Patent Application
20230393041
Biological systems contain complex networks of diverse molecules that work together to modulate cellular processes. While many technologies interrogate a single biomolecule class, such as proteins, substantially more information and value can be gained from performing multi-omic studies, which profile multiple biomolecule classes simultaneously.
Integrated analysis of biomolecules was once a lofty goal; however, multi-omic investigations are now feasible and increasingly applied across biological disciplines,1-3 in part due to improvements in mass spectrometry (MS) technologies. Evolving MS data acquisition strategies have increased the number of proteins, lipids, and metabolites surveyed;4-13 yet, the demand for faster and more efficient data collection persists. To meet this demand, simple, robust MS workflows must be developed that offer integrated analysis of multiple compound classes. While many recent advances have focused on improving integration of MS data acquisition and analysis strategies,14-18 other aspects of the MS multi-omic pipeline, such as sample preparation, are still in need of simplification and consolidation.
Sample preparation for published multi-omics studies involves either splitting samples into multiple aliquots for different -omes, or relying on extensive multi-step processes to isolate multiple compound classes.19-28 These multi-step methods often require extracting metabolites and lipids with a biphasic organic solvent system, precipitating proteins, and digesting proteins with trypsin (
And while robust and reproducible, these extraction methods require copious pipetting, vortexing, incubating, and centrifuging steps. Such steps are low throughput and susceptible to excessive sample loss. After phase separation, the lipid and metabolite layers are carefully aliquoted, and the protein pellet is then washed, dried, and resolubilized. Protein resolubilization in digestion buffer can be difficult and may require sonication or other facilitation methods. Subsequent overnight digestion of proteins adds 12-18 hours to the process, which is followed by desalting with solid phase extraction.
A more streamlined sample preparation would allow for simpler, faster, and more efficient ways to process lipids, metabolites, and proteins from a single sample and, when paired with an integrated acquisition method (e.g., multi-omic single-shot technology, MOST14), would allow a single lab or researcher to produce quality multi-omics data.
The present invention provides a method for fast and efficient extraction and separation of biomolecules, including but not limited to lipids, peptides, nucleic acids, carbohydrates, metabolites, and combinations thereof, from a single sample for subsequent analysis. In particular, one aspect of the present invention provides a method for fast and efficient extraction of the lipidome, metabolome, and proteome of a cell for analysis.
One embodiment of the invention provides a method for extracting biomolecules from a sample comprising mixing the sample with an extraction solvent and a plurality of immobilizing beads. The extraction solvent is able to solubilize a first portion of biomolecules, which optionally comprises lipids, carbohydrates, metabolites, and combinations thereof. In an embodiment, the first portion of biomolecules comprises a mixture of lipids and metabolites from the sample. The plurality of immobilizing beads are able to bind and immobilize a second portion of biomolecules, which optionally comprises nucleic acids, proteins, polypeptides, and combinations thereof. In an embodiment, the second portion of biomolecules comprises a mixture of proteins and polypeptides from the sample. Optionally, mixing the sample with the extraction solvent and the plurality of immobilizing beads comprises incubating the sample with the extraction solvent and the plurality of immobilizing beads for an incubation time period between 5 minutes and 1 hour, preferably between 5 minutes and 30 minutes, more preferably between 5 minutes and 20 minutes.
The mixing step generates an extraction solution comprising the first portion of biomolecules and bound immobilizing beads, which are insoluble, attached to the second portion of biomolecules. The bound immobilizing beads attached to the second portion of biomolecules are separated from the extraction solution comprising the first portion of biomolecules. The first portion of biomolecules are then separated from the extraction solution, thereby generating at least a first set of extracted biomolecules, and the second portion of biomolecules are separated from the bound immobilizing beads, thereby generating at least a second set of extracted biomolecules.
The first and second portion of biomolecules, independently from one another, are optionally further separated into additional fractions, thereby generating a third set (optionally, a fourth set, a fifth set, a sixth set, etc.) of extracted biomolecules. For example, the first portion of biomolecules may be separated from the extraction solution to generate a set of lipid molecules and a separate set of metabolite molecules. Similarly, the second portion of biomolecules may be separated from the immobilizing beads to generate a set of polypeptides and a separate set of nucleic acids.
In an embodiment, the extracted biomolecules are then analyzed, which includes but is not limited to performing mass spectrometry analysis on at least the first set and second set of extracted biomolecules. In an embodiment, the mass spectrometry analysis comprises MS1 analysis, MS2 analysis, and combinations thereof. Preferably, the method is able to provide mass spectrometry analysis of the proteins and polypeptides of the sample, as well as mass spectrometry analysis of one or more of the lipids of the sample, the metabolites of the sample, the nucleic acids of the sample, and combinations thereof.
