Patent Application
20120003680
The present invention relates generally to the fields of analytics of proteins and peptides. More particularly, the invention concerns generation of absolutely quantified peptide or protein solutions via element mass spectrometry used as internal standards in downstream proteomics experiments. Furthermore, the invention relates to the absolute quantification of phosphopeptides.
With the completion of the Human Genome Project, the emphasis is shifting to examining the protein complement in the human organism. This has given a rise to the science of proteomics, the study of the whole amount of the proteins produced by a cell type of an organism. At the same time, there has been a revival of interest in proteomics in many prokaryotes and eukaryotes as well.
The term proteome refers to all proteins expressed by a genome and comprises the identification of proteins in the body and the detection of their physiological and pathophysiological function. The about 30,000 genes, defined by the Human Genome Project translate into 30,000 up to 1 million of proteins, when alternate splicing and post-translational modifications are considered. While a specific genome remains unchanged to a high extent, the proteins in any particular cell change dramatically as genes are turned on and off, e.g. in response to extracellular stimulation.
As a reflection of the dynamic nature of the proteome, the term “functional proteome” is preferred by some scientists, which describes the entity of proteins produced by a cell at a certain time point. Finally, it is believed, that through proteomics, new disease markers and drug targets can be identified that will help to design new products to prevent, diagnose and treat disease.
Primary themes in proteomics are protein identification and the differentiation of physiological or pathological protein expression levels (comparative proteomics). The long-term goal of being able to define the entire proteome of a cell is still unrealized, but the characterization of many thousands of proteins in a single analysis is now attainable.
For proteomics to become a platform technology serving the emergent field of systems biology, there is a need for enhancement of quantification. Most comparative proteomic studies deliver a relative quantification, expressing the changes in concentrations of a protein in the context of a different cellular stage. However, the goal is to define the cellular concentrations of proteins absolutely, whether as molarities or as numbers of molecules per cell. Absolute quantification, which poses one of the greatest challenges in proteomics, draws on well-established concepts in analytical chemistry and requires either external standards or internal standards. Quantification by immunological methods is typically performed by external standardization e.g. on position-addressable antibody arrays. The second approach, reliant on internal standardization, is based on mass spectrometry (MS), wherein highly selective detection of molecular or fragment ions is performed.
In the most MS-analysis, stable isotope labeled variants of the analytes are used as internal standards. The key principle is that mass spectrometric signal intensities can be converted into absolute quantities of analytes by referencing to an authentic standard present in known concentration. The direct application of this approach to intact proteins is impractical and it is common to adopt the surrogate principle to quantify by referencing to a unique proteolytic peptide derived from the protein of interest.
Synthetic stable isotope labeled peptides used as internal standards have been named “AQUA” peptides (Absolute Quantification) (Gerber PNAS 100 (2003), 1940-1945). AQUA peptides are quantified by total hydrolysis, derivatisation of the amino acids and their quantification by UV spectroscopy, fluorescence spectroscopy or mass spectrometry. The accuracy of this analysis is dependent on the purity of the sample. Traces of peptides with deletion sequences, salts, metals or detergents can affect the accuracy of results. Conditions applied during hydrolysis are rigorous and destroy some of the amino acids (Met, Cys, Gln, Asn, Trp) and special conditions have to be applied for modified amino acids.
The international patent application WO 03/102220 provides methods to determine the absolute quantity of proteins present in a biological sample. The principle of WO 03/102220 is based on the generation of an ordered array of differently isotopically tagged pairs of peptides, wherein each pair represents a unique protein, as specific protein isoform or a specifically modified form of a protein. One element of the peptide pairs is a synthetically generated, external standard, and the other element of the pair is a peptide generated by enzymatic digestion of the proteins in a sample mixture. For performing the method of WO 03/102220, the standard peptides are calibrated so that absolute concentrations are known and added for comparison and quantification. A sample of interest is also labeled with the same chemical tag as used for the standard peptides except differing in the isotopic label. The signal pairs, which correspond to differently labeled samples and standard peptides are finally observed and related to a list of expected masses based on the particular standard peptides included. The disadvantages of this patent application are that both the sample and the standard peptides need to be specifically labeled separately, increasing the potential for variability between experiments.
