnospiraceae bacterium 9143BFAA
EES65026.1	butyryl-CoA dehydrogenase	Fusobacterium varium ATCC 27725
CBK80400.1	Acyl-CoA dehydrogenases	Coprococcus catus GD/7
YP_007771689.1	Acyl-CoA dehydrogenases	Eubacterium rectale DSM 17629
EEG92096.1	acyl-CoA dehydrogenase, C-terminal	Roseburia inulinivorans DSM 16841
	domain protein
YP_007789048.1	Acyl-CoA dehydrogenases	butyrate-producing bacterium SSC/2
EDO58999.1	acyl-CoA dehydrogenase, C-terminal	Clostridium sp. L2-50
	domain protein
EGB17912.1	acyl-CoA dehydrogenase, C-terminal	Clostridium symbiosum WAL-14673
	domain protein
WP_008981913.1	acyl-CoA dehydrogenase	Ruminococcaceae bacterium D16
CBL15057.1	hypothetical protein RBR_06960	Ruminococcus bromii L2-63
EEG90243.1	acetyl-CoA C-acetyltransferase	Coprococcus comes ATCC 27758
EGB17915.1	acetyl-CoA C-acetyltransferase	Clostridium symbiosum WAL-14673
EEG35102.1	pyridoxal-phosphate dependent TrpB-	Eubacterium hallii DSM 3353
	like enzyme
EGB18784.1	cell wall-binding repeat protein	Clostridium symbiosum WAL-14673
ADG61750.1	chaperonin protein Cpn60	Moraxella catarrhalis BBH18
EFU70513.1	chaperone GroEL	Arcobacter butzleri JV22
WP_010167160.1	molecular chaperone GroEL	Epulopiscium sp. ‘N.t. morphotype B’
YP_007849205.1	chaperonin GroL	Clostridium cf. saccharolyticum K10
EGA92696.1	hypothetical protein	Clostridium symbiosum WAL-14163
	HMPREF9474_03417
CBK80101.1	LSU ribosomal protein L10P	Coprococcus catus GD/7
YP_007785100.1	Formate-tetrahydrofolate ligase	Ruminococcus sp. SR1/5
EES75500.1	formate-tetrahydrofolate ligase	Ruminococcus sp. 5_1_39BFAA
CBL24477.1	Formate-tetrahydrofolate ligase	Ruminococcus obeum A2-162
WP_009644236.1	rubrerythrin domain protein	Mogibacterium sp. CM50
EGB17993.1	chaperonin GroL	Clostridium symbiosum WAL-14673
EDY32295.1	triose-phosphate isomerase	Ruminococcus lactaris ATCC 29176
YP_007786820.1	triosephosphate isomerase	Ruminococcus torques L2-14
ACV56754.1	ribosomal protein S13	Eggerthella lenta DSM 2243
EEX17882.1	glutamate dehydrogenase, NAD-	Prevotella veroralis F0319
	specific
EGN46805.1	50S ribosmal protein L7/L12	Lachnospiraceae bacterium 2_1_58FAA
EEG51161.1	Rubrerythrin, partial	Clostridium asparagiforme DSM 15981
EEX22906.1	formate-tetrahydrofolate ligase,	Blautia hansenii DSM 20583
	partial
YP_007782188.1	Glutamate dehydrogenase/leucine	Ruminococcus sp. SR1/5
	dehydrogenase
EEG47401.1	Glu/Leu/Phe/Val dehydrogenase,	Blautia hydrogenotrophica DSM 10507
	dimerization domain protein
EDQ97591.1	Glu/Leu/Phe/Val dehydrogenase,	Intestinibacter bartlettii DSM 16795
	dimerization domain protein
YP_007768517.1	Glutamate dehydrogenase/leucine	Coprococcus catus GD/7
	dehydrogenase
YP_007830561.1	Glutamate dehydrogenase/leucine	Roseburia intestinalis M50/1
	dehydrogenase
EHP49125.1	hypothetical protein	Clostridium perfringens WAL-14572
	HMPREF9476_01168
EFW89087.1	Glu/Leu/Phe/Val dehydrogenase,	Streptococcus equinus ATCC 9812
	dimerization domain protein
YP_007839796.1	glutamate dehydrogenase (NADP)	Eubacterium siraeum V10Sc8a
EEG31752.1	Glu/Leu/Phe/Val dehydrogenase,	Clostridium methylpentosum DSM 5476
	dimerization domain protein
EDS03463.1	glutamate dehydrogenase, NAD-	Alistipes putredinis DSM 17216
	specific
EKA95101.1	NADP-specific glutamate	Proteus mirabilis WGLW6
	dehydrogenase
EEG86298.1	Glu/Leu/Phe/Val dehydrogenase,	Proteus penned ATCC 35198
	dimerization domain protein
EES78421.1	hypothetical protein RSAG_00378	Ruminococcus sp. 5_1_39BFAA
EHG28402.1	hypothetical protein	Enterococcus saccharolyticus 30_1
	HMPREF9478_01803
EHO80265.1	hypothetical protein	Fusobacterium ulcerans 12-1B
	HMPREF0402_02090
CBK99448.1	Electron transfer flavoprotein, beta	Faecalibacterium prausnitzii L2-6
	subunit
EHJ31916.1	electron transfer flavoprotein subunit	Peptoclostridium difficile 002-P50-2011
	beta
EFB77236.1	electron transfer flavoprotein domain	Subdoligranulum variabile DSM 15176
	protein
WP_020989365.1	electron transfer flavoprotein beta	Ruminococcaceae bacterium D16
	subunit
YP_008664299.1	glutamate dehydrogenase	Adlercreutzia equolifaciens DSM 19450
YP_007801881.1	glutamate dehydrogenase (NADP)	Gordonibacter pamelaeae 7-10-1-b
CBL18303.1	glutamate dehydrogenase (NADP)	Ruminococcus champanellensis 18P13 =
		JCM 17042
WP_019893516.1	glutamate dehydrogenase	Allobaculum stercoricanis
EMZ41672.1	glutamate dehydrogenase (NADP+)	Atopobium minutum 10063974
YP_007837627.1	glutamate dehydrogenase (NADP)	Faecalibacterium prausnitzii L2-6
EDM50401.1	Glu/Leu/Phe/Val dehydrogenase,	Eubacterium ventriosum ATCC 27560
	dimerization domain protein
EFF68264.1	Glu/Leu/Phe/Val dehydrogenase,	Butyrivibrio crossotus DSM 2876
	dimerization domain protein
EEG37302.1	Glu/Leu/Phe/Val dehydrogenase,	Eubacterium hallii DSM 3353
	dimerization domain protein
EFR58785.1	glutamate dehydrogenase, NAD-	Alistipes sp. HGB5
	specific
CBK64456.1	glutamate dehydrogenase (NAD)	Alistipes shahii WAL 8301
EFW05566.1	Glutamate:leucine:phenylalanine:valine	Coprobacillus sp. 29_1
	dehydrogenase
CBL26527.1	Glutamate dehydrogenase/leucine	Ruminococcus torques L2-14
	dehydrogenase
EET58217.1	Glu/Leu/Phe/Val dehydrogenase,	Marvinbryantia formatexigens DSM 14469
	dimerization domain protein
EFK29209.1	Glu/Leu/Phe/Val dehydrogenase,	Lactobacillus plantarum subsp. plantarum
	dimerization domain protein	ATCC 14917
EEQ44915.1	NADP-specific glutamate	Candida albicans WO-1
	dehydrogenase
WP_009733626.1	glutamate dehydrogenase	Bilophila sp. 4_1_30
CBK74255.1	glutamate dehydrogenase (NADP)	Butyrivibrio fibrisolvens 16/4
EFC93334.1	Glu/Leu/Phe/Val dehydrogenase,	Methanobrevibacter smithii DSM 2374
	dimerization domain protein
EFI84299.1	Glu/Leu/Phe/Val dehydrogenase,	Listeria grayi DSM 20601
	dimerization domain protein
EEB34074.1	Glu/Leu/Phe/Val dehydrogenase,	Desulfovibrio piger ATCC 29098
	dimerization domain protein
EJF41864.1	Glu/Leu/Phe/Val dehydrogenase,	Actinomyces massiliensis F0489
	dimerization domain protein
YP_007781565.1	Glutamate dehydrogenase/leucine	Ruminococcus bromii L2-63
	dehydrogenase
EHL05236.1	NAD(P)-specific glutamate	Desulfitobacterium hafniense DP7
	dehydrogenase
EKX90337.1	Glu/Leu/Phe/Val dehydrogenase,	Corynebacterium durum F0235
	dimerization domain protein
EGG79390.1	NADP-specific glutamate	Lachnospiraceae bacterium 6_1_63FAA
	dehydrogenase
EJU21607.1	glutamate dehydrogenase, NAD-	Mogibacterium sp. CM50
	specific
EGX98849.1	glutamate dehydrogenase	Lactobacillus ruminis ATCC 25644
WP_002582046.1	glutamate dehydrogenase	Clostridium butyricum
EHN61579.1	Glu/Leu/Phe/Val dehydrogenase,	Listeria innocua ATCC 33091
	dimerization domain protein
YP_004374626.1	cryptic glutamate dehydrogenase	Carnobacterium sp. 17-4
CBL42834.1	Glutamate dehydrogenase/leucine	butyrate-producing bacterium SS3/4
	dehydrogenase
EES65252.1	translation elongation factor Ts	Fusobacterium varium ATCC 27725
EEG47205.1	formate--tetrahydrofolate ligase,	Blautia hydrogenotrophica DSM 10507
	partial
EFV22449.1	rubredoxin	Anaerostipes sp. 3_2_56FAA
EHO34579.1	hypothetical protein	Lachnospiraceae bacterium 7_1_58FAA
	HMPREF0995_01217
CBL23222.1	Carbon dioxide concentrating	Ruminococcus obeum A2-162
	mechanism/carboxysome shell protein
EHI57300.1	hypothetical protein	Clostridium hathewayi WAL-18680
	HMPREF9473_04409
CBL25604.1	Carbon dioxide concentrating	Ruminococcus torques L2-14
	mechanism/carboxysome shell protein
YP_007783749.1	Carbon dioxide concentrating	Ruminococcus sp. SR1/5
	mechanism/carboxysome shell protein
EFV41475.1	propanediol utilization protein PduA	Enterobacteriaceae bacterium 9_2_54FAA
EEG37602.1	BMC domain protein	Eubacterium hallii DSM 3353
EEB47897.1	ribosomal protein S4	Providencia alcalifaciens DSM 30120
EEQ62196.1	ribosomal protein S5	Clostridiales bacterium 1_7_47FAA
WP_009461300.1	30S ribosomal protein S5	Lachnospiraceae bacterium 2_1_46FAA
EEF93821.1	translation elongation factor Tu	Catenibacterium mitsuokai DSM 15897
EEY85351.1	translation elongation factor Tu	Acinetobacter radioresistens SH164
EFG16453.1	transporter, MotA/TolQ/ExbB proton	Bacteroides vulgatus PC510
	channel family protein
EFV65278.1	fructose-bisphosphate aldolase	Bacteroides sp. 3_1_40A
EEC95866.1	fructose-1,6-bisphosphate aldolase,	Parabacteroides johnsonii DSM 18315
	class II
CBK74269.1	phosphoenolpyruvate carboxykinase	Butyrivibrio fibrisolvens 16/4
	(ATP)
EEU32089.1	chaperonin	Fusobacterium nucleatum subsp. vincentii
		3_1_36A2
WP_018590022.1	molecular chaperone GroEL	Terrisporobacter glycolicus
EFS23154.1	chaperonin GroL	Fusobacterium necrophorum D12
EFY04737.1	B12 binding domain protein	Phascolarctobacterium succinatutens
		YIT 12067
EEQ65050.1	elongation factor Tu	Lactobacillus paracasei subsp. paracasei
		8700:2
CBL22274.1	phosphoenolpyruvate carboxykinase	Ruminococcus obeum A2-162
	(ATP)
CBL01371.1	phosphoenolpyruvate carboxykinase	Faecalibacterium prausnitzii SL3/3
	(ATP)
EDM63411.1	phosphoenolpyruvate carboxykinase	Dorea longicatena DSM 13814
	(ATP)
EEA80919.1	phosphoenolpyruvate carboxykinase	Tyzzerella nexilis DSM 1787
	(ATP)
EFV68881.1	plasminogen binding protein	Bacteroides sp. 3_1_40A
EEO45868.1	tetratricopeptide repeat protein	Bacteroides dorei 5_1_36/D4
EGN48017.1	50S ribosomal protein L7/L12	Lachnospiraceae bacterium
		3_1_57FAA_CT1
EGX69049.1	elongation factor Tu	Collinsella tanakaei YIT 12063
EEP44307.1	translation elongation factor Tu	Collinsella intestinalis DSM 13280
ACV51606.1	translation elongation factor Tu	Atopobium parvulum DSM 20469
YP_002294143.1	autonomous glycyl radical cofactor	Escherichia coli SE11
	GrcA
EER73923.1	translation elongation factor Tu	Weissella paramesenteroides ATCC 33313
EES64428.1	DNA-binding protein, YbaB/EbfC	Fusobacterium varium ATCC 27725
	family
EFK66259.1	phosphoenolpyruvate-protein	Escherichia coli MS 124-1
	phosphotransferase
EEX68873.1	ATP synthase F1, beta subunit	Mitsuokella multacida DSM 20544
EFY04736.1	methylmalonyl-CoA mutase domain	Phascolarctobacterium succinatutens
	protein	YIT 12067
ACV56138.1	ribosomal protein L2	Eggerthella lenta DSM 2243
EFY04939.1	ribosomal protein S3	Phascolarctobacterium succinatutens
		YIT 12067
EHR32371.1	ATP synthase subunit beta	Megamonas funiformis YIT 11815
EBA39887.1	translation elongation factor Tu	Collinsella aerofaciens ATCC 25986
EES64070.1	glyceraldehyde-3-phosphate	Fusobacterium varium ATCC 27725
	dehydrogenase, type I
EBA39912.1	ribosomal protein L7/L12	Collinsella aerofaciens ATCC 25986
EGX68988.1	30S ribosomal protein S10	Collinsella tanakaei YIT 12063

