WO2018042454A2

WO2018042454A2 - Method of hyperplexing in mass spectrometry to elucidate temporal dynamics of proteome

Info

Publication number: WO2018042454A2
Application number: PCT/IN2017/050369
Authority: WO
Inventors: Shilpa JAMWAL; Suruchi AGGARWAL; Amit Kumar Yadav; Kanury Venkata Subba Rao; Ajay Kumar
Original assignee: Translational Health Science And Technology Institute (Thsti)
Priority date: 2016-09-01
Filing date: 2017-08-31
Publication date: 2018-03-08
Also published as: WO2018042454A3

Abstract

The present invention relates to the method of novel combination of MS¹ and MS² labeling (hyperplexing) that enables better, reliable and efficient quantitation with reduced technical variability and increased multiplexing capacity at the same time.

Description

METHOD OF HYPERPLEXING IN MASS SPECTROMETRY TO ELUCIDATE TEMPORAL DYNAMICS OF PROTEOME

FIELD OF THE INVENTION

The present invention relates to field of biotechnology, particularly it relates to a method of mass spectrometry for studying multiple numbers of protein samples by a novel multiplexing technique.

BACKGROUND OF THE INVENTION

Proteins are large macro molecules or biomolecules made of one or more polypeptide chains. Polypeptide in turn consists of chain of amino acids linked by peptide or amide bonds. Proteins carry out important cellular functions. They are important structural components of cell, they catalyze various chemical reactions as enzymes, and play important role in signal transduction and cell signaling. In addition, proteins are the regulatory molecules bringing about a phenotypic manifestation; thus the effect of protein deregulation is quite marked and an indicator of pathophysiology. Understanding the cellular machinery not only requires their identification but also accurate quantification, isoform state and the stoichiometry. The post- translational modifications (PTMs) and subcellular localization of the proteins cannot be predicted from the genome, although these play vital role in protein function.

Mass spectrometry (MS) is a method for accurate determination of molecular weight of a compound. It allows elucidating protein structures and their identification. It is the only method available for large-scale characterization of protein alterations at the molecular level. The technique can be used for identification and quantitative profiling of proteomes of organism, protein modification and interactions. It has been increasingly applied to study post-translational modification state of proteins and their dynamics. The biological applications of MS include screening for metabolic disorders in newborns, comparative protein profiling between cells grown in different media, determining the bioavailability of minerals in food, and in vivo metabolization of pharmaceutical drugs. Since the last two decades, mass spectrometry has rapidly evolved from a method providing only a list of proteins; to a technology that can provide a quantitative readout of hundreds to thousands of proteins present in a sample. In MS, the ionization of the bio molecules results in formation of gas phase ions (positively or negatively charged). These ions can then be isolated electrically or magnetically based on their mass-to-charge ratio (m/z). The mass of these ions are measured by following their trajectories in the vacuum system.

The quantitative MS allows comparative analysis of proteomes to detect the relative and absolute protein abundance that result from perturbations in the system using approaches that require staining or labeling of proteins. Since MS is not innately quantitative technique, various labeling techniques are used for quantitation in MS. In stable isotope labeling of amino acids in cell culture (SILAC), a mass difference is introduced between two cell populations by growing the cells on identical culture media except one of them contains a 'light' and the other a 'heavy' form of a particular amino acid(s), Cells are harvested and samples are combined followed by protein identification by mass spectrometry. A mass shift in the mass spectrum is observed in MS between light and heavy forms due to metabolic incorporation of the amino acids into the proteins. The ratio of peak intensities in the mass spectrum gives the relative protein abundance. Metabolic labeling can be used to process up to three samples in one experiment using Light (0, 0), Medium (6, 6) and Heavy (8, 10) labels on Lysine and Arginine. The relative peak areas of MS I precursors are used to calculate quantitative ratios of the same analyte (protein) in different samples at high throughput. This quantitation applies to all analytes with labeled amino acids.

"Metabolic labeling with noncanonical amino acids tagging " (Bio Orthagonal Non Canonical Amino-acid Tagging, BONCAT) is a technique that allows identification of newly synthesized proteins in a proteome. The newly synthesized proteins are labeled using non- canonical amino acid (such as azide-bearing artificial amino acid azidohomoalanine). The small chemical group assigns unique chemical functionality to the target bio molecules. These molecules are then tagged by an affinity bearing tag via click chemistry. These tags can be then exploited for detection, affinity purification, and eventually MS identification. In "Isobaric quantitation like Isobaric tags for relative and absolute quantitation" (iTRAQ) and TMT (Tandem mass tags), isobaric tags are used to label primary amines of the peptides in the sample. "Quantification using isobaric tag " is a chemical labeling method that can use cell or tissue lysate as sample. The 114, 115, 116 and 117 are the four reagents used in the 4-plex version of "Quantification using isobaric tag " while 113, 118, 119 and 121 are used additionally in the 8-plex version. Each reagent is composed of a peptide reactive group and an isobaric tag that consists of a reporter group and a balancer group. The peptide-reactive group specifically reacts with primary amine groups of peptides. The reporter group gives strong signature ions in tandem mass spectrometry (MS/MS) and is used to determine the relative abundance of a peptide. These reporter ion areas can be used for relative quantitation between the samples.

In the experimental workflow for "Quantification using isobaric tag ", unlabelled protein samples are first trypsin-digested and labeled with different isobaric tags independently. These labeled peptides from different samples are then mixed together and separated by liquid chromatography. In MS¹, the mass of all peptides irrespective of sample is isobaric but during fragmentation, the reporter ion peaks are released at specific and unique known masses in MS spectrum.

Using software such as search engine which uses mass spectrometry data to identify proteins from primary sequence database, a protein database search can be performed on the fragmentation data to identify the labeled peptides and hence the corresponding proteins. The absolute abundance of low molecular mass reporter ions generated from the isobaric tags can then be used to quantify the relative abundance of peptides and proteins across the samples studied.

On the other hand, label-free methods allow theoretically infinite sample comparisons. Label- free approaches are based on the abundance of peptide ion intensity in the sample. Quantitation is estimated either by spectral counting or by comparing ion intensities for peptide peaks at specific retention times between different samples. It can also use the method of identification of peptides by MS/MS and sampling statistics such as spectral or sequence coverage and peptide count, to quantify the differences between samples. The label- free methods allow theoretically infinite sample comparisons but practical challenges in separation and run-to-run variability is a major limitation of mass spectrometry leading to practical problems in applying label-free methods to drug discovery and biomarker pipelines

However, using above-mentioned labeling techniques for multiple samples can still pose a challenge. "Metabolic labeling with stable isotope amino acids in cell culture " is limited to binary (2-plex) or ternary (3-plex) set of reagents. Isobaric quantitation by "Quantification using isobaric tag " allows 4/8-plex sample processing while TMT can be used for 6-10 samples.