Other methods for proteome analysis include quantitative mass spectrometry based on multidimensional peptide separation and isotope-coded affinity tagging of proteins. This method allows only relative quantitation. Also, chip technology using arrays of reagents with known specifity for target proteins, such as antibody arrays or arrays of aptamers, can be used for proteomic analysis. However, the use of such arrays can be limited by the need for selectivity, and for the preservation of the three-dimensional structure of the immobilized proteins.
Each quantitation approach has advantages and disadvantages. Quantification of protein expression ratios by metabolic labeling, such as a stable isotope labeling in cell culture (SILAC) (Oda Y., Huang K., Cross F. R., Cowburn D., Chait B. T., Proc. Natl. Acad. Sci. USA, 1999, Vol. 96, 6591-6596) provides the opportunity to quantify proteins, but has the limitation that primary tissue cannot be analyzed.
Thus, there is still an existing need to develop easy and convenient methods for determination of the absolute concentration and the degree of phosphorylation of a peptide which contain less sources of error and are less time consuming.
Accordingly, an aim of the present invention is to provide an accurate method for determining the degree of phosphorylation and the absolute concentration of a peptide.
This is achieved by a method for determining the degree of phosphorylation of a first peptide which is present in a sample in phosphorylated and/or unphosphorylated form or any mixtures thereof comprising:
It was found, that the method according to the invention is a surprisingly easy and sensitive way for generating a reference sample, containing either the isotope labeled non-phosphorylated or phosphorylated analogue of the first peptide or any mixtures thereof. In this way the determination of the absolute amount of each form of the first peptide respectively the degree of phosphorylation of the first peptide is performed. Advantageously, the method according to the invention incorporates fewer steps and thus contains less sources of error compared to existing methods for generating a reference sample which can be used for determining the absolute amount and degree of phosphorylation of peptides.
According to an embodiment of the invention, the degree of phosphorylation and the absolute concentration of the first peptide are determined by comparison of the respective signals in a further step (f) of the method of the invention. Hence, the present invention allows accurate determination of the absolute amount/concentration and degree of phosphorylation of a phosphopeptide. According to this aspect of the invention, a method for the absolute quantification of a phosphopeptide is disclosed, wherein only 0.01 to 0.1 nmol of the first peptide are required.
The term “first peptide” encompasses the unphosphorylated and/or phosphorylated form of a peptide or any mixtures thereof. As used herein, the term “peptide” refers to a peptide or a polypeptide of two or more amino acids. The polypeptide can additionally be modified by naturally occurring modifications, such as posttranslational modifications, including fatty acylation, sulfation, hydroxylation, acetylation, glycosylation, addition of prosthetic groups or cofactors, formation of disulfide bonds, assembly in molecular complexes and the like.
According to an embodiment of the invention, the expression “first peptide” encompasses a derivatized phosphopeptide, too. For peptides without serine (S), threonine (T) and/or tyrosine (Y) residues, the derivatisation comprises the addition of an extension carrying a phosphorylated serine, threonine and/or tyrosine residue. According to this aspect of the invention, the “first peptide” is obtained by the addition of a short, optionally enzyme-cleavable extension carrying a phosphorylated residue (pS, pT and/or pY). The addition can be performed at the N-terminus or at the C-terminus of the peptides, respectively. According to this aspect of the invention, in step (be) of the inventive method, this extension is cleaved by treatment with a protease and the third peptide with known concentration is obtained.
As used herein the term “sample” is intended to mean any biological fluid, cell tissue, organ or portion thereof that includes one or more different molecules, such as nucleic acid, polypeptides, or small molecules. A sample can be a tissue section obtained by a biopsy or a cell placed in or adapted to tissue culture. A sample can also be a biological fluid, specimen such as blood or plasma, cerebrospinal fluid, urine, saliva, seminal plasma, pancreatic juice, and the like. A sample can additionally be a cell extract from any species, including procaryotic and eucaryotic cells as well as viruses. A tissue or biological fluid specimen can be further fractionated, if desired, to a fraction containing particular cell types.
The method of the invention is to produce a reference sample of known concentration for determining the absolute amount and degree of phosphorylation of a phosphopeptide and/or for determining the absolute concentration of a peptide in a sample. The method is based on the identification of a distinct peptide which is unique for a polypeptide or protein which can therefore be used as surrogate to identify the degree of phosphorylation and/or the quantity of this polypeptide or protein in a sample.