Among the identified 187 significantly changed proteins (FIG. 6), eight orthologs of the translation elongation factor Tu (EF-Tu) from different bacterial species were found to be increased by FOS (up to 6 fold, FIG. 3B). Additionally, nine orthologs of glyceraldehyde-3-phosphate dehydrogenase protein from different species were also quantified, among which eight isoforms were decreased while the one from Fusobacterium varium was increased. It also found that the proteins involved in endotoxin synthesis (eg., ADP-glyceromanno-heptose 6-epimerase) were decreased, suggesting an inhibiting role of FOS on potential endotoxin-producing pathogens in the intestinal tract. This demonstrates that SILAMi can be used to quantitatively assess changes in metaproteomic expression following treatment with a compound and that these changes can indicate specific pathways and functions that are effected by the treatment of these compounds. This approach could be used to identify therapeutic targets, diagnostics and assess treatment response.

Finally, it was tested whether the SILAMi labelled-standard could be used to distinguish the effects of different monosaccharides on the microbiota. It will be understood that monosaccharides are used herein as an example of a compound that may have an effect on the microbiota. However, other compounds that have be introduced to a microbiota sample or to a subject of which a microbiota sample has been obtained, that may impact the microbiota, may be similarly analyzed as described herein.

Overall, 18 samples cultured with or without 2.5 g/L of each monosaccharide (N-acetyl glucosamine or GlcNAc, mannose, galactose, fucose, or glucose) were analyzed, by SILAMi-based metaproteomics which led to 3,158 quantified proteins. Two hundred and forty-six protein groups were identified as being differentially abundant as compared to the non-treated control group (Table 4):