High throughput studies aimed at identifying a large number of proteins (preferably, a complete proteome) require some quantitative readout from a panel of time profiles to study the temporal dynamics in response to a particular stress or drug. Such studies are however, limited by sample throughput and require quantitative values. Even when these criteria are met, not all proteins are equally amenable for liquid chromatography separation. Due to their physicochemical properties, some part of the proteome is always refractory to the conventional proteome analysis by MS. Strategies to enhance separation and detection help alleviate these problems at the cost of throughput, thereby forcing discovery and targeted proteomics into two separate categories. While discovery mode optimizes identification by with respect to time spent and effort per sample, the targeted on the other hand processes large number of samples to look for specific molecules through specifically optimized workflows.

The multiplexing capacity of labeled experiment helps in robust quantitative comparisons across the labeled samples. Since the samples are mixed before separation and mass spectrometry analyses, the technical variability is same for all samples irrespective of the labeled state. This makes the results interpretable, as the variation is likely due to biological state rather than measurement errors. For large number of samples, the experiments have to be conducted several times; and run-to-run variations arise, making biological interpretations less repeatable and reproducible, e.g., to process 18 samples, a triple Metabolic labeling with stable isotope amino acids experiment needs to be conducted six times, while a six-plex "Quantification using isobaric tag " experiment needs to be conducted thrice. The added complexity of variance between runs makes it difficult as well as time and resource consuming.

There is a recently developed MS ¹ labeling technique NeuCode which expands multiplexing capacity to 12-plex but it requires ultra-high resolution mass spectrometry making this hardware dependent and costly affair. It also needs significantly increased mass spectrometer run-time owing to ultra-high resolution requirements since the isotopic mass difference is in mDa, which may not be detectable even at good MS resolutions of most general-purpose instruments. The MS techniques may also not be able to multiplex beyond a limit due to practical constraints of designing the reporter masses in low mass region to prevent interference from MS/MS daughter ions representing the sequencing peaks. In another approach, Dephoure et al combined "Metabolic labeling with stable isotope amino acids in cell culture " triplex with TMT 6-plex to achieve 18-plex multiplexing. A flipside in their approach was an implicit requirement of MS so that reporter ions of TMT get accurately quantified.

It is desirable to have a multiplexing technique that is robust to variations, can work nearly as accurately as traditional labeling techniques, yet have high reproducibility and robustness of quantitation. Higher multiplexing theoretically can help alleviate the problem of run-to-run variation since all samples are challenged by the same amount of technical variation or systematic error. Since the experimental conditions are invariant, the changes in quantitation can be ascribed to biological variation rather than technical one. Analysis of data should be easier if only the precursor peaks are used for area calculation, but it is unwieldy to have high number of "Metabolic labeling stable isotope with amino acids in cell culture " -like MS¹ isotopic peaks and not get mixed up with other peptide precursor peaks.

To overcome these challenges, we have introduced a novel combination of MS 1 and MS 2 labeling that enables better, reliable and efficient quantitation with reduced technical variability and increased multiplexing capacity at the same time.

OBJECT OF THE INVENTION

An object of the present invention is to provide a method for hyperplexing, combining three powerful techniques "Metabolic labeling with noncanonical amino acid tagging ", triplex- " Metabolic labeling with stable isotope amino acids in cell culture " with 6-plex "Quantification using isobaric tag " to facilitate study of temporal dynamics of newly synthesized proteome.

SUMMARY OF THE INVENTION

The present invention relates to the method of novel combination of MS 1 and MS 2 labeling (hyperplexing) that enables better, reliable and efficient quantitation with reduced technical variability and increased multiplexing capacity at the same time. Hyperplexing is a simple yet efficient technique that draws from the advantages of both MS 1 and MS 2 labeling techniques and combines their power into a novel multiplexed technique. In this technique, we combined for the first time, a - triplex "Metabolic labeling with amino acids stable isotope in cell culture " with "Quantification using isobaric tag " 6-plex (can be 8-plex as well) to increase the multiplex capacity to 18-plex in a single shot of mass spectrometry run. This allows facile comparison of an analyte across 18 different samples on a global scale in high throughput shotgun proteomics manner. The dataset analysis was done using in-house developed computational analysis pipeline.

The present invention discloses a novel quantitative proteomics method for multiplex analysis with throughput accuracy comprising the steps of:

i. culturing the sample in a liquid culture medium at optimal cell density.

ii. labeling the protein sample obtained from step (i) with "Metabolic labeling with stable isotope amino acid in cell culture" to obtain the labeled protein sample.

iii. culturing the labeled sample obtained in step (ii) with non-canonical amino acid for "Metabolic labeling with noncanonical amino acid tagging" to obtain labeled newly synthesized protein sample.

iv. enrichment and digestion of the newly synthesized protein sample obtained in step (iii) to obtain peptide sample.

v. labeling the peptide sample obtained from step (iv) with "Quantification using isobaric tag "

vi. identification of the peptide sample obtained from step (v) by LC-Mass spectrometry analysis.

vii. identification of the peptide obtained from step (vi) by "Protein identification software ".

viii. quantification of "Quantification using isobaric tag " labeled peptide using "Tool for quantitation based on isobaric tag method in conjunction with software "

ix. integrating the result of step (vii) and step (viii) using "Computational software for integrating hyperplexing results " .

BRIEF DESCRIPTION OF FIGURES

Figure 1 depicts the comparison of protein identifications made by Sequest+ Vista (SQVS) vs. MaxQuant+HyperQuant (MQHQ) pipelines using Dephoure et al's, dataset.

Figure 2 depicts the distribution of peptide areas across the 6- " Quantification using isobaric tag " samples for peptides in uninfected condition (control) at different time points in one MS run. The x-axis, shows the peptide area of 113 reporter ion, while y-axis shows peptide areas of other "Quantification using isobaric tag" reporter ion- 114.1 (dark blue), 115.1 (red), 116.1(green), 117.1 (violet), 118.1 (light blue) with their equations and correlations represented in the same colour as the series.

Figure 3 depicts the distribution of ratios in biological replicates Linear 1 and Linear2 of Setl (linear 1 shown in green and linear 2 in blue). Five histogram panels are from 6-plex "Quantification using isobaric tag " from "Metabolic labeling with stable isotope amino acids in cell culture " light. The ratio bins are represented on x-axis and their frequency on y- axis compared for the two replicates LI (green) and L2 (blue).