Peptides uniquely identifying a protein can be selected experimentally or computationally. Experimentally such peptides are selected from databases that contain all the peptides, preferably phosphopeptides, from a species that have been previously observed, for example, by mass spectrometry experiments. Computationally such peptides are selected by translating the complete genomic sequence or the sequence of all predicted genes and their splice forms into the corresponding amino acid sequences, by applying the rules for cleavage that are predictable with each chemical or enzymatic protein cleavage reagent to these amino acid sequences, and by computing the sequence, mass and other properties for each of the general peptides. From this database of predicted peptides, a suitable selection of peptides that are unique for each target protein, in the context of the invention called first peptide, is the selected.
In the preferred embodiment of the invention, the first peptide has 5 to 100 amino acids, more preferred 5 to 50 amino acids, and most preferred 6 to 20 amino acids.
Once the first peptide is selected, a reference sample can be prepared by synthesizing a second peptide which contains at least one phosphate group and a known degree of phosphorylation and which is an isotope labelled form of the first peptide. The second peptide having an identical chemical structure like the first peptide is made by synthesis using a number of isotopic atoms for example 13C, 15N, 17O, 18O or 34S, so that the first and the second peptide can be distinguished using mass spectrometry. Peptide sequences, once selected, are preferably chemically synthesized by solid-phase step by step synthesis. Methods of peptides synthesis are well-known to those skilled in the art. The completely phosphorylated second peptide carries at least one isotopic atom selected from 13C, 15N, 17O, 18O or 34S. According to an embodiment of the invention, the second and third peptide contains 3 to 12, preferred 4 to 10 of these isotopic atoms.
The second peptide is subjected to absolute quantification by use of a LC-ICP mass spectrometer. For this purpose a known amount of a phosphorus standard having a known phosphorus concentration is combined with a solution containing the second peptide. Subsequently a signal of the phosphorus standard and of the second peptide is detected in LC-ICP-MS. The concentration of the second peptide is determined by comparison of the peak areas of the corresponding phosphorus signals within the LC-ICP-MS run. According to an embodiment of the invention a previously determined correction factor is used to account for signal intensity changes due to gradient elution (see Wind M. et al. Anal. Chem. 2001, 73, 29-35).
The phosphorus standard having known phosphorus concentration can be selected from various commercially available standards. According to an embodiment of the invention, a bis-4-nitrophenylphosphate (BNPP) standard of known concentration is used in the method of the invention. According to a further aspect of the invention aromatic mono- and diesters of phosphoric acid having a known concentration can be used as phosphorus standard.
After absolute quantification of the second peptide, an aliquot comprising the second peptide in known concentration is completely dephosphorylated to obtain a third peptide with known concentration. According to the invention, the third peptide is a completely dephosphorylated and isotope labeled form of the first peptide. For the purpose of dephosphorylation the second peptide is treated with a phosphatase e.g. selected from alkaline phosphatase and antarctic phosphatase to hydrolyse the phosphate ester bond(s) within the phosphopeptide. According to a further aspect of the invention, the second peptide can be dephosphorylated by the use of a reagent that contains at least one selected from the group consisting of hydrogen fluoride, hydrofluoric acid, and a hydrogen fluoride-containing compound. The use of the above mentioned reagents leaves the second peptide intact and is applicable to any kind of the type. This method is disclosed in the European patent application EP 1 595 889 A1. According to the embodiment of the invention if the “first peptide” does not contain a S, T or Y residue an enzyme cleavable extension can be introduced carrying a S, T or Y residue to obtain the second peptide which is quantified by LC-ICP-MS. The third peptide is then derived by treatment of the second peptide with a protease.
When phosphatase was used for dephosphorylation of the second peptide, usually a small amount of phosphatase remains in the sample containing the third peptide. In this case, prior combining a known amount of the second peptide and a known amount of the third peptide to obtain the reference sample, the phosphatase used for dephosphorylation has to be inactivated, to retain the accuracy of the quantification. Usually the used phosphatase was inactivated by acidification to pH≦3. For the preparation of acid-labile peptides, a different phosphatase, such as antarctic phosphatase, can be used. This phosphatase can be inactivated irreversibly by moderate heating (e.g. 5 min at 65° C. or extended incubation times at 55° C.). In this way, acidification of the solution and possible hydrolysis of acid-labile peptides is avoided.