TABLE 4
the two hundred and forty-six protein groups were identified as being differentially abundant
as compared to the non-treated control group following metaproteomics with SILAMi.
Protein_ID	Protein_name	taxonomy
EFY04736.1	methylmalonyl-CoA mutase domain protein	Phascolarctobacterium
		succinatutens YIT 12067
EFF50472.1	chaperonin GroL	Bacteroides ovatus SD CMC 3f
EFG25764.1	methylmalonyl-CoA mutase	Veillonella sp. 6_1_27
EFG19106.1	pyruvate, phosphate dikinase	Bacteroides vulgatus PC510
EFD84126.1	lactaldehyde reductase	Klebsiella sp. 1_1_55
AGJ89383.1	L-fucose isomerase	Raoultella omithinolytica B6
EFK03838.1	arabinose isomerase	Escherichia coli MS 182-1
EKN23949.1	hypothetical protein HMPREF1059_02856	Parabacteroides distasonis
		CL09T03C24
EEZ28128.1	pyruvate, phosphate dikinase	Bacteroides sp. 2_1_16
EET14319.1	phosphoenolpyruvate carboxykinase (ATP)	Bacteroides sp. 4_3_47FAA
EET60019.1	ribosomal protein L7 L12	Marvinbryantia
		formatexigens DSM 14469
EFK61747.1	hypothetical protein HMPREF9008_02015	Parabacteroides sp. 20_3
EKN23143.1	hypothetical protein HMPREF1059_03287	Parabacteroides distasonis
		CL09T03C24
YP_002292782.1	hypothetical protein ECSE_1507	Escherichia coli SE11
EEJ51224.1	arabinose isomerase	Oribacterium sinus F0268
EFJ64969.1	lactaldehyde reductase	Escherichia coli MS 175-1
EEG94073.1	ribosomal protein L7 L12	Roseburia inulinivorans
		DSM 16841
EFG23247.1	translation elongation factor G	Veillonella sp. 3_1_44
YP_003350581.1	L-fuculose-1-phosphate aldolase	Escherichia coli SE15
EHM50183.1	glutamine-fructose-6-phosphate transaminase	Yokenella regensburgei
		ATCC 43003
YP_002294335.1	L-fuculose phosphate aldolase	Escherichia coli SE11
EHP45389.1	phosphoenolpyruvate carboxykinase [ATP]	Odoribacter laneus YIT
		12061
EFY05605.1	translation elongation factor G	Phascolarctobacterium
		succinatutens YIT 12067
EEA80457.1	hypothetical protein CLONEX_03662	Tyzzerella nexilis DSM
		1787
EEO62927.1	Xaa-His dipeptidase	Bacteroides sp. 9_1_42FAA
EFK60382.1	phosphoenolpyruvate carboxykinase (ATP)	Parabacteroides sp. 20_3
EDV01624.1	ribosomal protein S12	Bacteroides coprocola DSM
		17136
EFU58095.1	L-fucose: H+ symporter permease	Escherichia coli MS 16-3
EFD04604.1	ribosomal protein S12	Peptostreptococcus
		anaerobius 653-L
EBA39920.1	ribosomal protein S12	Collinsella aerofaciens
		ATCC 25986
EHL76277.1	30S ribosomal protein S12	Bacillus smithii 7_3_47FAA
EFG23245.1	ribosomal protein S12	Veillonella sp. 3_1_44
EEU51870.1	phosphoglycerate kinase	Parabacteroides sp. D13
EEZ25115.1	phosphoglycerate kinase	Bacteroides sp. 2_1_16
EGB19009.1	pyruvate, phosphate dikinase	Clostridium symbiosum
		WAL-14673
EHP48549.1	pyruvate, phosphate dikinase	Clostridium perfringens
		WAL-14572
EFU52197.1	glutamine-fructose-6-phosphate transaminase	Escherichia coli MS 153-1
	(isomerizing)
CDM03263.1	SSU ribosomal protein S11p (S14e)	Bacteroides xylanisolvens
		SD CC 1b
EEU49302.1	tetratricopeptide repeat protein	Parabacteroides sp. D13
EDM19917.1	transporter, MotA TolQ ExbB proton channel	Bacteroides caccae
	family protein	ATCC 43185
EHE95587.1	ketol-acid reductoisomerase	Clostridium citroniae
		WAL-17108
EDM88222.1	pyruvate, phosphate dikinase	Ruminococcus obeum
		ATCC 29174
EFC98279.1	pyruvate, phosphate dikinase	Clostridium hathewayi
		DSM 13479
EHI58090.1	pyruvate, phosphate dikinase	Clostridium hathewayi
		WAL-18680
EFV23852.1	hypothetical protein HMPREF1011_00311	Anaerostipes sp.
		3_2_56FAA
ACA17149.1	pyruvate, phosphate dikinase	Methylobacterium sp. 4-46
EDS09607.1	pyruvate, phosphate dikinase	Anaerotruncus colihominis
		DSM 17241
EFJ97558.1	glycerol-3-phosphate dehydrogenase, anaerobic,	Escherichia coli MS 115-1
	A subunit
EDN88029.1	hypothetical protein PARMER_00607	Parabacteroides merdae
		ATCC 43184
EHP67661.1	succinate dehydrogenase flavoprotein subunit	Escherichia coli
		4_1_47FAA
EFK60341.1	tetratricopeptide repeat protein	Parabacteroides sp. 20_3
EFK61507.1	malate dehydrogenase, NAD-dependent	Parabacteroides sp. 20_3
EEQ57746.1	pyruvate, phosphate dikinase	Clostridiales bacterium
		1_7_47FAA
EDP13424.1	hypothetical protein CLOBOL_06339	Clostridium bolteae ATCC
		BAA-613
EEZ23361.1	rubredoxin	Bacteroides sp. 3_1_33FAA
EET16778.1	trigger factor	Bacteroides sp. 4_3_47FAA
EEY84376.1	TonB-linked outer membrane protein, SusC RagA	Bacteroides sp. 2_1_33B
	family
B6I7Z9.1	Succinyl-CoA ligase [ADP-forming] subunit beta	Escherichia coli SE11
EEZ25084.1	fructose-1,6-bisphosphate aldolase, class II	Bacteroides sp. 2_1_16
AGH75904.1	glycerol kinase	Xanthomonas axonopodis
		Xac29-1
EEZ20650.1	malate dehydrogenase, NAD-dependent	Bacteroides sp. 3_1_33FAA
EGN42283.1	phosphoenolpyruvate carboxykinase	Lachnospiraceae bacterium
		1_1_57FAA
EEU51305.1	rubredoxin	Parabacteroides sp. D13
YP_007794708.1	Rubrerythrin	Bacteroides xylanisolvens
		XB1A
EFI88374.1	transcriptional regulator, PadR family	Escherichia coli MS 196-1
EET17854.2	fructose-1,6-bisphosphate aldolase, class II	Bacteroides sp. 4_3_47FAA
EFU52050.1	glycerol kinase	Escherichia coli MS 153-1
EEH85689.1	L-asparaginase	Escherichia sp. 3_2_53FAA
EGB18784.1	cell wall-binding repeat protein	Clostridium symbiosum
		WAL-14673
EEH89155.1	glycerol kinase	Escherichia sp. 3_2_53FAA
EEO47368.1	succinate dehydrogenase flavoprotein subunit	Bacteroides dorei 5_1_36
		D4
EFR55977.1	succinate dehydrogenase flavoprotein subunit	Bacteroides fragilis 3_1_12
EFV69103.1	fumarate reductase flavoprotein subunit	Bacteroides sp. 3_1_40A
EFK60272.1	succinate dehydrogenase flavoprotein subunit	Parabacteroides sp. 20_3
YP_003350036.1	hypothetical protein ECSF_2046	Escherichia coli SE15
EES78774.1	triosephosphate isomerase	Ruminococcus sp.
		5_1_39BFAA
EFJ74438.1	glycerophosphodiester phosphodiesterase family	Escherichia coli MS 198-1
	protein
EFD83709.1	universal stress family protein	Klebsiella sp. 1_1_55
EFK02128.1	L-asparaginase, type II	Escherichia coli MS 182-1