Figure 4 depicts the effect of outlier removal on ratio concordance between replicates. The left panel column shows the replicates' comparison before outlier removal; the middle panel depicts enhanced concordance after removal of outlier peptides during protein ratio calculation; the right panel shows removal of outlier peptides combined with selection of highly confident protein identifications (>3 peptides).

Figure 5 depicts the identified (A) and quantified (B) proteins from replicates of Set 1 either individually (linear 1 in blue and linear 2 in yellow) or combined (green). This shows the combination of replicates helps select nearly all identified proteins (except 2) for quantification.

Figure 6 depicts the effect of replicate combination on quantitation accuracy. It shows LI (x- Axis) vs. L1L2 combined (black) and LI vs. L2 (red). We can observe several outliers in the data (in the red series). Upon combination, the protein quantitation errors are reduced and outliers are minimal (in the black series) as they are brought closer to the real value by replicate combination.

Figure 7 depicts the combined protein ratio comparisons between set 1 and set 2, where all replicates (linear 1 and 2 + nonlinear 1 and 2) were integrated. The left panel shows protein ratio comparisons for corresponding ratios from the 6plex "Quantification using isobaric tag " between set 1 and set 2 (for light label of "Metabolic labeling with stable isotope amino acids in cell culture "), before outlier removal and the right panel shows protein ratio comparisons between set 1 and set 2 upon outlier removal.

Figure 8 depicts the ratio comparisons between replicates when all reporter ions were compared in totality. The top panel shows comparison between Setl replicates - Linearl vs. Linear2 and Nonlinearl vs. Nonlinear2. The bottom panel depicts similar comparisons for second set of 6plex "Quantification using isobaric tag " (set 2). DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the method of novel combination of MS 1 and MS 2 labeling (hyperplexing) that enables better, reliable and efficient quantitation with reduced technical variability and increased multiplexing capacity at the same time.

Hyperplexing is used herein as a simple yet efficient technique that draws from the advantages of both MS 1 and MS 2 labeling techniques and combines their power into a novel multiplexed technique.

"Metabolic labeling with stable isotope amino acids in cell culture " is an approach where the cells are grown in a medium containing essential amino acids that carry light and heavy stable isotopes of a particular amino acid and thus this amino acid is incorporated in all newly synthesized proteins. Following repeated cell divisions, its isotope labeled analog will replace this particular amino acid.

"Metabolic labeling with noncanonical amino acid tagging" technique identifies the subpopulation of newly synthesized protein using bioorthogonal noncanonical amino acids by utilizing the cell's own translation machinery. This amino acid analog (azidohomoalanine or AHA) gets incorporated in all newly synthesized proteins instead of methionine. This helps in selective enrichment of AHA-tagged proteins by click chemistry.

"Quantification using isobaric tag " is a method in quantitative proteomics in which the samples are labeled with isobaric labels and detected by tandem mass spectrometry.

Temporal proteome dynamics of the present invention may comprise differential protein expression regulated in time in response to stimuli, (e.g. Infection of THPl macrophages by bacteria Mycobacterium tuberculosis in the present working example). Instead of time profiles, the technique may also capture any other biological state of interest.

Secretome in the present invention is the complete set of secreted protein by the THPl macrophages in response to the stimuli (infection by Mycobacterium tuberculosis).

The said method comprises the steps of:

i) "Metabolic labeling with stable isotope amino acids in cell culture ". ii) "Metabolic labeling with noncanonical amino acid tagging".

iii) "Quantification using isobaric tag " labeling.

iv) Nano LC-Mass Spectrometry Analysis.

v) Database Search using modification table of "Proteomics identification software ". vi) "Quantification using isobaric tag " labels were quantified using "Tool for quantitation based on isobaric tag method in conjunction with software ".

vii) Integrating result from "Proteomics identification software " and "Tool for quantitation based on isobaric tag method in conjunction with software " using "Computational software for integrating hyperplexing results" .

The present invention is a novel quantitative proteomics method for multiplex analysis with throughput accuracy comprising the steps of:

i. culturing the sample in a liquid culture medium at optimal cell density to obtain protein sample

iii. culturing the labeled protein sample obtained in step (ii) with non-canonical amino acid for "Metabolic labeling with noncanonical amino acid tagging" to obtain labeled newly synthesized protein sample.

iv. enrichment and digestion of the newly synthesized protein obtained in step (iii) to obtain peptide sample.

v. labeling the peptide sample obtained from step (iv) with "Quantification using isobaric tag ".

vi. identification of the peptide obtained from step (v) by LC-Mass spectrometry analysis.

vii. identification of "Quantification using isobaric tag" labeled peptide sample using "Tool for quantitation based on isobaric tag method in conjunction with software ". viii. identification of the peptide samples obtained from step (vi) by "Protein identification software "

ix. integrating the result from peptide identified in step (v) and step (vii) using "Computational software for integrating hyperplexing results ". Sample - The sample is selected from group comprising bacterial, mammalian, plant, tissues, fungal, microbial cell culture and the cell density of the cell culture is in the range of 0.5 to 2 million cells per mL. "Metabolic labeling with stable isotope amino acids in cell culture"- In the present invention the protein sample may be labeled by incorporating one or more nonradioactive stable isotope of essential amino acid from the group comprising heavy or medium isotopes of 2 H, 13 C, 15 N, and/or 18 O in the culture medium, more preferably with

C₆-Lysine

(Lys6) and 13

C6-arginine (Arg6) for medium isotopes. The cells may be first grown in the depleted medium (not containing essential amino acids), prior to replacing the medium with amino acids stable isotope in cell culture for metabolic labeling. The depletion media may be replaced by unlabeled media for control (containing natural amino acids Lys° and Arg⁰).

The protein sample is labeled by one or more non-radioactive stable isotope of essential amino acid selected from the group comprising heavy or medium isotopes of 2 H, 13 C,

1 ¹5^JN, 1 ^l8^o0, more preferably Light, medium, heavy isotopes of lysine and arginine, most preferably ¹⁵N₂ ¹³C₆-lysine (Lys8), ¹⁵N₄ ¹³C₆-arginine (ArglO), ¹³C₆-Lysine (Lys6) and 13

C6-arginine (Arg6). "Metabolic labeling with noncanonical amino acid tagging"- In the present invention all the newly synthesized proteome may be labeled with noncanonical amino acid from the group comprising of azide-bearing artificial amino acid azidohomoalanine (AHA) or homopropargylglycine (HPG) more preferably with AHA for each time point. The newly synthesized protein is labeled with a non-canonical amino acid selected from a group comprising azidohomoalanine (AHA) and homopropargylglycine (HPG), more preferably azidohomoalanine (AHA).

