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Phospho-iTRAQ data article: Assessing isobaric labels for the large-scale study of phosphopeptide stoichiometry.

ABSTRACT: The ability to distinguish between phosphopeptides of high and low stoichiometry is essential to discover the true extent of protein phosphorylation. We here extend the strategy whereby a peptide sample is briefly split in two identical parts and differentially labeled preceding the phosphatase treatment of one part (Pflieger et al., 2008. Mol. Cell. Proteomics, 7: 326-46 [1]; Wu et al., 2011. Nat. Methods, 8: 677-83 [2]). Our Phospho-iTRAQ method focuses on the unmodified counterparts of phosphorylated peptides, which thus circumvents the ionization, fragmentation, and phospho-enrichment difficulties that hamper quantitation of stoichiometry in most common phosphoproteomics methods. Since iTRAQ enables multiplexing, simultaneous (phospho)proteome comparison between internal replicates and multiple samples is possible. The technique was validated on multiple instrument platforms by adding internal standards of high stoichiometry to a complex lysate of control and EGF-stimulated HeLa cells. To demonstrate the flexibility of PhosphoiTRAQ with regards to the experimental setup and data mining, the proteome coverage was extended through gel fractionation, while an internal replicate measurement creates more stringent data analysis opportunities. The latter allows other researchers to set their own threshold for selecting potential phosphorylation events in the dataset presented here, depending on the biological question or corroboration under investigation. The latest developments in MS instrumentation promise to further increase the resolution of the stoichiometric measurement of Phospho-iTRAQ in the future. The data accompanying the manuscript on this approach (Glibert et al., 2015, J. Proteome Res. 14: 2015, 839-49 [5]) have been deposited to the ProteomeXchange with identifier PXD001574.

SUBMITTER: Glibert P

PROVIDER: S-EPMC4510381 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Phospho-iTRAQ data article: Assessing isobaric labels for the large-scale study of phosphopeptide stoichiometry.

Glibert Pieter P Meert Paulien P Van Steendam Katleen K Martens Lennart L Deforce Dieter D Dhaenens Maarten M

Data in brief 20150506

The ability to distinguish between phosphopeptides of high and low stoichiometry is essential to discover the true extent of protein phosphorylation. We here extend the strategy whereby a peptide sample is briefly split in two identical parts and differentially labeled preceding the phosphatase treatment of one part (Pflieger et al., 2008. Mol. Cell. Proteomics, 7: 326-46 [1]; Wu et al., 2011. Nat. Methods, 8: 677-83 [2]). Our Phospho-iTRAQ method focuses on the unmodified counterparts of phosph ...[more]

PMID: 26217764

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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.

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Phospho-iTRAQ data article: Assessing isobaric labels for the large-scale study of phosphopeptide stoichiometry.

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Phospho-iTRAQ data article: Assessing isobaric labels for the large-scale study of phosphopeptide stoichiometry.

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