EFU51004.1	phosphoenolpyruvate carboxykinase (ATP)	Escherichia coli MS 153-1
EFG23975.1	methylmalonyl-CoA carboxyltransferase 12S	Veillonella sp. 3_1_44
	subunit
EFK64468.1	transporter, MotA TolQ ExbB proton channel	Parabacteroides sp. 20_3
	family protein
EFJ83172.1	arylsulfatase	Escherichia coli MS 69-1
EEH87575.1	oxidoreductase, short chain dehydrogenase	Escherichia sp. 3_2_53FAA
	reductase family protein
EFY04740.1	methylmalonyl-CoA decarboxylase alpha subunit	Phascolarctobacterium
		succinatutens YIT 12067
EHP67477.1	galactokinase	Escherichia coli
		4_1_47FAA
EHI58078.1	glyceraldehyde-3-phosphate dehydrogenase	Clostridium hathewayi
		WAL-18680
EGB74319.1	carbon starvation protein CstA	Escherichia coli MS 57-2
EFJ62757.1	threonine ammonia-lyase	Escherichia coli MS 200-1
YP_007825311.1	Glutamate dehydrogenase leucine dehydrogenase	butyrate-producing bacterium SS3 4
EFJ73903.1	galactose mutarotase	Escherichia coli MS 198-1
AGJ85389.1	threonine dehydratase	Raoultella ornithinolytica B6
EFE22430.1	L-asparaginase, type II	Edwardsiella tarda ATCC
		23685
EHC27190.1	chaperonin 1	Propionibacterium sp.
		5_U_42AFAA
EDN83951.1	chaperonin GroL	Bifidobacterium
		adolescentis L2-32
YP_007766843.1	chaperonin GroL	Bifidobacterium longum
		subsp. longum F8
EEZ21779.1	ribosomal protein L22	Bacteroides sp. 3_1_33FAA
EFJ96310.1	UDP-glucose 4-epimerase	Escherichia coli MS 115-1
EEY85226.1	outer membrane protein 40	Bacteroides sp. 2_1_33B
EFK20636.1	PTS system, N-acetylglucosamine-specific IIBC	Escherichia coli MS 21-1
	component
EFJ79729.1	PTS system, N-acetylglucosamine-specific IIBC	Escherichia coli MS 69-1
	component
YP_003348608.1	glutaminyl-tRNA synthetase	Escherichia coli SE15
EEH84887.1	universal stress family protein	Escherichia sp. 3_2_53FAA
EDY33848.1	ribosomal protein L13	Ruminococcus lactaris
		ATCC 29176
EGB20384.1	ribosomal protein L13	Clostridium symbiosum
		WAL-14673
EDQ97629.1	hypothetical protein CLOBAR_00369	Intestinibacter bartlettii
		DSM 16795
EFJ75883.1	ABC transporter, substrate-binding protein, family	Escherichia coli MS 198-1
	5
EFU59091.1	Glu Leu Phe Val dehydrogenase, dimerization	Escherichia coli MS 16-3
	domain protein
B6I3D0.1	Glycine--tRNA ligase alpha subunit	Escherichia coli SE11
EEH85049.1	hypothetical protein ESAG_00761	Escherichia sp. 3_2_53FAA
B6I227.1	50S ribosomal protein L16	Escherichia coli SE11
EFK74482.1	tyrosine--tRNA ligase	Escherichia coli MS 78-1
WP_001295080.1	lysyl-tRNA synthetase	Escherichia sp. 4_1_40B
AFU19253.1	chaperonin GroEL	Actinobacillus suis H91-0380
YP_003349385.1	30S ribosomal protein S22	Escherichia coli SE15
EFV40188.1	2,3,4,5-tetrahydropyridine-2,6-dicarboxylate	Enterobacteriaceae bacterium
	N-succinyltransferase	9_2_54FAA
EFK88351.1	isoleucine--tRNA ligase	Escherichia coli MS 146-1
EFD82816.1	2,3,4,5-tetrahydropyridine-2,6-dicarboxylate	Klebsiella sp. 1_1_55
	N-succinyltransferase
EHP65572.1	phenylalanyl-tRNA synthetase alpha chain	Escherichia coli
		4_1_47FAA
EEH89206.1	LOW QUALITY PROTEIN: hypothetical protein	Escherichia sp. 3_2_53FAA
	ESAG_04918, partial
EFJ71500.1	GMP reductase	Escherichia coli MS 198-1
ADX45418.1	anti-sigma H sporulation factor, LonB	Acidovorax avenae subsp.
		avenae ATCC 19860
EGG53815.1	putative transcriptional regulatory protein FixJ	Parasutterella
		excrementihominis YIT 11859
B6I152.1	LPS-assembly lipoprotein LptE	Escherichia coli SE11
EFC57150.1	hypothetical protein ENTCAN_05663	Enterobacter cancerogenus
		ATCC 35316
EKA96042.1	adenylosuccinate synthetase	Proteus mirabilis WGLW6
EHM49114.1	lysine--tRNA ligase	Yokenella regensburgei
		ATCC 43003
EFJ58662.1	glutamate--tRNA ligase	Escherichia coli MS 200-1
YP_003351947.1	peptide ABC transporter substrate binding	Escherichia coli SE15
	component
EFU57211.1	hypothetical protein HMPREF9545_03054	Escherichia coli MS 16-3
EGK61283.1	chaperone GroEL	Enterobacter hormaechei
		ATCC 49162
EDU60353.1	peptidyl-prolyl cis-trans isomerase B	Providencia stuartii
		ATCC 25827
EFJ74879.1	Dyp-type peroxidase family protein	Escherichia coli MS 198-1
YP_003349700.1	glucose-6-phosphate dehydrogenase	Escherichia coli SE15
EHM47365.1	glyceraldehyde-3-phosphate dehydrogenase, type I	Yokenella regensburgei
		ATCC 43003
YP_003349494.1	outer membrane lipoprotein	Escherichia coli SE15
EEH72503.1	phosphopentomutase	Escherichia sp. 1_1_43
YP_003539075.1	pyruvate kinase II	Erwinia amylovora ATCC 49946
YP_003348183.1	hypothetical protein ECSF_0193	Escherichia coli SE15
EFJ68155.1	ATP-dependent protease HslVU, ATPase subunit	Escherichia coli MS 175-1
YP_003348995.1	hypothetical protein ECSF_1005	Escherichia coli SE15
YP_003537512.1	molecular chaperone GroEL	Erwinia amylovora ATCC 49946
YP_007847195.1	glutamyl-tRNA synthetase	Enterobacter cloacae subsp.
		cloacae NCTC 9394
EEH92664.2	glyceraldehyde-3-phosphate dehydrogenase A	Citrobacter sp. 30_2
EKA97512.1	glycyl-tRNA synthetase alpha subunit	Proteus mirabilis WGLW6
EFK92190.1	ADP-glyceromanno-heptose 6-epimerase	Escherichia coli MS 146-1
YP_003348413.1	acridine efflux pump protein AcrA	Escherichia coli SE15
B6I615.1	AltName: Full = GroEL protein; AltName:	0
	Full = Protein Cpn60; CH60_ECOSE RecName:
	Full = 60 kDa chaperonin
YP_003537198.1	serine acetyltransferase	Erwinia amylovora ATCC
		49946
EFK17770.1	co-chaperone GrpE	Escherichia coli MS 21-1
ADN76170.1	pyruvate kinase	Ferrimonas balearica DSM
		9799
YP_003350270.1	PTS system enzyme I	Escherichia coli SE15
EFP65709.1	phosphopyruvate hydratase	Ralstonia sp. 5_7_47FAA
B3PJB3.1	2-phospho-D-glycerate hydro-lyase	Cellvibrio japonicus
		Ueda107
EEH94022.2	autonomous glycyl radical cofactor	Citrobacter sp. 30_2
EEH95287.2	aspartate-semialdehyde dehydrogenase	Citrobacter sp. 30_2
EHP49497.1	enolase	Clostridium perfringens
		WAL-14572
EFJ73315.1	sporulation and cell division repeat protein	Escherichia coli MS 198-1
YP_002292075.1	translocation protein TolB	Escherichia coli SE11
EFU37439.1	hexose kinase, 1-phosphofructokinase family	Escherichia coli MS 85-1