Media from the infected samples, along with respective un-infected controls may be collected at different time points (6-26 hours). Media may be cleared of any cell debris by centrifugation and supernatant supplemented with protease inhibitor cocktail. Media from light, medium and heavy labels may be pooled and concentrated using ultra centrifugal filters (5-2-kDa cutoff) at 6-3°C, 4-8000 rpm and frozen at -80°C. Method of enrichment of newly synthesized proteins and on-bead digestion: Newly synthesized proteins from concentrated media were enriched using Click-iT Protein Enrichment kit (Invitrogen CI 0416) as per manufacturer's instructions with slight modifications. Sample and the catalyst solution were added to ΙΟΟμΙ of pre-washed agarose resin. The proteins were then reduced in the presence of SDS and dithiothreitol, followed by alkylation in presence of iodoacetamide. The resin was subsequently suspended in digestion and transferred to a fresh tube. On-bead digestion was mediated by incubating samples with O^g LysC, followed by overnight incubation with O^g Trypsin. The diluted digested samples were acidified with formic acid (FA) followed by desalting with pre-equilibrated C18 reversed-phase columns (Waters). Desalted peptides were finally eluted in acetonitrile ACN/0.1% FA, lyophilized and re-solubilized in ACN. The newly synthesized protein sample is concentrated, alkylated, reduced, and digested with 0.4 -1 μg/mL of trypsin. "Quantification using isobaric tag" labeling- In present invention multiplex "Quantification using isobaric tag " reagent kit may be used to label peptides from biological/protein samples, at different time points. The samples might be labeled with 6- 8 isobaric reagents, more preferably with 6- isobaric reagents.

The peptide sample is labeled with "Quantification using isobaric tag " with 6- 10 isobaric reagent more preferably with 6 isobaric reagent (113.1, 114.1, 115.1, 116.1, 117.1 118.1) at different time points .

Prior to labeling, all 6 isobaric tag reagents (113-118) may be allowed to reach RT and supplemented with isopropanol (30-60μ1). For each time point, sample may be labeled with a single isobaric tag by transferring the contents from respective isobaric reagent vials to sample tubes. The tagged peptide mixtures from all sample tubes may be pooled and dried by vacuum centrifugation followed by desalting. The desalted samples may be eluted with (30-70%) acetonitrile in formic acid. Elutes maybe pooled and further cleaned by strong cation exchange (SCX) chromatography. The tagged peptide mixtures may be reconstituted in SCX low ionic strength buffer (3-8mM ammonium formate, 20- 50% acetonitrile) pH 2-5 and loaded onto the cartridge and if so desired may be subsequently eluted with high cationic exchange buffer (400-600mM ammonium formate, 20-50% ACN) at a suitable pH. The samples may be lyophilized prior to LC- MS/MS analysis. Nano LC-Mass Spectrometry Analysis- All samples may be analyzed by reverse-phase high-pressure liquid chromatography electrospray ionization tandem mass spectrometry. The peptide samples is identified by Liquid chromatography - Mass spectrometry analysis more preferably nano-reverse phase high-pressure liquid chromatography electrospray ionization tandem mass spectrometry (RP-HPLC-ESI-MS/MS). Database Search using modification table of proteomics identification software-

The wiff files from mass spectrometry results may be searched using "Proteomics identification software " against Uniprot human database with cRAP sequences

(www.thegpm.org/crap/) and their corresponding reversed sequences may be appended to it. The parameters for search may include:

i. Triple- "Metabolic labeling with stable isotope amino acids in cell culture " on Lysine and Arginine with mass of "Quantification using isobaric tag " added to Lysine labels- more specifically - "Metabolic labeling with stable isotope amino acids in cell culture " modifications with "Quantification using isobaric tag " masses incorporated with each label i.e. the light (L), medium (M) and heavy (H) was defined for identifying hyperplexed masses. The search parameters used is "Metabolic labeling with stable isotope amino acids in cell culture " on lysine and arginine with mass of "Quantification using isobaric tag " added to Lysine labels. These modified masses may be incorporated in "Proteomics identification software " search engine by editing the modification XML/text file enabling identification of multiple-labeled peptides. ii. Fixed modifications may include- carbamidomethylation at cysteine & "Quantification using isobaric tag " at N-termimus.

iii. Variable modifications may include- methionine oxidation, "Metabolic labeling with noncanonical amino acid tagging " and deamidation at NQ residues.

iv. The results may be filtered at a desired false discovery rate (FDR), preferably 1% using "Proteomics identification software ". "Quantification using isobaric tag" labels were quantified using "Too/ for quantitation based on isobaric tag method in conjunction with software". All "Quantification using isobaric tag " (6-plex) labeled peptides generate a unique reporter ion peak in MS/MS mass region of 113.1, 114.1, 115.1, 116.1, 117.1, and 118.1 daltons (Da). Quantification of "Quantification using isobaric tag " labeled peptide sample was done using "Tool for quantitation based on isobaric tag method in conjunction with software " by using the summed intensity (SI) algorithm.

These peaks may be used to quantitate peptides in 'Too/ for quantitation based on of Isobaric tag method in conjunction with software" by using the summed intensity (SI) algorithm in which the peaks for each reporter ion are summed up to calculate area if they fall within a desired mass window (in the example, 0.05 Da window) around particular reporter mass. The SI area is used to calculate the relative ratios between different reporter ions to compare quantities across samples labeled by the respective reporters. Only first 6 tags of the eight (113.1 - 118.1) of "Quantification using isobaric tag " were used, thus making the experiment 6-plexed. 8-plex/10-plex may also be applicable for this technique. Integrating result from "Proteomics identification software" and "Tool for quantitation based on isobaric tag method in conjunction with software" using "Computational software for integrating hyperplexing results"

Peptides identified by "Proteomics identification software " and peptide quantified by "Tool for quantitation based on isobaric tag method in conjunction with software " are integrated using "Computational software for integrating hyperplexing results ".

"Computational software for integrating hyperplexing results" works in two steps using the mapper and combiner modules. This computational software is an integration pipeline to map quantitation values of isobaric tags for every type of metabolically labeled stable isotope amino acid peptides; and it also calculates protein ratios when replicates are combined directly from spectrum level quantitation data. This pipeline runs in two steps:

a. Maps spectrum identification with quantitation for every different type of label.

b. Combines all replicates and calculates protein ratios from PSM level data. The integration of results is carried out by "Computational software for integrating hyperplexing results" in two steps using following modules:

i. mapper module -mapping of spectrum identification with quantitation for each different labels after matching mass-to-charge (m/z) and retention time (RT).

i. combiner module- combines the protein level information from these mapped files across any number of replicates, removes statistical outliers and outputs a list of protein with final quantitation ratios integrated from input files.