Conventional separation methods typically utilize a biphasic extraction solution. Preferably the extraction solution of the present invention is a monophasic solution. In an embodiment, the monophasic solution is able to solubilize at least 50% of both the lipids and metabolites of the sample, preferably at least 60% of both the lipids and metabolites of the sample, more preferably at least 75% of both the lipids and metabolites of the sample, more preferably at least 90% of both the lipids and metabolites of the sample.
The extraction solvent may be any solvent or combination of solvents able to solubilize the desired biomolecules from the sample for subsequent analysis. Optionally, the extraction solvent is a polar solvent or an amphiphilic solvent. In an embodiment, the extraction solvent comprises methanol, ethanol, propanol, butanol, water, acetone, ethyl acetate, dimethyl sulfoxide, hexane, methylene chloride, chloroform, diethyl ether, acetonitrile, or combinations thereof. Preferably, the extraction solvent comprises n-butanol and one or more co-solvents selected from the group consisting of methanol, ethanol, water, acetone, and acetonitrile. In an embodiment, the extraction solvent comprises n-butanol, preferably between 20% and 80% of n-butanol (by volume). Preferably, the extraction solvent comprises n-butanol and one or more co-solvents selected from the group consisting of methanol, ethanol, water, acetone, and acetonitrile. In an embodiment, the extraction solvent comprises, by volume, between 40%-70% n-butanol and 30%-60% water, and optionally one or more additional co-solvents. In a further embodiment, the extraction solvent comprises, by volume, between 50%-70% n-butanol, between 10%-30% acetonitrile, and between 10%-30% water. In a further embodiment, the extraction solvent comprises, by volume, between 55%-65% n-butanol, between 15%-25% acetonitrile, and between 15%-25% water.
In an embodiment, the immobilizing beads may be any type of bead or particle able to bind to proteins, polypeptides, nucleic acids, and desired negatively charged biomolecules as is known in the art (see as non-limiting examples, Hughes et al., Mol. Syst. Biol., 2014, 10:757; and Berensmeier, S., Appl. Microbiol. Biotechnol., 2006, 73(3): 495). In an embodiment, the immobilizing beads are magnetic or paramagnetic. In an embodiment, the immobilizing beads are silica beads or metal beads that are optionally coated. Optionally, the beads are functionalized, such as to contain antibodies, succinimide or carboxylate ligands, polymers, or carbohydrates. Preferably, the immobilizing beads are unmodified silica beads.
Optionally, the method further comprises digesting the proteins and polypeptides attached to the bound immobilizing beads. In an embodiment, the digestion step comprises mixing the bound immobilizing beads attached to the proteins, polypeptides, and combinations thereof, with a protein digestion enzyme, chemical agent, or combinations thereof. The protein digestion enzymes and chemical agents used herein may be any enzyme or chemical agent known in the art to digest or denature proteins and polypeptides for analysis, including but not limited to trypsin, pepsin, chymotrypsin, papain, calpain, serrapeptase, thermolysin, carboxypeptidase, acids, oxidizing or reducing agents, and combinations thereof. In an embodiment, the digestion step is performed for a time period between 15 minutes and 24 hours, between 2-24 hours, between 6-22 hours, between 8-20 hours, between 12-18 hours, between 10-16 hours, or between 8-12 hours. Preferably, the digestion step is a rapid digestion step performed for a time period between 20 minutes and 120 minutes, between 25 minutes and 90 minutes, between 30 minutes and 80 minutes, between 30 minutes and 60 minutes, between 35 minutes and 50 minutes, or between 35 minutes and 45 minutes. Preferably, the digestion step is performed at a temperature between 30° C. and 90° C., between 40° C. and 80° C., between 45° C. and 75° C., between 50° C. and 70° C., or between 55° C. and 65° C.
In an embodiment, the digestion step comprises mixing the bound immobilizing beads attached to the proteins, polypeptides, and combinations thereof, with a protein digestion enzyme, chemical agent, or combinations thereof for a digestion time period between 30 minutes and 60 minutes at a digestion temperature between 40° C. and 80° C., preferably for a digestion time period between 35 minutes and 45 minutes at a digestion temperature between 55° C. and 65° C. Optionally, the digestion step comprises using a protein digestion enzyme.
In an embodiment, the methods of the present invention (optionally including the digestion steps) are able to be completed within six hours, preferably within five hours, more preferably within four hours, more preferably within three hours, and more preferably within two hours. This is in contrast to conventional extraction and sample preparation methods which typically require 1-2 days.
Preferably, the sample comprises the proteome as well as the lipidome and/or metabolome of a cell, tissue, biological fluid (including but not limited to whole blood, plasma, saliva, cerebral spinal fluid, amniotic fluid, and synovial fluid), or combinations thereof. For example, the first portion of biomolecules (i.e., the portion solubilized by the extraction solvent) comprises the lipidome and metabolome of a cell, and the second portion of biomolecules (i.e., the portion attached to the immobilizing beads) comprises the proteome of the cell. In an embodiment, the sample is a whole cell lysate of one or more cells. In an embodiment, the sample is a solution or biological fluid, where the solution or biological fluid may or may not contain cells or components of lysed cells.