B6I4S5.1	Heat shock protein HslV	Escherichia coli SE11
EFU37381.1	glyceraldehyde-3-phosphate dehydrogenase, type	Escherichia coli MS 85-1
	I
YP_003349138.1	putative PTS system enzyme I	Escherichia coli SE15
EGJ08498.1	6-phosphofructokinase	Shigella sp. D9
EJZ47828.1	fructose-bisphosphate aldolase class 1	Escherichia sp. 1_1_43
EFK73994.1	NAD(P)H: quinone oxidoreductase, type IV	Escherichia coli MS 78-1
YP_003348173.1	methionine aminopeptidase	Escherichia coli SE15
WP_018592092.1	hypothetical protein	Terrisporobacter glycolicus
EDO60098.1	formate C-acetyltransferase	Clostridium leptum DSM 753
WP_024039107.1	Formate acetyltransferase	Clostridium butyricum
EGU99618.1	2,3-bisphosphoglycerate-independent	Escherichia coli MS 79-10
	phosphoglycerate mutase
EFU56329.1	2,3-bisphosphoglycerate-independent	Escherichia coli MS 16-3
	phosphoglycerate mutase
B6I2X8.1	Fe S biogenesis protein NfuA	Escherichia coli SE11
EEH87714.1	autonomous glycyl radical cofactor	Escherichia sp. 3_2_53FAA
A6VUU9.1	2-phospho-D-glycerate hydro-lyase	Marinomonas sp. MWYL1
YP_003348538.1	alkyl hydroperoxide reductase subunit F	Escherichia coli SE15
YP_002291949.1	alkyl hydroperoxide reductase subunit F	Escherichia coli SE11
EFJ56595.1	stringent starvation protein A	Escherichia coli MS 185-1
YP_003348537.1	alkyl hydroperoxide reductase subunit C	Escherichia coli SE15
EFK73356.1	chaperonin GroS	Escherichia coli MS 78-1
WP_009008039.1	autonomous glycyl radical cofactor GrcA	Shigella sp. D9
EFK71210.1	glucose-6-phosphate isomerase	Escherichia coli MS 78-1
AGJ88568.1	ADP-L-glycero-D-mannoheptose-6-epimerase	Raoultella ornithinolytica B6
EKB82759.1	chaperonin	Klebsiella pneumoniae
		subsp. pneumoniae WGLW5
YP_003348095.1	cell division protein FtsZ	Escherichia coli SE15
YP_002291773.1	heat shock protein 90	Escherichia coli SE11
EFK25863.1	deoxyribose-phosphate aldolase	Escherichia coli MS 187-1
EFU57607.1	deoxyribose-phosphate aldolase	Escherichia coli MS 16-3
EFJ62792.1	hypothetical protein HMPREF9553_01102	Escherichia coli MS 200-1
YP_003350475.1	autoinducer-2 production protein	Escherichia coli SE15
EFU58344.1	peptidyl-prolyl cis-trans isomerase B	Escherichia coli MS 16-3
EGF14772.1	formate acetyltransferase	Haemophilus aegyptius
		ATCC 11116
YP_003348814.1	formate acetyltransferase 1	Escherichia coli SE15
EFE22433.1	formate C-acetyltransferase	Edwardsiella tarda ATCC
		23685
EGU97855.1	formate acetyltransferase	Escherichia coli MS 79-10
EFK20865.1	phosphoserine transaminase	Escherichia coli MS 21-1
A6VZ92.1	Phosphohydroxythreonine aminotransferase	Marinomonas sp. MWYL1
EFD85361.1	formate C-acetyltransferase	Klebsiella sp. 1_1_55
EFJ53806.1	rhodanese-like protein	Escherichia coli MS 185-1
EFK4491E1	formate C-acetyltransferase	Escherichia coli MS 119-7
YP_004220428.1	formate acetyltransferase	Bifidobacterium longum
		subsp. longum JCM 1217
AGJ89504.1	S-ribosylhomocysteinase	Raoultella omithinolytica B6
YP_003349716.1	aspartyl-tRNA synthetase	Escherichia coli SE15
EEH86836.1	aspartate--tRNA ligase	Escherichia sp. 3_2_53FAA
EDP16360.1	hypothetical protein CLOBOL_03126	Clostridium bolteae
		ATCC BAA-613
EFR46519.1	chaperone protein ClpB	Helicobacter cinaedi
		CCUG 18818
EFK89002.1	glycerol-3-phosphate dehydrogenase, anaerobic,	Escherichia coli MS 146-1
	C subunit
EFK00860.1	beta-aspartyl peptidase	Escherichia coli MS 182-1
EFO55869.1	chaperone protein DnaK	Escherichia coli MS 145-7
YP_003350137.1	hypothetical protein ECSF_2147	Escherichia coli SE15
YP_003351949.1	truncated formate dehydrogenase H, partial	Escherichia coli SE15
EGU96599.1	ATP-dependent chaperone protein ClpB	Escherichia coli MS 79-10
EFE53197.1	methionine adenosyltransferase	Providencia rettgeri
		DSM 1131
YP_003537739.1	chaperone protein	Erwinia amylovora ATCC 49946
EEH84693.1	outer membrane protein X	Escherichia sp. 3_2_53FAA
EFU58863.1	pyrroline-5-carboxylate reductase	Escherichia coli MS 16-3
EFK20818.1	curved DNA-binding protein	Escherichia coli MS 21-1
EFD82978.1	chaperone protein DnaK	Klebsiella sp. 1_1_55
YP_003351331.1	putative lipoprotein	Escherichia coli SE15
EEH85525.1	TIGR00156 family protein	Escherichia sp. 3_2_53FAA
EFO56144.1	outer membrane protein slp	Escherichia coli MS 145-7
EHP49037.1	chaperone ClpB	Clostridium perfringens
		WAL-14572
EFB70971.1	chaperone protein DnaK	Providencia rustigianii DSM 4541
EEZ20102.1	hypothetical protein HMPREF0105_3492	Bacteroides sp. 3_1_33FAA
EFJ65716.1	putative protein HdeB	Escherichia coli MS 175-1
EFU51921.1	DNA protection during starvation protein	Escherichia coli MS 153-1
EJZ49312.1	N-acetylglucosamine-6-phosphate deacetylase	Escherichia sp. 1_1_43
EFU58593.1	DNA protection during starvation protein	Escherichia coli MS 16-3
EFO58101.1	N-acetylglucosamine-6-phosphate deacetylase	Escherichia coli MS 145-7
YP_003349426.1	hypothetical protein ECSF_1436	Escherichia coli SE15
EFU51394.1	glucosamine-6-phosphate deaminase	Escherichia coli MS 153-1
EEH70955.1	aspartate-ammonia ligase	Escherichia sp. 1_1_43
YP_003349391.1	amino acid antiporter	Escherichia coli SE15
EFK92597.1	oxygen-insensitive NAD(P)H nitroreductase	Escherichia coli MS 146-1
EFK24351.1	aminotransferase AlaT	Escherichia coli MS 187-1
EFK90487.1	glutamate decarboxylase	Escherichia coli MS 146-1
YP_003351335.1	glutamate decarboxylase	Escherichia coli SE15
EFC55519.1	asparagine synthase (glutamine-hydrolyzing)	Enterobacter cancerogenus
		ATCC 35316
EFK50572.1	asparagine synthase (glutamine-hydrolyzing)	Escherichia coli MS 107-1
EFR55608.1	ribosomal protein S10	Bacteroides fragilis 3_1_12
EFR55609.1	50S ribosomal protein L3	Bacteroides fragilis 3_1_12
EFK61943.1	SusD family protein	Parabacteroides sp. 20_3
EBA38760.1	ribosomal protein S20	Collinsella aerofaciens
		ATCC 25986
EGB75313.1	indole-3-glycerol phosphate synthase	Escherichia coli MS 57-2
EFK61740.1	Tat pathway signal sequence domain protein	Parabacteroides sp. 20_3
EET16977.1	phosphoglucomutase	Bacteroides sp. 4_3_47FAA