"Proteomics identification software " provides the output results in form of several tab delimited tables. The scan file outputted by "Proteomics identification software " (scan.txt) contains information about scan indexes, while the identification results after statistical correction of <\% false discovery rate (FDR) contain scan wise list of assigned peptide sequence. The mapper module finds the scan index from "Proteomics identification software " file and maps the identified results to the scans after matching mass-to-charge (m/z) and retention time (RT). The combiner then combines the protein level information from these mapped files across any number of replicates, removes statistical outliers which are shown to have a significantly different ratio after a z-test at a = 0.05 and outputs a list of proteins with final quantitation ratios integrated from several input files. High accuracy and sensitivity of "Computational software for integrating hyperplexing results"

In the present invention, "Computational software for integrating hyperplexing results" used the information from raw results to calculate the quantitation areas separately from each combination of "Metabolic labeling with stable isotope amino acids in cell culture " and "Quantification using isobaric tag " labels separately (leading to 18 areas instead of 3 or 6 only). The high degree of specificity is achieved by correctly mapping the scans for "Quantification using isobaric tag " labels in each light, medium and heavy "Metabolic labeling with stable isotope amino acids in cell culture " labeled peptides. The sensitivity is achieved by statistical outlier removal and using high confidence proteins (those identified with >3 peptides) which will have enough statistical sampling for the mean quantitation to be closer to the real value.

Replicate combination is a complex step while doing protein quantitation analysis. Usually the median of protein ratios is taken to combine protein ratios across replicates. Protein ratios are a central measure of peptide ratios. When low number of spectra is used to calculate average ratios, the values may not be close to the real value. Since the errors follow multiplicative model, this will inflate the errors in combined ratios. Peptides show heterogeneity in measurement of ratios based on their intensities. Peptides with lower intensities have higher error.

In "Computational software for integrating hyperplexing results", instead of averaging the protein ratios from replicates, we used the ratio data of all spectra from the replicates combined together for calculating protein ratios directly. This enhances result quality due to two reasons - (i) increased statistical sampling for each protein by combining replicates' raw ratios and therefore, after outlier removal the calculated ratio value is more accurate (ii) prevents loss of proteins for which the peptide counts is lower than 3. On combination, the summed peptide count allows the proteins to pass and has enough sampling to get accurate ratios. This ensures high specificity and sensitivity of the present method.

The protein is quantified using "Computational software for integrating hyperplexing results" after outlier peptide ratio removal and filtering out proteins with peptide count lower than 3.

The present invention discloses multiplex capacity that enables 10 to 50, more preferably 10-40, most preferably 18-30 sample quantitation in a single mass spectrometry run. The present invention may be used to study the temporal dynamics of a proteome.

ADVANTAGES OF THE PRESENT INVENTION

1. The present invention is a new variant of hyperplexing method, combining triplex "Metabolic labeling with stable isotope amino acids in cell culture " with "Metabolic labeling with noncanonical amino acid tagging " and 6-plex "Quantification using isobaric tag " to achieve 18-plex quantitation in a single MS run to selectively enrich and temporally capture the newly synthesized and secreted proteome.

2. The present invention combines "Metabolic labeling with noncanonical amino acid tagging " labeling technique with "Metabolic labeling with stable isotope amino acids in cell culture " and "Quantification using isobaric tag " enabling selective enriching of the newly translated proteins, which are in lower abundance as compared to existing proteins. This allows selectively studying the newly translated protein in combination with the temporal dynamics (In the present invention demonstrated by example, secretome of the THP1 cells) of the cells' response to stimuli (In the present invention demonstrated by example, infection with Mycobacterium tuberculosis).

3. In the present invention, samples are mixed before separation by mass spectrometry analyses, thus the technical variability is same for all samples irrespective of the labeled state. This makes the results more interpretable as the variation is likely due to biological state rather than measurement errors. This also allows better statistical sampling for proteomics results.

4. The present invention eliminates the need for the experiments to be conducted several times for large number of samples, and thus reducing run-to-run variations, making biological interpretations more reproducible.

5. The present invention allows studying temporal dynamics of proteome that might be very difficult with present MS techniques when large number of samples involved.

6. The present invention greatly reduces the time required for running large number of biological/ protein samples.

7. The present invention a "Computational software for integrating hyperplexing results " also helps in combining protein ratios from different replicates based on user defined criteria after outlier peptide ratios removal and filtering proteins with low peptide counts giving highly accurate results.

The present invention is illustrated by the following examples, which are not to be construed as limiting the scope of the invention:

Example 1

Method of culturing bacterial Cell

All bacterial cultures were grown and maintained in Middlebrooke 7H9 broth (Difco) supplemented with 10% ADC (Becton Dickinson), 0.4% glycerol and 0.05% Tween 80 until the mid-log phase. Bacteria were harvested by centrifugation, washed with and re-suspended in RPMI 1640 (GIBCO). Cultures were prepared for infection by passing the bacterial suspension through 23, 26 and 30 gauge needles in tandem. Dispersed bacterial cultures were allowed to settle for 5 minutes and suspension from top was used for infecting THP1 cells. Bacteria were quantified by measuring absorbance at 600nm wavelength (an optical density value of 0.6 corresponds to approximately 100 million bacteria). The mycobacterial strains included in the study are H37Ra, H37Rv, BND433 and JAL2287.

Example 2

Method of culturing host cell human monocytic cell line (THP1)

Human monocytic cell line, THP1, was cultured at 37°C and 5% C02 in RPMI 1640, supplemented with 10% FBS (Hyclone) and lx penicillin and streptomycin (penicillin, lOOU/ml; streptomycin, lOOug/ml; GIBCO). Cell density was maintained around 0.5 - 1 million cells per ml. Cell viability and counts were determined by trypan blue staining.

