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

Abstract

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.

Involvement of Lyn and the Atypical Kinase SgK269/PEAK1 in a Basal Breast Cancer Signaling Pathway

“…SgK269 kinase activity may represent a target for therapeutic intervention with small-molecule kinase inhibitors, and if SgK269 activates Stat3 via an indirect mechanism, then Stat3 kinases that act downstream of SgK269 may also represent therapeutic targets. Finally, given its signaling role downstream of multiple tyrosine kinases (6)(7)(8)(9)(10)(11), and recent data showing that SgK269 modulates cellular sensitivity to trastuzumab (12), it will be interesting to determine whether SgK269 represents a biomarker of therapeutic responsiveness that could be used for improved patient stratification for targeted therapy.…”

“…Although both proteins exhibit substitutions within the critical DFG, a triplet of the kinase domain activation loop, leading to their initial classification as pseudokinases (4,5), the Klemke group has reported that SgK269 possesses weak tyrosine kinase activity (3). In addition, SgK269 is tyrosine-phosphorylated downstream of several tyrosine kinases (6)(7)(8)(9)(10)(11). SgK269 localizes to the actin cytoskeleton and focal adhesions and associates with p130Cas and Crk, indicating that it may regulate cytoskeletal organization (3).…”

Deep proteome profiling of Trichoplax adhaerens reveals remarkable features at the origin of metazoan multicellularity

“…5a), especially when taking into account that no common tyrosine phosphorylation enrichment procedures 23 were used. To further independently confirm these high levels of tyrosine phosphorylation, we performed phosphopeptide enrichment experiments using Ti-IMAC affinity beads 14 .…”

In-depth Analyses of Kinase-dependent Tyrosine Phosphoproteomes Based on Metal Ion-functionalized Soluble Nanopolymers

“…S9. Although there have been reports that tyrosine phosphopeptides can be analyzed with single stage affinity purification based on anti-Tyr(P) antibodies (7,34), including recent large scale phosphotyrosine profiling (34,35), tandem affinity approaches combining antiTyr(P) antibodies and metal oxide improve the selectivity and confidence in the identification of tyrosine phosphopeptides (5,8). We carried out parallel experiments to compare three methods using anti-tyrosine phosphopeptide PT66 alone, PT66 ϩ TiO 2 , and PT66 ϩ PolyMAC purifications.…”