In an embodiment, the present invention provides a method for extracting biomolecules from a sample comprising the steps of: a) mixing the sample with an extraction solvent and a plurality of unmodified immobilizing beads, wherein the extraction solvent comprises, by volume, between 20% and 80% of n-butanol and is able to solubilize a first portion of biomolecules comprising lipids and metabolites, and wherein the plurality of unmodified immobilizing beads are able to bind and immobilize a second portion of biomolecules comprising proteins and polypeptides, thereby generating a monophasic extraction solution comprising the first portion of biomolecules and generating bound immobilizing beads attached to the second portion of biomolecules; b) separating the bound immobilizing beads attached to the second portion of biomolecules from the extraction solution comprising the first portion of biomolecules; c) mixing the bound immobilizing beads attached to the proteins and polypeptides with a protein digestion enzyme or chemical agent for a digestion time period preferably between 20 minutes and 120 minutes at a digestion temperature between 30° C. and 90° C.; and d) separating the first portion of biomolecules from the extraction solution, thereby generating at least a first set of extracted biomolecules, and separating the second portion of biomolecules from the bound immobilizing beads, thereby generating at least a second set of extracted biomolecules. In an embodiment, the digestion is performed for a time period between 30 minutes and 60 minutes at a digestion temperature between 40° C. and 80° C., more preferably for a digestion time period between 35 minutes and 45 minutes at a digestion temperature between 55° C. and 65° C. In an embodiment, steps a) through d) are performed within four hours or less, preferably within three hours or less, or preferably within two hours or less. In an embodiment, the method does not comprise any additional extraction steps performed on the sample, or additional separation steps.
In an embodiment, the present invention also provides a kit comprising the reagents and buffers described herein used to extract the desired biomolecules from the sample, including but not limited to the extraction solvent and immobilizing beads. For example, an embodiment of the invention provides a kit comprising an extraction solvent able to at least partially solubilize lipids, carbohydrates, biological metabolites, and combinations thereof, a plurality of immobilizing beads able to bind and immobilize polypeptides, and a digestion solution able to digest polypeptides, where the digestion solution comprises a protein digestion enzyme and/or a chemical agent. Optionally, kit further comprises one or more washing solutions, desalting solutions, buffers, and combinations thereof.
In an embodiment, the extraction solvent comprises methanol, ethanol, propanol, butanol, water, acetone, ethyl acetate, dimethyl sulfoxide, hexane, methylene chloride, chloroform, diethyl ether, acetonitrile, or combinations thereof. Preferably, the extraction solvent comprises 20-80% n-butanol and one or more co-solvents selected from the group consisting of methanol, ethanol, water, acetone, and acetonitrile. Preferably, the plurality of immobilizing beads comprise magnetic beads, paramagnetic beads, or silica beads. The protein digestion enzymes and chemical agents used herein may be any enzyme or chemical agent known in the art to digest or denature proteins and polypeptides for analysis, including but not limited to trypsin, pepsin, chymotrypsin, papain, calpain, serrapeptase, thermolysin, carboxypeptidase, acids, oxidizing or reducing agents, and combinations thereof. In an embodiment, the components of the kit are contained in separate containers from one another so that they may be added to one or more samples in separate steps. In an alternative embodiment, the extraction solvent and plurality of immobilizing beads are stored together and may be added to the one or more samples at the same time.
The analysis of proteins, lipids, and metabolites—multi-omics—is a powerful approach for gaining insights into complex biological networks and is increasingly applied across multiple disciplines (Krassowski et al., Frontiers in Genetics, 2020: 1598). Mass spectrometry (MS) is a prominent tool for multi-omic studies, offering robust and reproducible profiling of the proteome, lipidome, and metabolome. Preparing samples for MS-based multi-omic analysis broadly involves extraction of metabolites and lipids with a biphasic organic solvent system, precipitation of proteins, and overnight trypsin digestion (Stefely st al., Nat. Biotechnol. 2016, 34: 1191-1197). However, existing sample preparation for MS-based multi-omic analysis are laborious, disparate, and difficult to automate, requiring numerous pipetting, vortexing, and centrifugation steps along with protein resolubilization and solid phase extraction. Conventional sample preparation methods can further take 1-2 days to perform.
The present invention provides streamlined and efficient methods for preparing biomolecules, including but not limited to lipids, peptides, nucleic acids, carbohydrates, metabolites, and combinations thereof, from a single sample for mass spectrometry (MS) and other types of analysis.
