Dataset Information

ITRAQ LLC-PK1 CnI - Sum of peak intensities outperforms peak area integration in iTRAQ protein expression measurement by LC-MS/MS using a TripleTOF 5600+ platform

ABSTRACT: In the field of quantitative proteomics, the Isobaric Tags for Relative & Absolute Quanti-tation (iTRAQ) technology has demonstrated efficacy for proteome monitoring despite its lack of a consensus for data handling. In this study, after peptide and protein identification, we com-pared the widespread quantitation method based on the calculation of MS/MS reporter ion peaks areas ratios (Protein Pilot) to the alternative method based on the calculation of ratios of the sum of peak intensities (jTRAQx (Quant)) and we processed output data with the in-house Customi-zable iTRAQ Ratios Calculator (CiR-C) algorithm. Quantitation based on peak area ratios dis-played no significant linear correlation with Western blot quantitation. On the contrary, quantita-tion based on the sum of peak intensities displayed a significant linear association with Western blot quantitation (non-zero slope; Pearson correlation coefficient test, r = 0.2962, p = 0.0099 **) with an average bias of 0.08747 ± 0.5004 and 95% Limits of Agreement from - 0.8932 to 1.068. We proposed the Mascot-jTRAQx-CiR-C strategy as a simple yet powerful data processing ad-junct to the iTRAQ technology

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Sus Scrofa Domesticus (domestic Pig)

TISSUE(S): Epithelial Cell, Kidney

SUBMITTER: Bastien Burat

LAB HEAD: Marie Essig

PROVIDER: PXD007891 | Pride | 2019-08-19

REPOSITORIES: Pride

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Sum of peak intensities outperforms peak area integration in iTRAQ protein expression measurement by LC-MS/MS using a TripleTOF 5600+ platform.

Burat Bastien B Gonzalez Julien J Sauvage François-Ludovic FL Aouad Hassan H Arnion Hélène H Pinault Emilie E Marquet Pierre P Essig Marie M

Bioscience reports 20190607 6

In the field of quantitative proteomics, the Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) technology has demonstrated efficacy for proteome monitoring despite its lack of a consensus for data handling. In the present study, after peptide and protein identification, we compared the widespread quantitation method based on the calculation of MS/MS reporter ion peaks areas ratios (ProteinPilot) to the alternative method based on the calculation of ratios of the sum of peak intensitie ...[more]

PMID: 31110078

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Project description:Dynamic tyrosine phosphorylation is fundamental to a myriad of cellular processes. However, the inherently low abundance of tyrosine phosphorylation in the proteome and the inefficient enrichment of phosphotyrosine(pTyr)-containing peptides has led to poor pTyr peptide identification and quantitation, critically hindering researchers’ ability to elucidate signaling pathways regulated by tyrosine phosphorylation in systems where cellular material is limited. The most popular approaches to wide-scale characterization of the tyrosine phosphoproteome use pTyr enrichment with pan-specific, anti-pTyr antibodies from a large amount of starting material. Methods that decrease the amount of starting material and increase the characterization depth of the tyrosine phosphoproteome while maintaining quantitative accuracy and precision would enable the discovery of tyrosine phosphorylation networks in rarer cell populations. To achieve these goals, a novel method leveraging the multiplexing capability of tandem mass tags (TMT) and the use of trigger channels – cells treated with the promiscuous tyrosine phosphatase inhibitor pervanadate (PV) – selectively increased the relative abundance of pTyr-containing peptides. After PV facilitated selective fragmentation of pTyr-containing peptides, TMT reporter ions delivered sensitive quantitation of each peptide for the experimental samples while the quantitation from PV trigger channels was ignored. This method yielded up to 6.3-fold boost in quantification depth of statistically significant data derived from contrived ratios, compared to TMT without trigger channels or intensity-based label-free (LF) quantitation while maintaining quantitative accuracy and precision, allowing quantitation of over 2300 unique pTyr peptides from only 1 mg of T cell receptor-stimulated Jurkat T cells. The trigger channel strategy can potentially be applied in analyses of other post-translational modifications where treatments that broadly boost the levels of those modifications across the proteome are available.

