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Estimate phosphopeptide identification rate by tandem mass spectrometry

ABSTRACT: Mass spectrometry has played a significant role in the identification of unknown phosphoproteins and sites of phosphorylation in biological samples. Analysis of protein phosphorylation, in particular large scale phosphorylation analysis – phosphoproteomics, has recently been enhanced by efficient enrichment, fast and accurate instrument, and better software, but challenges remain because of low stoichiometry of phosphorylation and poor efficiency in ionization and fragmentation due to neutral loss. To evaluate current mass spectrometric performance, we present here a method to estimate phosphopeptide identification efficiency by tandem mass spectrometry. Phosphopeptides were directly isolated from whole plant cell extracts, dephosphorylated, and then incubated with three purified kinases, CK2, MAPK6, and SnRK2.6, along with 16O4- and 18O4-ATP separately, for in vitro kinase reactions. Phosphopeptides were further enriched and analyzed by LC-MS. Phosphopeptide identification rate was estimated by comparing phosphopeptides identified by tandem mass spectrometry with phosphopeptide pairs generated by stable isotope labeled kinase reactions. Overall we found that the current high speed and high accurate mass spectrometry can only identify 20-40% of total phosphopeptides mainly due to relatively poor fragmentation and low abundance.

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

SUBMITTER: Dr. W. Andy Tao

PROVIDER: PXD005079 | JPOST Repository | Sun Oct 02 00:00:00 BST 2016

REPOSITORIES: jPOST

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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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Estimate phosphopeptide identification rate by tandem mass spectrometry

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