In particular, the examples below describe a faster and simpler method to prepare samples for MS-based multi-omic analysis. In the specific examples described below, an n-butanol-based monophasic extraction solvent is used that efficiently extracts lipids as well as both polar and non-polar metabolites. The monophasic extraction is paired with paramagnetic bead technology for on-bead protein aggregation that requires only a short incubation time. Furthermore, the present methods may be used with a heated and rapid protein digestion step. After digestion, the protein solution is acidified, desalted, and dried down. The separate metabolite, lipid, and protein fractions are able to be resuspended for MS analysis.
Thus, the examples described below provide an improved multi-omic sample preparation method that enables faster (reduces preparation time by ˜94%) and simpler preparation (approximately ten steps vs. over twenty). The simplicity and time savings make it more amenable to a single lab technician preparing samples for same-day MS analysis. In addition, this process is more compatible with robotic automation and multi-well plate formats, which could significantly increase throughput.
Overall, this new strategy facilitates preparation of lipids, metabolites, and proteins in approximately three hours or less in some embodiments. This strategy eliminates several manual manipulations, centrifugations, tedious phase separation, and protein resolubilization steps. Despite the simplified steps, it was demonstrated that the performance of each part of the new workflow compares well to standard multi-omic workflows.
Generally speaking, monophasic solvent extraction is combined with magnetic bead-peptide technology and an optional rapid digestion step, in order to expedite small molecule recovery and protein digestion. A sample comprising a mixture of lipids, peptides, metabolites and optionally nucleic acids is mixed with unmodified magnetic beads and a monophasic extraction solvent. A short incubation period facilitates protein aggregation on the beads, and the bead-bound proteins are separated from the monophasic solution containing unbound small molecules (such as lipids and metabolites). After small molecule removal, bead-bound proteins are enzymatically digested without the need for typical wash steps. The resulting peptides are then optionally desalted and purified, while the unbound molecules from the monophasic solution are similarly purified.
Compared to standard workflows, this new method reduced the total number of processing steps, eliminating several manual manipulations, centrifugations, tedious phase separation, and protein resolubilization steps. Despite the simplification of the process, biomolecule coverage and data quality were not compromised for any sample type. Prepared lipid, metabolite, and peptide samples are ready for MS analysis in approximately two to four hours, compared to approximately 1-2 days for standard workflows. Furthermore, compared to standard workflows, this method is more amenable to robotic automation and multi-well plate formats for increased throughput.
Published multi-omic sample preparation workflows vary widely in terms of their number of steps, solvent systems, digestion conditions, and overall throughput (see Kang et al.26, Stefely et al.24, Nakayasu et al.23, and Coman et al.22).
One major drawback to such workflows is the standard biphasic solvent extraction. The two most common extraction systems are Matyash29 (MTBE, methanol, water) and Folch/Bligh-Dyer30-31 (chloroform, methanol, water). While robust, these extraction methods require multiple pipetting, vortexing, incubating, and centrifuging steps to achieve phase separation. After phase separation, the lipid and metabolite layers are removed, and the protein pellet is then washed, dried, and resolubilized.
Resolubilization of the proteins in digestion buffer can be difficult and may require sonication or other facilitation methods. Additionally, workflows relying on centrifugation to pellet the protein are not particularly amenable to limited amounts of starting material, as miniscule protein pellets are not easily visible. Subsequent digestion of proteins with Lys-C and trypsin typically adds 12-18 hours to the process, followed by desalting of peptides. In general, prepared lipids, metabolites, and peptides are ready for analysis after about a day or two. Overall, innovative strategies are needed to simplify this highly manual, tedious, and lengthy process to enable additional labs to prepare and analyze lipidomics, metabolomics, and proteomics data in-house.
This example describes a faster, simpler method to prepare samples for multi-omic analysis that maintains similar biomolecular coverage and data quality as published methods.24-26 To simplify the preparation, a monophasic extraction system is used leveraging n-butanol's diverse miscibility32, with the goal of efficiently recovering both polar and non-polar metabolites. Next, a monophasic extraction is paired with paramagnetic bead technology for on-bead protein aggregation. In recent years, functionalized magnetic bead-based protocols have been introduced as an effective way to improve scalability, throughput, and flexibility for proteomics sample preparation, but they have not yet been tested for compatibility with metabolite and lipid extractions.33-37 Lastly, proteomic sample preparation time is reduced by implementing a heated, accelerated on-bead protein digestion with trypsin. As described further below, the strategy is able to eliminate several manual manipulations and reduces sample preparation time from 18+ hours to −3 hours. This particular embodiment is referred to as the Bead-enabled Accelerated Monophasic Multi-omics (BAMM) sample preparation for multi-omics analysis, and is generally illustrated in
Mouse Brain: All experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee at the University of Wisconsin-Madison. Brains were harvested from C57BL/6J adult female mice after euthanasia and immediately frozen in liquid nitrogen. Tissues from several mice were combined and pulverized in liquid nitrogen; 15±2 mg of frozen pulverized brain was aliquoted into separate 1.5 mL microcentrifuge tubes and maintained at −80° C. until the time of extraction.