Project description:Background The goat (Capra hircus) represents one of the most important farm animal species. It is reared in all continents with an estimated world population of about 800 million of animals. Despite its importance, studies on the goat genome are still in their infancy compared to those in other farm animal species. Comparative mapping between cattle and goat showed only a few rearrangements in agreement with the similarity of chromosome banding. We carried out a cross species cattle-goat array comparative genome hybridization (aCGH) experiment in order to identify copy number variations (CNVs) in the goat genome analysing animals of different breeds (Saanen, Camosciata delle Alpi, Girgentana, and Murciano-Granadina) using a tiling oligonucleotide array with ~385,000 probes designed on the bovine genome. Results We identified a total of 161 CNVs (an average of 17.9 CNVs per goat), with the largest number in the Saanen breed and the lowest in the Camosciata delle Alpi goat. By aggregating overlapping CNVs identified in different animals we determined CNV regions (CNVRs): on the whole, we identified 127 CNVRs covering about 11.47 Mb of the virtual goat genome referred to the bovine genome (0.435% of the latter genome). These 127 CNVRs included 86 loss and 41 gain and ranged from about 24 kb to about 1.07 Mb with a mean and median equal to 90,292 bp and 49,530 bp, respectively. To evaluate whether the identified goat CNVRs overlap with those reported in the cattle genome, we compared our results with those obtained in four independent cattle experiments. Overlapping between goat and cattle CNVRs was highly significant (P<0.0001) suggesting that several chromosome regions might contain recurrent interspecies CNVRs. Genes with environmental functions were over-represented in goat CNVRs as reported in other mammals. Conclusions We describe a first map of goat CNVRs. This provides information on a comparative basis with the cattle genome by identifying putative recurrent interspecies CNVs between these two ruminant species. Several goat CNVs affect genes with important biological functions. Further studies are needed to evaluate the functional relevance of these CNVs and their effects on behavior, production, and disease resistance traits in goats. We analysed CNVs in the goat genome by means of a cross-species aCGH experiment using the Roche NimbleGen platform (Roche NimbleGen Inc., Madison, WI; http://www.nimblegen.com) based on custom tiling arrays designed on the bovine (Bos taurus) genome, Btau_4.0 version, including a fraction of BTA13 of the University of Maryland (UMD) Bos taurus v. 2.0 assembly (ftp://ftp.cbcb.umd.edu/pub/data/Bos_taurus/Bos_taurus_UMD_2.0/). Arrays contained ~385,000 probes on a single slide to provide an evenly distributed coverage with an average interval of ~6 kb for the Btau_4.0 genome. The BTA13 of the UMD v. 2.0 assembly was included from nucleotide position 48 M bp to nucleotide position 78 M bp (4,673 oligonucleotides and average spacing of ~6 kb). This chromosome region was analysed as internal control because it contains the ASIP gene, not assembled in the BTA13 of the Btau_4.0 version Goat genomic DNA was extracted from blood of 2 Camosciata delle Alpi, 3 Girgentana, 3 Saanen, 1 black and 1 brown Murciano-Granadina goats using the Wizard® Genomic DNA Purification kit (Promega Corporation, Madison, WI). All analysed animals were females. Reference DNA sample of one (C1) Camosciata delle Alpi goat was labeled with Cy5 and co-hybridised with the other test DNA samples labelled with Cy3 on 9 different arrays. A self hybridisation (reference labelled by both Cy5 and Cy3) was carried out in another array. Hybridization and array scanning were performed by Roche NimbleGen as previously described. Data normalization was conducted using the normalize.qsline method from the Bioconductor package in R. Then data were analysed for each hybridization using normalized log2 ratios using the CGHweb server (http://compbio.med.harvard.edu/CGHweb/) that includes multiple algorithms.