Example 3

Method of infecting THP1 with Mycobacteria (described in example 1 &2) and "Metabolic labeling with stable isotope of amino acids" and noncanonical amino acid in cell culture THP1 cells were differentiated in the presence of Phorbol 12-myristate 13-acetate (PMA), and seeded in T-175 flasks @ 30 million cells per flask. Cells were either left uninfected or infected with mycobacterial strains (H37Ra and H37Rv; BND433 and JAL2287) at MOI of 10 per cell. After 48 hours of seeding, the cells were infected in two separate batches; each batch comprising of an un-infected control along with two of the targeted mycobacterial strains (H37Ra and H37Rv; BND433 and JAL2287). To deplete cells of methionine, lysine and arginine, the cells were incubated for 1 hour in depletion medium (RPMI non-GMP formulation without methionine, arginine and lysine; GIBCO) with 10% dialyzed FBS (GIBCO). Thereafter, depletion media was replaced with unlabeled media for the uninfected cells (Lys°Arg°) (40ug/ml of normal lysine and 200ug/ml of normal arginine), medium labeled media for H37Ra and BND433 infected cells (Lys6 Arg6) (61.68ug/ml of (4,4,5,5,- D4) L- lysine and 62.26ug/ml of (13C6) L- arginine) and heavy labeled media for cells infected with H37Rv and JAL2287 (Lys8 ArglO) (52.22ug/ml of (13C615N2) L-lysine and 63.42ug/ml of (13C615N4) L-arginine) (Cambridge Isotope Laboratories, Inc.). O.lmM L- Azidohomoalanine (L-AHA) (AnaSpec, Inc) was supplemented to all three media i.e. unlabeled (Lys°Arg°), medium (Lys6 Arg6) and heavy (Lys8 ArglO). All newly synthesized proteins were tagged by AHA incorporation, in each of the time windows starting from six hours post-infection till 26 hours, at a difference of 4 hours each. Media from the infected samples, along with respective un-infected controls were collected at 6, 10, 14, 18, 22 and 26 hours respectively. Collected media were cleared of any cell debris by centrifugation at l,000g for 5 minutes and the supernatant was supplemented with 2x Protease inhibitor cocktail (Peirce 78437). Media from flasks containing light, medium and heavy labels were pooled and concentrated using Amicon Ultra Centrifugal filters (15ml, 3-kDa cutoff) at 4°C, 6,000rpm and frozen at -80°C. The technique was recently referred to as BONLAC to represent the combination of "Metabolic labeling with noncanonical amino acid tagging " with "Metabolic labeling with amino acids stable isotope in cell culture " .

Example 4

Method of enrichment of newly synthesized proteins and on-head digestion

Newly synthesized proteins (described in example 3) from concentrated media were enriched using Click-iT Protein Enrichment kit (Invitrogen C10416) as per manufacturer's instructions with slight modifications. For each reaction, the suggested reagent volumes were reduced to half. Sample and the catalyst solution were added to ΙΟΟμΙ of pre-washed agarose resin and kept at room temperature (RT) with constant shaking for 18-20 hours incubation. The proteins were then reduced in the presence of SDS and dithiothreitol (DTT) (Bio-Rad) at 70°C for 15 minutes, followed by alkylation in presence of iodoacetamide (Bio-Rad). The resin was transferred into a spin column and washed with 10ml of SDS buffer, 10ml of 8M urea in lOOmM Tris, pH8 and 20ml of 20% acetonitrile (ACN). The resin was subsequently suspended in digestion buffer (lOOmM Tris, pH8, 2mM CaC12 and 10% ACN) and transferred to a fresh tube. On-bead digestion was mediated by incubating samples with 0^g LysC at 37°C for 4 hours followed by overnight incubation with 0^g Trypsin (Promega). ACN was diluted to 2% with water and the diluted digested samples were acidified with formic acid (FA) followed by desalting with pre-equilibrated C18 reversed- phase columns (Waters). The pre-equilibration step included activation with 50% ACN/0.1% FA, followed by equilibration with 0.1% FA. After adding the sample, the column was washed twice with 0.1% FA. Desalted peptides were finally eluted in 50% ACN/0.1% FA, lyophilized and re-solubilized in ACN. Example 5

Method of Quantification using isobaric labeling of the proteins described in example 4

To enable simultaneous identification and quantification, multi-Plex "Quantification using isobaric tag " reagent kit, from Sciex, was used to label peptides from 6 different biological samples, representing time points 6hrs, lOhrs, 14hrs, 18hrs, 22hrs and 26hrs per mycobacterial strain. Prior to labeling, all 6 "Quantification using isobaric tag " reagents (113-118) were allowed to reach RT and supplemented with 50μ1 isopropanol. Each time point sample was labeled with a single isobaric tag in the aforementioned order by transferring the contents from "Quantification using isobaric" reagent vials to respective sample tubes. Samples, mixed with respective "Quantification using isobaric" reagents, were subjected to 2 hours incubation at RT. The tagged peptide mixtures from all sample tubes were pooled and dried by vacuum centrifugation followed by desalting through C-18 columns (Waters). The desalted samples were eluted in 40% and 60% ACN in 0.1% FA, respectively. Elutes were pooled and further cleaned by off-line strong cation exchange (SCX) chromatography using the SCX cartridge (ThermoFischer Scientific). The tagged peptide mixtures were reconstituted in SCX low ionic strength buffer (5mM ammonium formate, 30% ACN) pH 3 and loaded onto the cartridge and subsequently eluted with high cationic exchange buffer (500mM ammonium formate, 30% ACN) pH 3. The samples were finally lyophilized prior to LC-MS/MS analysis. The combined MS I and MS2 labeling, also called hyperplexing, enables 18-plex multiplexing in a single run of mass spectrometer.

Example 6

Method of peptide identification from the samples described in example 5 by Nano IX - Mass Spectrometry Analysis

All samples were analyzed by reverse-phase high-pressure liquid chromatography electrospray ionization tandem mass spectrometry (RP-HPLC-ESI-MS/MS) using a Nano LC-Ultra ID plus (Eksigent; Dublin, CA) and nanoFlexcHiPLC system (Eksigent) which is directly connected to an ABSCIEX 5600 Triple-TOF (AB SCIEX; Concord, Canada) mass spectrometer.

Reverse Phase-HPLC was performed via an elute configuration using two Nano cHiPLC columns (Eksigent) in tandem to make a long column set up for better separation with good resolution; the analytical column (75 μιη x 15cm) were manufacturer (Eksigent)-packed with 3 μιηΟΐΓθΓηΧΡ C-18 (120A). Reverse-phase LC solvents included: mobile phase A: 2% ACN/98% 0.1% FA (v/v) in water, and mobile phase B: 98% ACN/2% 0.1% FA (v/v) in water. The auto-sampler was operated in full injection mode overfilling a Ιμΐ loop with 3μ1 analyte for optimal sample delivery reproducibility. All samples were eluted in replicates from the analytical column at a flow rate of 300nL/min by two different elution gradient modes: Linear and Step wise elution modes. Using linear gradient mode of 5% solvent B to 50% solvent B over duration of 300 minutes, separation was done. The column was regenerated by washing with 90% solvent B for 20 minutes and re-equilibrated with 5% solvent B for 40 minutes. During Non-Linear gradient mode, separation was done in step wise manner with 5% solvent B to 20% solvent B over duration of 180 minutes; 20% B to 30% B for 80 minutes; 30% B to 50% B for 30 minutes. The column was regenerated by washing with 90% solvent B for 20 minutes and re-equilibrated with 5% solvent B for 40 minutes. Auto-calibration of spectra occurred after acquisition of every 2 samples using dynamic LC-MS and MS/MS acquisitions of 50 fmol β-galactosidase.