Human Plasma: Pooled, mixed-gender plasma sample was purchased from BioIVT (Human Plasma NaHep Lot #HMN378062) and used for all plasma experiments except for
Yeast: The Saccharomyces cerevisiae haploid W303 strain was grown in YPGD repository medium for 25 hours as previously described.14 Prior to extraction, the yeast cell pellets were flash-frozen in liquid nitrogen and maintained at −80° C. Each yeast pellet yielded about 500 μg of protein.
Adipocytes: Cultured adipocytes were prepared as previously described.38 Briefly, differentiated adipocytes derived from murine mesenchymal precursors were trypsinized, seeded on cell culture microplates, and cultured for two days. Cell plates were then flash frozen in liquid nitrogen and maintained at −80° C. prior to extraction.
HEK293. In a standard tissue culture incubator, female HEK293 cells were cultured in DMEM (Thermo Fisher; high glucose with pyruvate and 4 mM glutamine) supplemented with 10% FBS at 37° C. and 5% CO2. At approximately 70% confluency, cells were harvested by gentle washing with DPBS, scraping on ice in DPBS with 5 mM EDTA, and pelleting at 200×g for 4 minutes at 4° C. The resulting cell pellets were flash frozen in liquid nitrogen and stored at −80° C.
Metabolite and Lipid Standards: For recovery analysis, 5 μL of SPLASH Lipidomix internal standard mixture (Avanti Polar Lipids, Inc.) and 5 μL of Cell Free 13C, 15N Amino Acid Mixture (Sigma; diluted 1:100 from the stock) were added to samples for lipidomics and metabolomics analyses.
Preparation of Beads: Prior to performing experiments, Cytiva SeraSil-Mag 700 bead stock (or as specified, see
Biomolecule Extractions: For all metabolite/lipid extraction methods, samples were removed from −80° C. conditions and immediately placed on ice to thaw (plasma) or directly extracted from frozen material (cells and tissue). All extraction solvents were chilled and of liquid chromatography (LC)-MS grade.
For monophasic extraction with beads, extraction solvent (60% n-butanol/20% ACN/20% H2O) was added to each sample along with washed bead stock to achieve a 10:1 bead-to-protein ratio (or as specified, see
For extraction without beads, many n-butanol solvent systems were tested (see
Accelerated Protein Digestion with Paramagnetic Beads: For on-bead accelerated protein digestion (see for example,
Overnight Protein Digestion with Paramagnetic Beads: When on-bead overnight protein digestion was performed (
Protein Digestion without Magnetic Beads: For protein digestion without beads, the method was dependent on whether all -omes were analyzed (
Lipidomics Data Acquisition and Analysis: For untargeted lipidomics LC-MS/MS analysis, dried supernatant aliquots were reconstituted in a 9:1 v/v methanol:toluene solution. To perform chromatographic separations, a Vanquish Split Sampler HT autosampler (Thermo Scientific) was used to inject 10 μL of reconstituted extract onto a Waters Acquity CSH C18 column (2.1×100 mm, 1.7 μm particle size) held at 50° C. throughout the analysis. Flow rate was maintained at 400 μL/min using a Vanquish Binary Pump (Thermo Scientific). The mobile phases consisted of 10 mM ammonium acetate in 70% ACN/30% H2O (v/v) with 250 μL/L acetic acid (mobile phase A) and 10 mM ammonium acetate in 90% IPA/10% ACN (v/v) with the same additives (mobile phase B). A Q Exactive HF Orbitrap mass spectrometer (Thermo Scientific) coupled to a heated electrospray ionization (HESI-II) source was used for mass spectrometric detection. The source conditions were set as follows: HESI-II probe and capillary temperature, 350° C.; auxiliary gas temperature, 350° C.; sheath gas flow rate, 25 units; auxiliary gas flow rate, 15 units; sweep gas flow rate, 5 units; spray voltage, |3.5 kV| for both positive and negative ionization modes; and S-lens RF at 90 units. Data were acquired via polarity switching mode, acquiring full MS and MS/MS (Top2) spectra in both positive and negative ionization modes within the same injection. The acquisition parameters for full MS in both modes were set as follows: resolution of 30,000; automatic gain control (AGC) target of 1×106; ion accumulation time (max IT) of 100 ms; and a scan range of 200-2000 m/z. MS/MS scans in both modes were then performed as follows: resolution of 30,000; AGC target of 1×105; max IT of 50 ms; isolation window of 1.0 m/z; stepped normalized collision energy (NCE) at 20, 30, 40; and a dynamic exclusion of 30 seconds.
