A probability-based approach for high-throughput protein phosphorylation analysis and site localization

  title={A probability-based approach for high-throughput protein phosphorylation analysis and site localization},
  author={Sean A. Beausoleil and Judit Vill{\'e}n and Scott A. Gerber and John Rush and Steven P. Gygi},
  journal={Nature Biotechnology},
Data analysis and interpretation remain major logistical challenges when attempting to identify large numbers of protein phosphorylation sites by nanoscale reverse-phase liquid chromatography/tandem mass spectrometry (LC-MS/MS) (Supplementary Figure 1 online). In this report we address challenges that are often only addressable by laborious manual validation, including data set error, data set sensitivity and phosphorylation site localization. We provide a large-scale phosphorylation data set… 

An efficient dynamic programming algorithm for phosphorylation site assignment of large-scale mass spectrometry data

The algorithm, referred to as PhosSA, optimizes the objective function defined as the summation of peak intensities that are associated with theoretical phosphopeptide fragmentation ions and is shown to achieve > 99% accuracy with a high degree of sensitivity.

Comparison of alternative MS/MS and bioinformatics approaches for confident phosphorylation site localization.

Alternative fragmentation and different bioinformatics approaches are used for the identification and confident localization of phosphorylation sites and underscore the need for the systematic assessment of FLRs for different score values to report confident modification site localization.

Pinpointing phosphorylation sites: Quantitative filtering and a novel site-specific x-ion fragment.

Analyzing a large set of HCD spectra of SILAC-labeled phosphopeptides, a novel fragmentation mechanism is identified that generates a phosphorylation site-specific neutral loss derived x-ion, which directly pinpoints the phosphorylated residue.

Determining In Vivo Phosphorylation Sites Using Mass Spectrometry

The protocols described here focus on two common strategies: (1) identifying phosphorylation sites from individual proteins and small protein complexes, and (2) identifying global phosphorylated sites from whole‐cell and tissue extracts.

Phosphorylation-specific MS/MS scoring for rapid and accurate phosphoproteome analysis.

A new scoring function is presented for the Inspect software for phosphorylated peptide tandem mass spectra for ion-trap instruments, without the need for manual validation, and runs much faster than either SEQUEST or X!Tandem, making desktop phosphoproteomics feasible.

PhosSA: Fast and accurate phosphorylation site assignment algorithm for mass spectrometry data

The main contribution of this study is the design and implementation of a linear time and space dynamic programming strategy for phosphorylation site assignment referred to as PhosSA, which uses summation of peak intensities associated with theoretical spectra as an objective function.

Confident Phosphorylation Site Localization Using the Mascot Delta Score

The Mascot Delta Score is validated as a simple method that achieves similar sensitivity and specificity for phosphosite localization as the published Ascore and it is shown that the ability to call sites correctly increases with increasing distance of two candidate sites within a peptide sequence.

Automated and high confidence protein phosphorylation site localization using complementary collision-activated dissociation and electron transfer dissociation tandem mass spectrometry.

A novel approach for phospho-site localization by the combined use of peptide tandem mass spectrometry data obtained using both collision-activated dissociation and electron transfer dissociation, an approach termed the Cscore is implemented and evaluated.

Reduction of Ambiguity in Phosphorylation-site Localization in Large-scale Phosphopeptide Profiling by Data Filter using Unique Mass Class Information

The Phospho-UMC filter, which is a simple method of localizing the site of phosphorylation using unique mass classes (UMCs) information to differentiate phosphopeptides with differentosphorylation sites, wasveloped and was demonstrated to be effective in the reducing ambiguity associated with the tandem mass spectrometric data analysis of phosphopePTides.



An iterative statistical approach to the identification of protein phosphorylation motifs from large-scale data sets

This method, in addition to shedding light on the consensus sequences of identified and as yet unidentified kinases and modular protein domains, may also eventually be used as a tool to determine potential phosphorylation sites in proteins of interest.

Large-scale characterization of HeLa cell nuclear phosphoproteins.

Using a strategy based on strong cation exchange chromatography, phosphopeptides were enriched from the nuclear fraction of HeLa cell lysate and determined 2,002 phosphorylation sites, an unprecedented large collection of sites permitted a detailed accounting of known and unknown kinase motifs and substrates.

Large-scale Analysis of in Vivo Phosphorylated Membrane Proteins by Immobilized Metal Ion Affinity Chromatography and Mass Spectrometry*

A scheme for two-dimensional peptide separation using strong anion exchange chromatography prior to IMAC that both decreases the complexity of IMAC-purified phosphopeptides and yields a far greater coverage of monophosphorylated peptides is presented.

Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae

Protein kinases are coded by more than 2,000 genes and thus constitute the largest single enzyme family in the human genome. Most cellular processes are in fact regulated by the reversible

Global phosphoproteome of HT-29 human colon adenocarcinoma cells.

The list of protein phosphorylation sites identified in the present experiment provides broad information on phosphorylated proteins under normal (asynchronous) cell culture conditions and may be utilized as surrogate bio-markers to assess the activity of selected kinases and signaling pathways from different cell states and exogenous stimuli.

Comprehensive Identification of Phosphorylation Sites in Postsynaptic Density Preparations*S

To characterize the phosphorylation state of proteins in PSD samples, strong cation exchange (SCX) chromatography with IMAC was combined with reverse phase chromatography coupled to tandem mass spectrometry, which led to the identification of phosphopeptides in most SCX fractions.

Proteomic Analysis of in Vivo Phosphorylated Synaptic Proteins*

Bioinformatic and in vitro phosphorylation assays of peptide arrays suggest that a small number of kinases phosphorylate many proteins and that each substrate is phosphorylated by many kinases, which support a model where the synapse phosphoproteome is functionally organized into a highly interconnected signaling network.

Quantitative Phosphoproteomics Applied to the Yeast Pheromone Signaling Pathway*S

Of more than 700 identified phosphopeptides, 139 were differentially regulated at least 2-fold in response to mating pheromone and among these regulated proteins were components belonging to the mitogen-activated protein kinase signaling pathway and to downstream processes including transcriptional regulation, the establishment of polarized growth, and the regulation of the cell cycle.

Comparative evaluation of mass spectrometry platforms used in large-scale proteomics investigations

Comparisons indicate that Mascot and SEQUEST yield similar results for LTQ-acquired spectra but less so for QSTAR spectra, and low reproducibility between replicate data acquisitions made on one or both instrument platforms can be exploited to increase sensitivity and confidence in large-scale protein identifications.

Optimization and Use of Peptide Mass Measurement Accuracy in Shotgun Proteomics*S

It was shown that improving mass accuracy at a cost to the MS/MS acquisition rate substantially lowered the sensitivity of LC-MS/MS analyses and a simple mass calibration strategy exploiting polydimethylcyclosiloxane background ions as calibrant ions was developed.