Unique metaproteome patterns were observed in response to the different monosaccharide treatments (FIG. 3C). The monosaccharide fucose showed the smallest effect on the metaproteome, however cluster 206, mainly consisting of fucose utilization-related proteins, was increased only in the fucose-treated microbiota (FIG. 3C). Moreover, all of the six quantified fucose utilizing proteins in the fucose utilization pathway were increased upon the supplementation of fucose (FIG. 3D) [12]. On the other hand, GlcNAc resulted in the most dramatic alterations of the metaproteome with significant increase of GlcNAc degrading proteins (FIG. 3A-C), which produce fructose 6-phosphate and NH3. The latter may be used for asparagine synthesis since asparagine synthase was also significantly increased (FIG. 3D).

As a result, it is shown that use of the heavy-labelled standard obtained via SILAMi may be used to assess changes in a microbiome as a result of a given compound. More specifically, this approach allows for identification of specific pathways and metabolic processes which may be altered in a treated microbiome sample. This data could be used by one of skill in the art to determine who changes in composition effect function as well as identify pathways effected in disease or by drug and chemical treatment. It will be understood that such compounds may include xenobiotics, but also drugs, chemicals, therapeutic agents, toxins, poisons, beverages, food additives, cosmetics, cosmetic ingredients, packaging materials, pesticides, herbicides, consumer products. A skilled person recognizes that a given microbiome is very sensitive to change, and therefore such a compound may have an impact upon the microbiome. Such an impact is now quantifiable as a result of the heavy-labelled standard developed using the SILAMi technique.