Lipidomics raw files were processed with Compound Discoverer 2.1 or higher (Thermo Fisher Scientific) and LipiDex.13
Metabolomics Data Acquisition and Analysis: For metabolomics LC-MS/MS analysis, dried supernatant aliquots were reconstituted in a 1:1 v/v acetonitrile:water solution. To perform chromatographic separations, a Vanquish Split Sampler HT autosampler (Thermo Scientific) was used to inject 2 μL of reconstituted extract onto a Millipore SeQuant ZIC-pHILIC column (2.1×100 mm, 5 μm particle size) held at 50° C. throughout the analysis. The flow rate was maintained at 150 μL/min using a Vanquish Binary Pump (Thermo Scientific). The mobile phases consisted of 10 mM ammonium acetate in 10% ACN/90% H2O (v/v) with 0.1% ammonium hydroxide (mobile phase A) and 10 mM ammonium acetate in 95% ACN/5% H2O (v/v) with 0.1% ammonium hydroxide (mobile phase B). A Q Exactive HF Orbitrap mass spectrometer (Thermo Scientific) coupled to a HESI-II source was used for mass spectrometric detection. The source conditions were set as follows: HESI II and capillary temperature, 350° C.; sheath gas flow rate, 40 units; auxiliary gas flow rate, 15 units; sweep gas flow rate, 1 unit; spray voltage, |3.0 kV| for both positive and negative ionization modes; and S-lens RF at 50 units. Data were acquired via polarity switching mode, acquiring full MS and MS/MS (Top10) spectra in both positive and negative ionization modes within the same injection. The acquisition parameters for full MS in both modes were set as follows: resolution of 60,000; AGC target of 1×106; max IT of 100 ms; and a scan range of 70-900 m/z. MS/MS scans in both modes were then performed as follows: resolution of 45,000; AGC target of 1×105; max IT of 100 ms; isolation window of 1.0 m/z; stepped NCE at 20, 30, 40; and a dynamic exclusion of 30 seconds.
Metabolomics raw files were processed with TraceFinder 3.3 (Thermo Fisher Scientific) using m/z and retention time tolerances for integration of specific metabolite features (see Table 1).
Proteomics Data Acquisition and Analysis: For proteomics LC-MS/MS analysis, dried peptide samples were reconstituted in 0.2% formic acid in water. To perform chromatographic separations, a Dionex UltiMate WPS-3000RS autosampler (Thermo Fisher Scientific) was used to inject 1 μg of peptides onto a PicoFrit fused silica capillary column (New Objective) that was packed in-house5 to 30 cm with 1.7 μm, 130 Å pore size C18 BEH particles (75×360 μm). The column was held at 50° C. with an in-house built heater. The flow rate was maintained at 300 nVmin. The mobile phases consisted of 0.2% formic acid in water (mobile phase A) and 0.2% formic acid in 80% ACN/20% H2O (v/v, mobile phase B). The LC was coupled to an Orbitrap Eclipse (Thermo Fisher Scientific) via a nano ESI source for mass spectrometric detection. The transfer capillary temperature was set to 275° C., and the positive spray voltage was 2.5 kV. A one-second cycle time was used to acquire full MS and MS/MS scans in “Top Speed” mode. The acquisition parameters for full MS were set as follows: resolution of 240,000 in the Orbitrap, AGC target of 1×106; max IT of 50 ms, and a scan range of 300-1350 m/z. The APD algorithm was toggled on.7 MS/MS scans were performed as follows: turbo scanning mode in the ion trap, AGC target of 3×10, max IT of 14 ms, isolation window of 0.5 m/z, NCE of 25%, and dynamic exclusion of 10 s. Charge states 2-5 were included, and the default charge state was 2.
MaxQuant4 (version 1.5.2.8) was used to search all proteomics raw files. The appropriate references database was downloaded from UniProt (human, mouse, or yeast; canonical and isoforms). Searches were performed using the Andromeda42 search algorithm and label-free quantification.43 Default parameters were used. Match between runs was not applied unless noted.
Dilution corrections: To compare extraction solvents, metabolite and lipid extractions were corrected for differences in dilutions between monophasic and biphasic extractions. Correction factors were calculated as the sum of the measured volumes (aqueous phase+organic phase) divided by the volume of the phase used for analysis, and normalized total volumes across all samples. Actual biphasic volumes were estimated using a glass Hamilton pipettor
Monophasic Solvent System for Lipid and Polar Metabolite Extraction: Initial efforts toward a simplified multi-omics workflow involved optimizing a monophasic solvent system for lipid and metabolite extraction. In standard biphasic solvent systems, lipids partition into a strongly lipophilic solvent (e.g. chloroform,30-31 MTBE29), while polar metabolites partition into the aqueous phase. The biphasic systems successfully extract a wide range of compound classes. In contrast, current monophasic extraction methods, although simpler, tend to preferentially extract either lipophilic or polar metabolites.39,44-48 With the goal of developing a monophasic solvent system that recovers both lipids and small molecules with high efficiency, aqueous n-butanol mixtures were explored, as they have been described32 as containing properties compatible with both polar and non-polar compounds.