According to the invention, the reference sample containing a known amount of the second peptide (isotope labeled phosphopeptide) and a known amount of the third peptide (corresponding isotope labeled unphosphorylated peptide) is combined with the sample containing the first peptide, and molecular ion and/or fragment ion signals of each of the peptides are detected in an analyzer that is an ESI, LC-ESI-MS or MALDI analyzer. Since the isotope label of the second peptide leads to a mass shift, the signals of the three peptides can be identified by use of an MS analyzer. Since the amount of phosphorylated and unphosphorylated second and third peptide is known, the degree of phosphorylation of the first peptide can be determined by comparing the respective molecular or fragment ion signals. According to a further aspect of the invention, the absolute concentration of the first peptide is determined by comparison of respective molecular or fragment ion signals.
The invention further refers to a reference sample containing a second peptide which contains at least one phosphate group and a known degree of phosphorylation and which is an isotope labelled form of a first peptide, and a third peptide, that is a completely dephosphorylated and isotope labelled form of the first peptide derived from the second peptide in a known concentration prepared obtained by a method comprising:
The method of the invention can be used in a variety of different applications. For example, the method of the invention can be used for profiling blood serum. The possibility to analyze readily accessible specimens such as blood serum is particularly useful in clinical applications. Hence, the method is also applicable to basic biology and clinical analysis.
The accuracy of the method of the invention is shown in table 1. Table 1 shows the recoveries of experiments, where three calibrated solutions of sulfur-containing phosphopeptides were prepared via the method described above. In all three independent experiments phosphorus and sulfur were monitored by LC-ICP-MS. The concentrations of the phosphopeptides (also determined by S detection) and of the dephosphorylated peptides (determined by S detection) were found to be identical within the experimental error. These data support the practicability of the concept introduced, namely the preparation of calibrated peptide solutions via dephosphorylation of phosphopeptides.
The method of the invention is advantageous because it incorporates significantly less steps and thus contains less sources of error. A single analysis requires 0.01 to 0.1 nmol of the first peptide and at least three replicates are recommended. A detection limit of the preferably used LC-ICP-MS analyzer is below 0.1 pmol, a sample amount≧10 pmol is recommended to obtain a reproducible and accurate quantification in a single analysis. Further, the quantification by LC-ICP-MS with subsequent phosphatase treatment is less time consuming than the classical aforementioned amino acid analysis connected with the AQUA quantification method and less sensitive to common buffers and detergents used during peptide synthesis. Additionally, the quantification is carried out by a peptide-specific signal, which ensures that only the synthetic peptide is quantified while any impurities resolvable by LC are not measured. In contrast, amino acid analysis quantifies all amino acids and peptides present in the sample besides the selected synthetic peptide.
The following Figures and examples are included to further illustrate various aspects of the present invention. It should be appreciated by those of skilled in the art that the techniques disclosed in the examples which follow the presented techniques and/or compositions discovered by the inventor to function well in the practise of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skilled in the art should, in light of a present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain alike or a similar result without departing from the spirit and the scope of the invention.
In
After enzymatic dephosphorylation, a small amount of phosphatase used for dephosphorylation remains in the calibrated solution. To ensure a general applicability of this solution as internal standard, the phosphatase has to be inactivated. Alkaline phosphatase was inactivated by acidification to pH≦3. To confirm the inactivation of the alkaline phosphatase an equimolar solution of phosphatase-treated and untreated phosphopeptide was prepared and analysed after an incubation time of about 20 hours (see
For the preparation of absolutely quantified acid-labile peptides, a different phosphatase, such as antarctic phosphatase can be used. This phosphatase can be inactivated irreversibly by moderate heating (e.g. 5 min at 65° C. or extended incubation times at 55° C.). In this way, acidification of the solution and possible hydrolysis of acid-labile peptides is avoided. nanoESI mass spectra of a phosphopeptide solution before and after antarctic phosphatase treatment are shown in
The method of the invention is based on the presence of serine, threonine and tyrosine residues within the selected peptide (first peptide). The probability for the presence of at least one of these residues in an average peptide sequence is shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 09150634.5 | Jan 2009 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2010/050225 | 1/11/2010 | WO | 00 | 9/26/2011 |