Taken together, a fast and cost-effective approach is provided, namely SILAMi, to perform accurate and large-scale quantitative metaproteomic studies on the microbiota. Moreover, it was successfully applied to screen and evaluate the effects of different compounds on human microbiota. More interestingly, new insights on the interactions between drug, microbe and host may be acquired through experiments benefiting from the heavy-labelled standard obtained with SILAMi. Thus, the application of SILAMi can help to improve the accuracy of metaproteomics, thereby largely promoting its application in studying the microbiota in the context of health and disease. It will be understood that such study in the context and disease may include determining for a given patient if the disease is in remission or if the disease is worsening in severity. The study may also involve determining if a patient is responding to a given treatment, or even determining which treatment should be given for a specific patient. Furthermore, diagnosis of disease is also possible with SILAMi. It is known in that changes in health and disease often yield a change in the microbiota of the patient. These changes, in particular in depth metaproteomic changes, can now be quantified and analyzed as a result of the heavy-labelled standard obtained using SILAMi.

Example 2: RapidAIM, a High Throughput Screening Platform to Assess the Effect of Drugs

Reference is now made to RapidAIM, an experimental and computational framework to rapidly assay an individual's microbiome (called RapidAIM), a platform to assess the effects of compounds including but not limited to drugs on the microbiome and drug metabolism is described. The use of RapidAIM to validate the platform for compounds, specifically, in this example, those used in IBD, is described (FIGS. 8, 9). Briefly, RapidAIM consists of panel of microbiomes derived from multiple individuals that are treated with selected compounds and screened in a multi-well format; This approach allows for rapid classification of compounds that have no affect or affect the microbiota composition (biota-affectors), or compounds that are affected by the microbiome (biota-altered) using metagenomic (¹⁶S-based sequencing), and/or fast-pass metaproteomics, and/or metabolomics assessment of the microbiome's metabolic activities on the compound; Optionally, the RapidAIM platform will be utilized to gain mechanistic insights on these compounds in combination with functional metagenomics, metatranscriptomics and in depth metaproteomics. It will be appreciated that certain steps of the RapidAIM can be performed separately. Finally, this bioinformatics platform can be used to rapidly guide the selection of positive hit compounds based on the metaOMICS analyses of an individual's microbiome or broad screening of many microbiomes. Currently, no technology exists to rapidly assay individual microbiome, particularly with respect to metaproteomics. The RapidAIM project is transformative to the pharmaceutical and biotechnology development and microbiology field, as it allows for the rapid screening of candidate drugs against human microbiome before the drugs are commercialized, to screen current drugs for potential adverse microbiome effects, to stratify patients based on their response to drug treatment, and to screen compounds that would have efficacy across a wider population.

In another embodiment, RapidAIM can be used to screen a panel of microbiomes derived from IBD and control patients in multi-well plates against selected xenobiotics. However, it will be understood that RapidAIM may also be used in the context of selected therapeutics, amino acids, and dietary supplements, etc. Assessment of the changes in the metaproteome upon treatment with any such compounds in the microbiota of healthy individuals or those associated with a disease other than inflammatory bowel disease may be similarly performed without departing from the present teachings. Biota-affectors can be selected by metagenomic (¹⁶S-based sequencing) analysis of microbial composition changes and fast-pass metaproteomics to identify impacts on the top 1,500 most abundant proteins. Biota-altered compounds can be identified by metabolomics. Each multi-well plate takes approximately 2 days for screening and can identify compounds that either target specific microbes or group of microbes and/or their metabolic activities. Furthermore, this screening can be done to determine the effect of any compound upon the microbiome. The assay can be repeated on a reduced pool of compounds to generate functional metagenomics, metatranscriptomics and more in-depth metaproteomics (4000-5000 proteins/sample). A modeling algorithm can be used to rapidly guide selection of compounds based on the metaOMICS analyses, and pathway databases.

Developing RapidAIM in a multi-well plate format: microbiota can be inoculated and grown in culture media. Assays, performed in any multiwall format (e.g. 6 well to 96 well plate formats, or any other type of format, for example, using tubes) and can be titrated, examining at each stage whether the yield per well provides sufficient material for downstream analyses. The analysis can be performed using a workflow for metaproteome as described in Zhang et al. “MetaPro-IQ: a universal metaproteomic approach to studying human and mouse gut microbiota”, Microbiome, 2016 Jun. 24:4(1):31, doi: 10.1186/s40168-016-0176-z. The workflow uses the close-to-complete human or mouse gut microbial gene catalog as a database and uses an iterative database search strategy. An example of a high-through put experimental workflow for the RapidAIM has been established based on a 96-well format (FIG. 9). This workflow includes, (A) 96-well based microbiome culturing, and (B) 96-well based metaproteomics analysis.

As shown in FIG. 9, one of the steps in the workflow describes how “the SILAMi spike in can be added here”, where the SILAMi spike may be optionally added. The preparation of the “SILAMi spike” or the “SILAMi Reference Standard” can be prepared as described herein. Alternatively, a representative superSILAMi standard for quantitative metaproteomics can be used. To prepare a superSILAMi standard, multiple SILAMi reference samples can be combined using strategic additions to the culture media and samples from many individuals. The superSILAMi reference can be used as a comprehensive quantitative spike-in for diverse samples.

The performance of RapidAIM may also involve parameter setting for time in order to measure (i) microbiota changes and (ii) generation of drug metabolites. These can be guided for example by current literature including from in vitro liver system drug metabolism tests [13]. Briefly, microbiota can be inoculated and grown in basal culture media with or without compounds for different times (ranging from 30 min to 24 hrs), and samples collected for analyses.