First, a range of n-butanol formulations (0-80% n-butanol) were tested for suitability to extract lipids and polar metabolites. The proportion of water was maintained at 20% (v/v), and acetonitrile was used to balance the proportion of n-butanol. From 0-60% n-butanol, the solvents remained miscible, yielding the desired monophasic extraction solvent. However, at 70% and 80% n-butanol, slight and moderate phase separation was induced, respectively. Using 500 μL of each solvent mixture, 10 μL of human plasma were extracted and analyzed with equal portions of extract (100 μL) by LC-MS/MS for lipids and metabolites. For the phase-separated n-butanol extracts, the upper layer was used for both metabolite and lipid analyses. All extracts were dried by vacuum centrifugation and resuspended in the same solvent for analysis. The n-butanol formulations were compared to a common metabolomics monophasic solvent48 (2:2:1 MeOH:ACN:H2O, “MAW”) and the traditional biphasic Matyash solvent system29 (10:3:2.5 MTBE:MeOH:H2O, “MTBE”), using the same ratio of plasma to solvent.
To evaluate the extraction solvents, the number of lipids and metabolites identified were first assessed (
After determining that 60% n-butanol was optimal, class distributions of extracted lipids and metabolites were assessed (
Finally, quantitative correlation (relative abundance) was examined between 60% n-butanol and MTBEor for lipids (
Magnetic Beads to Facilitate Integrated Sample Preparation: Expanding on the simplicity of the monophasic solvent for metabolomics and lipidomics sample preparation, this extraction was integrated with paramagnetic bead technology to expedite proteomics preparation. The use of magnetic beads for proteomics (termed the SP3 approach33-36) was introduced in recent years as a universal sample preparation platform. The SP3 protocol uses carboxylate-coated hydrophilic magnetic beads in the presence of high organic solvent to induce protein-bead aggregation. Once proteins are immobilized on the surface of the beads, they can be rinsed of contaminants (e.g. chaotropes, detergents), released, and digested. Here, a modified SP3 approach was envisioned that would be amenable to multi-omics. First, magnetic beads were added to monophasic solvent and sample, and proteins were allowed to aggregate around the beads during a short incubation period. Unbound metabolites and lipids were removed for further analysis, and bead-bound proteins were rinsed, digested, and desalted. Overall, the goal was to eliminate centrifugation and protein resolubilization by combining a bead-based protocol with the monophasic solvent extraction. However, because this SP3 method has not been demonstrated for compatibility with metabolite or lipid analyses, it was hypothesized that different functional groups on the beads may influence extractions of metabolites and/or lipids.
Four different types of magnetic beads were obtained to test with multi-omic extractions: 1 μm hydrophilic carboxylate functionalized beads (Cytiva), 1 μm hydrophobic carboxylate functionalized beads (Cytiva), 3 μm unmodified silica beads (G-Biosciences), and 700 nm unmodified silica beads (Cytiva). Even though the SP3 protocol is typically performed with carboxylate functionalized beads, it has been shown33 that proteins are not influenced by specific bead properties and thus aggregate on any available surface upon conditions known to induce aggregation. To examine the performance of the four bead types, metabolites, lipids, and proteins were extracted from plasma with each bead and without beads. The log2 fold changes in intensity were compared between each bead type and the no-bead control for common metabolites, lipid classes, and peptide GRAVY (grand average of hydropathicity index; a measure of hydrophobicity) score range49 (
The reduction in the recovery of certain metabolites when using functionalized beads is likely due to inadvertent capture of those metabolites by the bead surface. Interestingly, the 700 nm nonfunctionalized beads avoid this problem, but the 3 μm nonfunctionalized beads avoid it only partially. Some metabolites likely still have a partial interaction with the silica surface50 of the 3 μm beads, and size may potentially play a role. Regardless, the 700 nm unmodified beads were clearly optimal over the other bead types for metabolites; therefore, these beads were chosen for subsequent experiments and final multi-omics workflow. After establishing the bead type, it was verified that bead surface interaction with biomolecules was not time-dependent, as little to no difference in metabolite, lipid, and peptide recovery from plasma were seen when varying the incubation period of the beads with the sample from 5 to 60 minutes (
These experiments confirmed that the SP3 method for proteomics can be expanded for multi-omics, preferably with a nonfunctionalized bead. A bead-based multi-omics workflow not only consolidates the preparation but also eliminates the need for centrifugation and protein resolubilization, as aggregated proteins are digested directly on-bead.