Mass spectra and tandem mass spectra were recorded in positive-ion and "high-sensitivity" mode with a resolution of -35,000 full-width half-maximum. Peptides were injected into the mass spectrometer using ΙΟμιη SilicaTip electrospray PicoTip emitter (New Objective Cat. No. FS360-20-10-N-5-C7-CT), and the ion source was operated with the following parameters: ISVF = 2200; GS 1 = 20; CUR = 25.

The data acquisition mode in DDA experiments was set to obtain a high resolution TOF-MS scan over a mass range 350-1250 m/z, followed by MS/MS scans of 15 ion candidates per cycle with activated rolling collision energy, operating the instrument in high sensitivity mode. The selection criteria for the parent ions included the intensity, where ions had to be greater than 150 cps, with a charge state between +2 to +5, mass tolerance of 50 mDa and were present on a dynamic exclusion list. Once an ion had been fragmented by MS/MS, its mass and isotopes were excluded from further MS/MS fragmentation for 10s. Collision- induced dissociation was triggered by rolling collision energy. The ion accumulation time was set to 500 ms (MS) and to 200 ms (MS/MS).

Example 7

Method of database Search and relative quantification for the peptides identified as described in example 6 The wiff files from ABSCIEX 5600 were searched using "Proteomics identification software " against Uniprot human database with cRAP sequences (www.thegpm.org/crap/) and their corresponding reversed sequences appended to it. The parameters for search were as follows - triple "Metabolic labeling with stable isotope amino acids in cell culture " on Lysine and Arginine with mass of "Quantification using isobaric tag " added to Lysine labels, fixed modifications used were- carbamidomethylation at cysteine & "Quantification using isobaric tag " at N-term; variable modifications used were- methionine oxidation, "Metabolic labeling with noncanonical amino acid tagging " and deamidation at NQ residues. The results were filtered at 1% false discovery rate (FDR) using "Proteomics identification software ".

Example 8

Method of dataset analysis

The wiff files were converted to mgf files using msconvert and "Quantification using isobaric tag " labels was quantified using in-house developed "Tool for quantitation based on isobaric tag method in conjunction with software ". Using "Proteomics identification software " and "Tool for quantitation based on isobaric tag method in conjunction with software " and "Computational software for integrating hyperplexing results ", we have mapped the temporal and strain specific dynamics of newly synthesized proteins in host.

Example 9

Testing the accuracy of "Computational software for integrating hyperplexing results"

Dephoure et al's dataset on "Metabolic labeling with stable isotope amino acids in cell culture " and TMT which was also 18-plex, was used to test the accuracy of "Computational software for integrating hyperplexing results " pipeline. The data was searched using MaxQuant with modified Lysine labels with the TMT masses incorporated. The data was searched using the parameters as described in the original publication. Figure 1 shows the comparison of the proteins identified using the two pipelines (Sequest+ Vista vs MaxQuant+HyperQuant). Although the two analyses used different search engines, and are expected to differ based on their quantitation tools as well, we still observed a high degree of correlation in the ratios obtained for the proteins that matched between the two outputs. The top left panel shows the comparison of proteins identified by the two pipelines. The other panels depict the concordance between the ratios for different reporter ions. Example 10

Distribution of peptide areas across different time points in one MS run of "Computational software for integrating hyperplexing results".

The peptide areas in the first time point was compared against the other time points in the control to check if our technique with the analysis pipeline can demonstrate robust linearity in the data across the whole dynamic range of quantitation. Since the data used is from control samples to test this assumption (Figure 2), it is expected that most protein areas should behave similarly when those are not regulated by developmental process or external stress (which was absent). Any deviation from an expected linearity can be attributed to developmental stage (biological) changes.

Hyperplexing enables us to run 18 different samples in a single run reducing the errors owing to run to run variation. We compared the 6 "Quantification using isobaric tag " samples for peptides in uninfected condition (Figure 2). Since all the six samples were run in a single shot, the technical variability inherent in one should also reflect in others. When the peptides' "Quantification using isobaric tag " areas were compared across the time-course samples, it was expected that similar proteome profiles for all proteins would be observed. It means that the complete proteome will follow similar quantitative trends of abundance and are likely to be linear in nature even if the amounts vary. We observed a strong correlation (R >0.93) confirming the hypothesis we posited. On the x-axis, the peptide area of 113 reporter ion is plotted while y-axis has other "Quantification using isobaric tag " reporter ion areas- 114.1 (dark blue), 115.1 (red), 116.1 (green), 117.1 (violet), 118.1 (light blue) with their equations and correlations represented in the same colour as the series.

In other words, this shows that uninfected cells from time-point 0 to time-point 6 may have biological variations but the technical variation in the data is negligible. Instead of ratios, areas were used for this comparison to visualize the variation in data across the complete dynamic range of values.

Example 11

Comparison of variability and dispersion in the distribution pattern of protein ratios in replicates

Figure 3 shows the comparison of histograms for the replicates. As is clear from the figure 3, high concordance in ratio distributions (linear 1 and linear 2 shown in green and blue respectively) was observed. A similar distribution pattern of protein ratios suggests that broadly the variability and dispersion is nearly same for both replicates.

Example 12

Effect of Outlier removal

Since the heterogeneity of variance for the area measurement is variable and dependent on the intensity of precursor peak in a nonlinear manner, not all peptides depict accurate quantitative values.

Therefore, it is suggested to remove outliers when enough values of peptide measurements are present so as to prevent ratio skewing due to outliers. The effect of outlier removal (OR) on ratio concordance between replicates was tested and found that removing outlier peptides while integrating peptides to protein ratios can enhance the overall performance of quantitation (figure 4). The left panel column shows the replicates comparison before outlier removal (labeled as OR in figure 4) ; the middle panel depicts enhanced concordance after removal of outlier peptide during protein ratio calculation; the right panel shows removal of outlier peptide combined with selection of highly confident protein identifications (>3 peptides).

Example 13

Effect of replicate combination on quantitation yield

When the replicates are compared individually (LI and L2) or together (L1L2), the identification yields may not be affected by replicate combination in totality (Figure 5A) but the quantitation yield approaches the identification yield when replicates are combined together rather than individually analyzed (Figure 5B). Figure 5 depicts the identified (A) and quantified (B) proteins from replicates of Set 1 either individually (linear 1 in blue and linear 2 in yellow) or when combined (green). This shows the combination of replicates helps select nearly all identified proteins (except 2) for quantification. There are 136 proteins in L1L2 combined, 22 proteins from LI, and 23 proteins from L2, which would have been left out due to either low number of spectra in one replicate or both the replicates. Due to combination, the statistical sampling rescues these proteins and enhances quantification.

