Phospho.ELM: a database of phosphorylation sites update 2008

Diella, Francesca; Gould, Cathryn M.; Chica, Claudia; Via, Allegra; Gibson, Toby J.

doi:10.1093/nar/gkm772

Cited by 209 publications

(249 citation statements)

References 26 publications

(27 reference statements)

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“…This is one of the largest experimental data sets of phosphotyrosine peptides detected in a single experiment without any further TiO 2 or IMAC enrichment. We compared our data set with phosphotyrosine peptides and sites reported in the recent update of the Phospho.ELM (28) database and with a recently reported large phosphotyrosine peptide data set of lung cancer cell lines consisting of the phosphotyrosine peptides cumulatively identified from 20 different cell lines and 30 different tissues samples (10). Approximately 28% of the phosphotyrosine sites that were identified in the present study overlapped with Rikova et al (10) (supplemental Fig.…”

Section: Resultssupporting

confidence: 73%

“…The different constitutions of their and our cells, together with a different antibody used for phosphotyrosine peptide enrichment, might explain the relatively small overlap in tyrosine phosphopeptides, whereas the greater protein amount and larger number of LC-MS analyses might have allowed them an even deeper penetration into the tyrosine phosphoproteome. The overlap between LTP and HTP phosphosites in the Phospho.ELM database is surprisingly low, indicating that the tyrosine phosphoproteome has not yet been fully mapped (28). Not unexpectedly, the overlap between our data set and the HTP data set is larger than with the smaller LTP data set.…”

Section: Discussionmentioning

confidence: 81%

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In-depth Qualitative and Quantitative Profiling of Tyrosine Phosphorylation Using a Combination of Phosphopeptide Immunoaffinity Purification and Stable Isotope Dimethyl Labeling

Boersema

Foong

Ding

et al. 2010

Molecular & Cellular Proteomics

156

121

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Several mass spectrometry-based assays have emerged for the quantitative profiling of cellular tyrosine phosphorylation. Ideally, these methods should reveal the exact sites of tyrosine phosphorylation, be quantitative, and not be cost-prohibitive. The latter is often an issue as typically several milligrams of (stable isotope-labeled) starting protein material are required to enable the detection of low abundance phosphotyrosine peptides. Here, we adopted and refined a peptidecentric immunoaffinity purification approach for the quantitative analysis of tyrosine phosphorylation by combining it with a cost-effective stable isotope dimethyl labeling method. We were able to identify by mass spectrometry, using just two LC-MS/MS runs, more than 1100 unique non-redundant phosphopeptides in HeLa cells from about 4 mg of starting material without requiring any further affinity enrichment as close to 80% of the identified peptides were tyrosine phosphorylated peptides. Stable isotope dimethyl labeling could be incorporated prior to the immunoaffinity purification, even for the large quantities (mg) of peptide material used, enabling the quantification of differences in tyrosine phosphorylation upon pervanadate treatment or epidermal growth factor stimulation. Analysis of the epidermal growth factor-stimulated HeLa cells, a frequently used model system for tyrosine phosphorylation, resulted in the quantification of 73 regulated unique phosphotyrosine peptides. The quantitative data were found to be exceptionally consistent with the literature, evidencing that such a targeted quantitative phosphoproteomics approach can provide reproducible results. In general, the combination of immunoaffinity purification of tyrosine phosphorylated peptides with large scale stable isotope dimethyl labeling provides a cost-effective approach that can alleviate variation in sample preparation and analysis as samples can be combined early on. Using this approach, a rather complete qualitative and quantitative picture of tyrosine phosphorylation signaling events can be generated. Molecular & Cellular Proteomics 9:84 -99, 2010.

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Section: Resultssupporting

confidence: 73%

Section: Discussionmentioning

confidence: 81%

In-depth Qualitative and Quantitative Profiling of Tyrosine Phosphorylation Using a Combination of Phosphopeptide Immunoaffinity Purification and Stable Isotope Dimethyl Labeling

Boersema

Foong

Ding

et al. 2010

Molecular & Cellular Proteomics

156

121

View full text Add to dashboard Cite

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“…Italics indicate the exact site of phosphorylation could not be determined. ‡ Phosphosites searched in the Phospho-ELM database (41). Values in parentheses show the score from the NetPhos 2.0 Server (22) indicating the probability for the site to be phosphorylated, scale 0 -1.0.…”

Section: Resultsmentioning

confidence: 99%

“…Source proteins for the identified phosphopeptides were searched in the Phospho-ELM database (http://phospho. elm.eu.org) (41) to determine whether the phosphoresidue was previously described or novel. Phosphopeptide sequences were analyzed in the NetPhos 2.0 Server (www.cbs.dtu.dk/services/NetPhos) (25), to obtain a prediction score indicating the confidence in occurrence of the phosphorylation site.…”

Section: (Si Materials and Methods)mentioning

confidence: 99%

Identification of tumor-associated, MHC class II-restricted phosphopeptides as targets for immunotherapy