Reducing Overall Preparation Time By Accelerating Proteomics Preparation: As a final simplification to the multi-omics workflow, opportunities to reduce the overall time taken for proteomics sample preparation were explored, which is the lengthiest portion of the process. First, it was attempted to remove any unnecessary steps, such as wash steps between removal of the metabolite and lipid supernatant and addition of digestion buffer. In current SP3 protocols, it is typical to perform 2-3 wash steps (often with acetonitrile and/or ethanol) before digestion.38,37 The intent of the washes is to remove detergents and contaminants; however, detergents are largely incompatible with MS-based metabolomics and lipidomics, and therefore it was reasoned that wash steps are not as necessary with multi-omics samples. The experiments in
Second, enabling same-day analysis of all three -omes was explored. Metabolite and lipid samples can typically be prepared and analyzed on the instrument within the same day; however, the typical overnight digestion of proteins stalls peptide analysis until at least the following day. To expedite digestion, it was attempted to combine Promega Rapid Trypsin with on-bead protein digestion. The Rapid Trypsin platform reduces protein digestion time to one hour or less by heating samples up to 70° C., which requires a specialized non-urea buffer and thermostable trypsin.51 The Rapid Trypsin protocol was optimized to be compatible with magnetic beads, which required the sample to be shaken throughout the digestion period (˜35% increase vs. not shaken) and the temperature to be lowered to 60° C. from 70° C. (
Interestingly, while the peptide GRAVY score distributions of the rapid bead and overnight no-bead workflows were similar, the bead workflow appears to extract hydrophilic peptides to a greater extent than without beads (
Final Streamlined Multi-omic Workflow for Mass Spectrometry Analysis: Overall, these improvements led to a significantly streamlined multi-omics sample preparation workflow (
After developing the simplified and consolidated BAMM method, it was validated for multiple sample types and formats (cell pellets, biofluids, cell culture plates) for widespread use (
Conclusion: The present example describes a simple and consolidated method (named BAMM) to prepare metabolites, lipids, and proteins from a single sample. This method combines an n-butanol-based monophasic extraction with paramagnetic bead technology, expediting small molecule extraction and protein digestion. This new strategy produces quality multi-omics data comparable to classic methodologies at a fraction of the time and effort. Prepared metabolites, lipids, and peptides are ready for MS analysis in about 3 hours, compared to about a day on average for current workflows. It was also noted that due to the use of magnetic beads, this method is potentially more amenable to robotic automation and multi-well plate formats for increased throughput. Additionally, this BAMM sample preparation method is optimized for LC-MS analysis.
Furthermore, the components of this workflow can also be adapted as necessary and used individually. For example, after validating the monophasic solvent extraction, this method was applied to a large-scale COVID-19 study for fast lipidomics sample preparation.25 It is envision that this expedient method or its individual components will be particularly beneficial for specific applications wherein turnaround time is an important consideration, such as clinical screening, iterative process optimization, rapid process analytics, and large sample screens. These benefits are amplified even further when pairing this streamlined multi-omics sample preparation with an integrated acquisition method such as MOST.14
Having now fully described the present invention in some detail by way of illustration and examples for purposes of clarity of understanding, it will be obvious to one of ordinary skill in the art that the same can be performed by modifying or changing the invention within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any specific embodiment thereof, and that such modifications or changes are intended to be encompassed within the scope of the appended claims.
When a group of materials, compositions, components or compounds is disclosed herein, it is understood that all individual members of those groups and all subgroups thereof are disclosed separately. Every formulation or combination of components described or exemplified herein can be used to practice the invention, unless otherwise stated. Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. Additionally, the end points in a given range are to be included within the range. In the disclosure and the claims, “and/or” means additionally or alternatively. Moreover, any use of a term in the singular also encompasses plural forms.
As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of” excludes any element, step, or ingredient not specified in the claim element. As used herein, “consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any recitation herein of the term “comprising”, particularly in a description of components of a composition or in a description of elements of a device, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or elements.
One of ordinary skill in the art will appreciate that starting materials, device elements, analytical methods, mixtures and combinations of components other than those specifically exemplified can be employed in the practice of the invention without resort to undue experimentation. All art-known functional equivalents, of any such materials and methods are intended to be included in this invention. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Headings are used herein for convenience only.
All publications referred to herein are incorporated herein to the extent not inconsistent herewith. Some references provided herein are incorporated by reference to provide details of additional uses of the invention. All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. References cited herein are incorporated by reference herein in their entirety to indicate the state of the art as of their filing date and it is intended that this information can be employed herein, if needed, to exclude specific embodiments that are in the prior art.
This application claims priority from U.S. Provisional Patent Application No. 63/348,357, filed Jun. 2, 2022, which is incorporated by reference herein to the extent that there is no inconsistency with the present disclosure.
This invention was made with government support under GM108538 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63348357 | Jun 2022 | US |