Example 14

Effect of replicate combination on accuracy of quantitation When the replicates are analyzed individually, the quantitative ratio for some proteins may not be accurate due to low number of sampled peptide ratios. When combined together, the sampling statistics get better and the new combined ratio is more accurate due to proper sampling from the combined peptide ratios. Figure 6 depicts the effect of replicate combination on quantitation accuracy. It shows LI (x-Axis) vs. L1L2 combined (black) and LI vs. L2 (red). We can observe several outliers in the data (values away from the diagonal in the red series). Upon combination, the protein quantitation errors are reduced and outliers are minimal (in the black series) as they are brought closer to the real value (towards the diagonal) by replicate combination.

Example 15

Comparison of Ratios between Set 1 and Set 2 before and after outlier removal

We know that having few peptides per protein may not give statistically viable quantitation values and sometimes false positive identifications are made when we rely on less than three peptides per protein. So we tested that if we select only the highly confident protein identifications (>3 peptides), can ratio concordance between replicates (right panel) be improved. Figure 7 shows the combined protein ratio comparisons between set 1 and set 2, where all replicates (linear 1 and 2 + nonlinear 1 and 2) were already integrated. The concordance between the two suggests that despite different replicate runs, the two light labels of "Metabolic labeling with stable isotope amino acids in cell culture " experiments performed similarly for "Quantification using isobaric tag " reporters (left panel) and upon outlier removal, the performance was enhanced (right panel). Some points away from diagonal in left panel are brought closer to the diagonal in right panel depicting better concordance, supported by better regression coefficient.

Example 16

Comparison of ratios across all reporters for different replicates

Despite the known reproducibility concerns with shotgun proteomics, the quantitative data faithfully depicts concordant ratios across different technical MS replicates, suggesting high quality of data as shown in figure 8. The figure shows ratio comparisons between replicates when all reporter ions were compared in totality. The top panel shows comparison between Setl replicates - Linearl vs. Linear2 and Nonlinearl vs. Nonlinear2. The bottom panel depicts similar comparisons for second set of 6plex "Quantification using isobaric tag " (set 2).

Claims

We claim

1. A novel quantitative proteomics method for multiplex analysis with throughput accuracy comprising the steps of:

i. culturing the sample in a liquid culture medium at optimal cell density to obtain protein sample.

v. labeling the peptide sample obtained from step (iv) with "Quantification using isobaric tag " .

vii. identification of the peptide obtained from step (vi) by "Protein identification software "

viii. quantification of "Quantification using isobaric tag " labeled peptide using "Tool for quantitation based on isobaric tag method in conjunction with software ".

2. The method as claimed in claim 1 step (i) wherein, the sample is selected from group comprising bacterial, mammalian, plant, tissues, fungal, microbial cell culture and the cell density of the cell culture is in the range of 0.5 to 2 million cells per mL.

3. The method as claimed in claim 1 step (ii) wherein, the protein sample is labeled by one or more non-radioactive stable isotope of essential amino acid selected from the group comprising heavy or medium isotopes of 2 H, 13 C, 15 N, 18 O, more preferably Light, medium, heavy isotopes of lysine and arginine, most preferably 1⁵N₂ ¹³C₆-lysine (Lys8), ¹⁵N₄ ¹³C₆-arginine (ArglO), ¹³C₆-Lysine (Lys6) and ¹³C₆- arginine (Arg6).

4. The method as claimed in claim 1 step (iii) wherein, the newly synthesized protein is labeled with a non-canonical amino acid selected from a group comprising azidohomoalanine (AHA) and homopropargylglycine (HPG), more preferably azidohomoalanine (AHA).

5. The method as claimed in claim 1 step (iv) wherein, the newly synthesized protein sample is concentrated, alkylated, reduced, and digested with 0.4 -1 μg/mL of trypsin.

6. The method as claimed in claim 1 step (v) wherein, the peptide sample is labeled with "Quantification using isobaric tag " with 6- 10 isobaric reagent more preferably with 6 isobaric reagent (113.1, 114.1, 115.1, 116.1, 117.1 118.1) at different time points.

7. The method as claimed in claim 1 step (vi) wherein peptide sample is identified by Liquid chromatography - Mass spectrometry analysis more preferably nano- reverse phase high-pressure liquid chromatography electrospray ionization tandem mass spectrometry (RP-HPLC-ESI-MS/MS).

8. The method as claimed in claim 1 step (vii) wherein peptide sample is identified using "Proteomics identification software" against Uniprot human database with Common Repository of Adventitious Proteins-cRAP sequences using search parameters:

i. "Metabolic labeling with stable isotope amino acids in cell culture " on lysine and arginine with mass of "Quantification using isobaric tag " added to lysine labels.

ii. fixed modifications - carbamidomethylation at cysteine & "Quantification using isobaric tag " at N-terminus.

iii. variable modifications - methionine oxidation, "Metabolic labeling with noncanonical amino acid tagging ".

iv. deamidation at NQ residues.

9. The method as claimed in claim 1 step (viii) wherein, quantitation of "Quantification using isobaric tag " labeled peptide sample is done using "Tool for quantitation based on isobaric tag method in conjunction with software" using summed intensity (SI).

10. The method as claimed in claim 1 step (ix) wherein, peptide identified by "Proteomics identification software " and peptide quantified by "Tool for quantitation based on isobaric tag method in conjunction with software " is integrated using "Computational software for integrating hyperplexing results ".

11. The method as claimed in claim 1 step (ix) wherein, the integration of results is carried out by "Computational software for integrating hyperplexing results" in two steps using following modules:

i. mapper module -mapping of spectrum identification with quantitation for each different labels after matching mass-to-charge (m/z) and retention time (RT). ii. combiner module- combines the protein level information from these mapped files across any number of replicates, removes statistical outliers and outputs a list of protein with final quantitation ratios integrated from input files.

12. The method as claimed in claim 1 wherein, step (ix), protein is quantified using "Computational software for integrating hyperplexing results" after outlier peptide ratio removal and filtering out proteins with peptide count lower than 3.

13. The method as claimed in claim 1 wherein, the multiplex capacity enables 10 to 50, more preferably 10-40, most preferably 18-30 sample quantitation in a single mass spectrometry run.

14. Use of the method as claimed in claim 1 to study the temporal dynamics of a proteome.