Project description:One of the most extensively studied protein modifications is phosphorylation, a reversible posttranslational modification (PTM) with a central role in a broad range of cellular processes such as cell metabolism, homeostasis, transcription and apoptosis. Due to their transient character, phosphorylations initiate and propagate signal transduction pathways, making these fundamental to inter- and intracellular communication. Detection of phosphorylation is nevertheless challenging considering the dynamic regulation, low stoichiometry and heterogeneous character of the phosphosites. Numerous dedicated phosphoproteomics workflows have therefore been developed in order to unravel phosphorylation networks and the activity of their modulating enzymes. In these workflows, phosphopeptides are typically enriched prior to mass spectrometry analysis to remove the strong background interference caused by the bulk of non-phosphorylated peptides (3). Apart from requiring thorough optimization and a corresponding high level of expertise of the analyst however, enrichment is also responsible for sample loss and limited quantitation accuracy (4). Subsequent mass spectrometry analysis is furthermore confounded by low ionization efficiency and inferior fragmentation of phosphopeptides. Conventional MS/MS analysis with collision-induced dissociation predominantly results in the neutral loss of the phosphate group, leading to poor sequence coverage due to reduced backbone fragmentation. An additional stage of fragmentation in MS3 or the use of alternative fragmentation technologies such as higher energy C-trap dissociation or electron transfer dissociation can partially facilitate the detection and identification of phosphopeptides (5, 6). In order to draw accurate biological conclusions it is mandatory to obtain quantitative information on the phospho-modifications. Most quantitative approaches aim to elucidate the relative phosphorylation changes between samples, employing direct label-free comparisons of phospho-enriched fractions, or metabolic or chemical labeling methods such as SILAC, iTRAQ or tandem mass tag (TMT) (4, 6, 7). Such methods however, forego the investigation of phosphorylation stoichiometry, also known as occupancy, since such analysis requires the simultaneous monitoring of the unphosphorylated counterpart (8). One interesting labeling strategy that is specifically designed to quantify occupancy circumvents the common difficulties of standard phosphopeptide detection by focusing on the non-phosphorylated counterparts of the phosphopeptides (11). Herein, a peptide sample is briefly split in two identical parts which are differentially labeled, preceding a phosphatase treatment of one part and mock-treatment of the other. Immediately afterwards the two parts are recombined for the subsequent LC-MS/MS analysis. Dephosphorylation of a phosphopeptide will induce a 79.979 mass shift resulting in a skewed label ratio in the unphosphorylated peptide’s reporter region which now specifies the phosphorylation stoichiometry (12). Although the principle has been described previously (12-19), only one phosphatase-based workflow was adapted specifically for the large-scale study of a complete lysate whereby known phosphopeptides present in a database were quantified by isotopic labels (20). Here we extend for the first time the isobaric tag equivalent of this approach for the large-scale quantitative phospho-analysis of complex mixtures. The technique was validated on three different instrument platforms on epidermal growth factor (EGF)-stimulated HeLa cells (23). We discuss a novel data analysis approach for the discovery of high stoichiometry phosphopeptides in complex peptide mixtures and present a way to interpret the sizable dataset without matching the peptide identifications to a phospho-database. Because all peptides and not only the phosphorylated fraction of the proteome is measured in this approach, instruments with high-speed acquisition capabilities and adequate resolution for accurate reporter quantitation are indispensable. Still, because of the minimal technical variation that is introduced throughout the workflow (as for Wu et.al. (20)), implementation of extensive fractionation and purification steps such as protein gel-electrophoresis and 2DLC at the peptide level allows for more in-depth coverage of the (phospho) proteome. What makes this approach even more attractive, is that the iTRAQ multiplexing allows for a replicate measurement within one experiment, creating a new data analysis workflow that meets the stringent requirements of peptide quantitation. All three instrument panels were able to annotate a large set of known phosphoproteins. Extending the latter sample fractionation and using novel methodologies to avoid dilution of reporter ratios in MSMS will allow for the annotation of increasing numbers of phosphopeptide with increasingly low stoichometries in the future.

Dataset Information

ITRAQ LLC-PK1 CnI - Sum of peak intensities outperforms peak area integration in iTRAQ protein expression measurement by LC-MS/MS using a TripleTOF 5600+ platform

Publications

Sum of peak intensities outperforms peak area integration in iTRAQ protein expression measurement by LC-MS/MS using a TripleTOF 5600+ platform.

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