The performance of RapidAIM may also involve parameter setting for the dosage of each compound in the pool which can be tested and pre-determined using the clinical dosage or reported concentrations for culturing as guidance. Microbiota from multiple individuals (including both male and female) can be used to negate inter-individual variability of intestinal microbiota.

As an example, RapidAIM was used to assay an individual a microbiome treated with a high, medium or low dose of berberine compared to the sample cultured without drug treatment (FIG. 10). The taxonomic composition at the species level were quantified in each of the cultured microbiome sample on the MetaLab bioinformatics platform. FIG. 10A shows the results from the loadings plot of a principal component analysis (PCA) with all bacterial species. FIG. 10B shows that the abundances of the species originating from the Akkermansia genus (identified in FIG. 10B as Akkermansia species 1, Akkermansia species 2 and Akkermansia species 3) were significantly increased when treated with high concentration of Berberine. Akkermansia spp has been reported to be beneficial bacterial in the gut microbiome, which has been shown to be increased by another antidiabetic drug, Metformin.

The description of the present invention has been presented for purposes of illustration but is not intended to be exhaustive or limited to the disclosed embodiments. Many modifications and variations will be apparent to those of ordinary skill in the art.

REFERENCES

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• 4. Verberkmoes, N. C., et al. ISME J, 2009. 3 (2), 179-89.
• 5. Juste, C., et al. Gut, 2014. 63 (10), 1566-77.
• 6. Jagtap, P., et al. J Proteomics, 2013. 13, 1352-1357.
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Claims

1-73. (canceled)

74. A method of high throughput screening of multiple microbiota samples from a human subject, soil, or animal for metaproteomic analysis of said samples comprising: providing proteome extracts from a plurality of microbiota samples from one or more human subjects, soil, or animals in culture;spiking said proteome extracts with an isotope-labelled standard, which comprises a proteome extract of microbiota from a given human, soil, or animal microbiota sample labelled with an isotope by culturing said microbiota from a given human, soil, or animal microbiota in an isotope enriched medium;performing a pre-screening using a metaproteomic analysis to identify changes in microbiomes of said plurality of microbiota samples;selecting said microbiomes exhibiting changes; andanalyzing the selected microbiomes to characterize the changes.

75. The method of claim 74, wherein said analyzing comprises using a microbial gene catalog of a given subject type and an iterative database search strategy.

76. The method of claim 74, wherein said analyzing comprises performing a metaproteomic analysis combined with a metagenomic analysis.

77. The method of claim 75, wherein said microbial gene catalog of a given subject type is a microbial gene catalog of an animal or human.

78. The method of claim 74, wherein said spiking comprises adding sufficient isotope-labelled standard to reach a 1:1 protein mass ratio with the protein contained in said proteome extracts from the plurality of microbiota samples.

79. The method of claim 74, wherein the plurality of microbiota samples are from one or more human subjects or animals, and wherein the method further comprises assessing the results of said analysis of the selected microbiomes to perform at least one of: disease diagnosis in a target human subject or an animal;assessing treatment response in a target human subject or an animal;assessing remission in a human subject or an animal receiving treatment;screening for xenobiotic effects on a microbiome of a target human subject or an animal;screening for effects of a compound on a microbiome of a target human subject or an animal, wherein said compound is one of a food, a drug, a chemical, a therapeutic agent, a toxin, a poison, a beverage, a food additive, a cosmetic, a cosmetic ingredient, packaging material, a pesticide, a herbicide, a consumer product; andscreening a microbiome to identify the responsiveness of a human subject or an animal to a therapy or treatment.

80. The method of claim 74, wherein said isotope-labelled standard comprises labelled proteins representative of a metaproteome from the given human, soil, or animal microbiota sample and having at least 90% average heavy isotopic enrichment rate.

81. The method of claim 80, wherein the given human microbiota sample is from an intestinal microbiota.

82. The method of claim 74, wherein said isotope-labelled standard is taxon-specific for at least about 50% of the microbe populations present in the plurality of microbiota samples from the one or more human subjects, soil, or animals.

83. The method of claim 74, wherein said isotope-labelled standard is taxon-specific for at least about 85% of the microbe populations present in the plurality of microbiota samples from the one or more human subjects, soil, or animals.

84. The method of claim 74, wherein the plurality of microbiota samples from the one or more human subjects are from an intestinal microbiota, a vaginal microbiota, an oral microbiota, a cutis microbiota, a bladder microbiota, a kidney microbiota, a lung microbiota, an eye microbiota, a breast microbiota, a penile microbiota, or any combination thereof.

85. The method of claim 74, wherein the isotope comprises a stable isotope, a radioactive isotope, or both.

86. The method of claim 85, wherein the isotope comprises 13C, 14C, 15N, 32S, 35S, 32P, deuterium, or any combination thereof.

87. A method of performing a compositional analysis of a microbiota sample comprising providing proteome extract from a microbiota sample from a human subject, soil, or animal in culture;spiking said proteome extract with an isotope-labelled standard, which comprises a proteome extract of microbiota from a first given human, soil, or animal microbiota sample cultured in an isotope enriched medium;performing metaproteomic analysis, and wherein said metaproteomic analysis is for measuring an amount of one or more protein in said microbiota sample, said metaproteomic analysis comprising:obtaining labelled/unlabelled protein ratios of proteins in said labelled standard and said proteome extract.

88. The method of claim 87, wherein said metaproteomic analysis further comprises obtaining a label free quantification (LFQ) of said proteome extract.

89. The method of claim 87, wherein said method further comprises performing metagenomic analysis.

90. The method of claim 89, wherein said metagenomic analysis comprises 16S-based sequencing or shotgun sequencing.

91. The method of claim 87, wherein said compositional analysis is performed to achieve at least one of: disease diagnosis in a target subject;assessing treatment response in a target subject;assessing remission in a subject receiving treatment;screening for xenobiotic effects on a microbiome of a target subject;screening for effects of a compound on a microbiome of a target subject, wherein said compound is one of a food, a drug, a chemical, a therapeutic agent, a toxin, a poison, a beverage, a food additive, a cosmetic, a cosmetic ingredient, packaging material, a pesticide, a herbicide, a consumer product; andscreening a microbiome to identify the responsiveness of a subject to a therapy or treatment.

92. The method of claim 87, wherein said compositional analysis is performed to achieve said disease diagnosis in a target subject, wherein said using a labelled microbiota sample as a labelled standard to perform compositional analysis further comprises measuring an amount of said one or more protein in said second microbiota sample and wherein deviation from normal is indicative of said disease.

93. The method of claim 92, wherein said metaproteomic analysis is performed at one or more time points using a time-point microbiota sample taken at said one or more time points, and wherein, following a metaproteomic analysis performed on said time-point microbiota sample, a measured one or more proteins from said time-point microbiota sample is compared to a control sample, wherein said control sample is at least one of: a standard control sample taken from a subject in a predetermined state of health; and a control sample obtained at a predetermined time in the life of said target subject.