Depontieu

Qian²,

Zarling

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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The activation and recruitment of CD4 ؉ T cells are critical for the development of efficient antitumor immunity and may allow for the optimization of current cancer immunotherapy strategies. Searching for more optimal and selective targets for CD4 ؉ T cells, we have investigated phosphopeptides, a new category of tumorderived epitopes linked to proteins with vital cellular functions. Although MHC I-restricted phosphopeptides have been identified, it was previously unknown whether human MHC II molecules present phosphopeptides for specific CD4 ؉ T cell recognition. We first demonstrated the fine specificity of human CD4 ؉ T cells to discriminate a phosphoresidue by using cells raised against the candidate melanoma antigen mutant B-Raf or its phosphorylated counterpart. Then, we assessed the presence and complexity of human MHC II-associated phosphopeptides by analyzing 2 autologous pairs of melanoma and EBV-transformed B lymphoblastoid lines. By using sequential affinity isolation, biochemical enrichment, mass spectrometric sequencing, and comparative analysis, a total of 175 HLA-DR-associated phosphopeptides were characterized. Many were derived from source proteins that may have roles in cancer development, growth, and metastasis. Most were expressed exclusively by either melanomas or transformed B cells, suggesting the potential to define cell type-specific phosphatome ''fingerprints.'' We then generated HLA-DR␤1 * 0101-restricted CD4 ؉ T cells specific for a phospho-MART-1 peptide identified in both melanoma cell lines. These T cells showed specificity for phosphopeptide-pulsed antigen-presenting cells as well as for intact melanoma cells. This previously undescribed demonstration of MHC II-restricted phosphopeptides recognizable by human CD4 ؉ T cells provides potential new targets for cancer immunotherapy.tumor antigen ͉ tumor immunology

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“…Reported protein interactions were collected from the following protein-protein interaction databases: Human Protein Reference Database (29) and STRING (30). Reported phosphorylation data was obtained from Phospho.ELM (31,32) and PhosphoSite (33).…”

Section: Methodsmentioning

confidence: 99%

SH2 Domains Recognize Contextual Peptide Sequence Information to Determine Selectivity

Liu¹,

Jablonowski²,

Shah³

et al. 2010

Molecular & Cellular Proteomics

106

131

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Selective ligand recognition by modular protein interaction domains is a primary determinant of specificity in signaling pathways. Src homology 2 (SH2) domains fulfill this capacity immediately downstream of tyrosine kinases, acting to recruit their host polypeptides to ligand proteins harboring phosphorylated tyrosine residues. The degree to which SH2 domains are selective and the mechanisms underlying selectivity are fundamental to understanding phosphotyrosine signaling networks. An examination of interactions between 50 SH2 domains and a set of 192 phosphotyrosine peptides corresponding to physiological motifs within FGF, insulin, and IGF-1 receptor pathways indicates that individual SH2 domains have distinct recognition properties and exhibit a remarkable degree of selectivity beyond that predicted by previously described binding motifs. The underlying basis for such selectivity is the ability of SH2 domains to recognize both permissive amino acid residues that enhance binding and non-permissive amino acid residues that oppose binding in the vicinity of the essential phosphotyrosine. Neighboring positions affect one another so local sequence context matters to SH2 domains. This complex linguistics allows SH2 domains to distinguish subtle differences in peptide ligands. This newly appreciated contextual dependence substantially increases the accessible information content embedded in the peptide ligands that can be effectively integrated to determine binding. This concept may serve more broadly as a paradigm for subtle recognition of physiological ligands by protein interaction domains. Molecular & Cellular Proteomics 9:2391-2404, 2010. The Src homology 2 (SH2)1 domain is the classic archetype for the large family of modular protein interaction domains that serve to organize a diverse array of cellular processes. As its name suggests, the SH2 domain was identified in the regulatory regions of non-receptor protein-tyrosine kinases (PTKs) of the Src family (1). The human genome encodes 110 SH2 domain proteins (2) that represent the primary mechanism for cellular signal transduction immediately downstream of PTKs. SH2 domains interact with phosphorylated tyrosinecontaining peptide sequences (3-6). In doing so, they couple activated PTKs to intracellular pathways that regulate many aspects of cellular communication in metazoans (7,8). In this manner, SH2 domains function alongside PTKs and proteintyrosine phosphatases to direct specificity in phosphotyrosine signaling. SH2 domain proteins play a critical role in development and have been linked to a wide range of human diseases including cancers, diabetes, and immunodeficiencies (2).Activation and recruitment of PTKs and protein-tyrosine phosphatases can control the spatial and temporal organization of phosphotyrosine signaling. However, signaling specificity in terms of downstream targets is entirely determined by phosphotyrosine-mediated recruitment events for which SH2 domains are responsible. Therefore, the binding selectivity of SH2 domains is critical for ...

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Phospho.ELM: a database of phosphorylation sites update 2008

Cited by 209 publications

References 26 publications

In-depth Qualitative and Quantitative Profiling of Tyrosine Phosphorylation Using a Combination of Phosphopeptide Immunoaffinity Purification and Stable Isotope Dimethyl Labeling

In-depth Qualitative and Quantitative Profiling of Tyrosine Phosphorylation Using a Combination of Phosphopeptide Immunoaffinity Purification and Stable Isotope Dimethyl Labeling

Identification of tumor-associated, MHC class II-restricted phosphopeptides as targets for immunotherapy

SH2 Domains Recognize Contextual Peptide Sequence Information to Determine Selectivity

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