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.

Fig. 1.

A, LC-MS base peak chromatogram of the eluate from the phosphotyrosine-immunoprecipitated pervanadate-treated HeLa digest. The numbers indicate intense peaks representing phosphotyrosine-containing peptides. Only a few non-phosphorylated peptides were detected in this eluate. A numbered list of these abundant phosphopeptides is available as supplemental Table 1. * represents a peak of an ion that could not be identified and most likely is a non-peptide species. B, number of identified phosphotyrosine (pY) and non-phosphorylated peptides as a function of the applied Mascot threshold score revealing the apparent specificity of the immunopurification. With decreasing Mascot score threshold, the number of non-modified peptides in the data set increases exponentially below 20. The inflection point at score 20 indicates that at an even lower score relatively more false positive identifications are introduced in the data set.

Fig. 2.

motif-X (29) analysis of here acquired HeLa phosphotyrosine data set identified six conserved motifs, here displayed as WebLogos (30). The numbers indicate the number of peptides exhibiting this motif in the full data set.

Fig. 3.

Experimental scheme for quantitative phosphotyrosine proteome studies. Cell cultures were mock treated or stimulated with pervanadate or EGF. After lysis and enzymatic digestion, peptides were differentially stable isotope dimethyl-labeled and combined before immunoprecipitation with a phosphotyrosine-specific antibody. The precipitate was analyzed by LC-MS followed by quantification using the triplet peaks originating from the different isotopes. AmBi, ammonium bicarbonate.

Fig. 4.

Representative examples of mass spectra and fragmentation spectra of peptides identified and quantified after pervanadate treatment. A, the abundance of SHIP-2 peptide TLSEVDpYAPAGPAR (where pY is phosphotyrosine) (fragmentation spectrum shown of m/z 781.8848, +2, heavy dimethyl-labeled) is dramatically increased by pervanadate treatment. B, the abundance of peptide IGEGTpYGVVYK (fragmentation spectrum shown of m/z 665.3498, +2, intermediate dimethyl-labeled) is not affected by pervanadate treatment. * indicates the site of stable isotope dimethyl labeling. perv., pervanadate.

Fig. 5.

A, clustering of tyrosine phosphorylation profiles. Cluster 1 (dark gray), no or only a small change in phosphorylation levels; cluster 2 (light gray), an immediate up-regulation that remains after 30 min; cluster 3 (black), strong and quick increase in phosphorylation. B, representative examples of mass spectra and fragmentation spectra of peptides identified and quantified after EGF stimulation from each of the clusters. B, BCAR1 peptide HLLAPGPQDIpYDVPPVR (where pY is phosphotyrosine) (fragmentation spectrum shown of m/z 1002.0319, +2, heavy dimethyl-labeled) is from cluster 1 with only a slight increase in phosphorylation upon EGF stimulation. C, SGK269 peptide SSAIRpYQEVWTSSTSPR (fragmentation spectrum shown of m/z 689.6682, +3, intermediate dimethyl-labeled) is from cluster 2 with a larger increase after EGF stimulation. D, RBCK1 peptide NSQEAEVSCPFIDNTpYSCSGK (fragmentation spectrum shown of m/z 1273.0631, +2, heavy dimethyl-labeled) is from cluster 3 showing an extensive increase in abundance after EGF stimulation. * indicates the site of stable isotope dimethyl labeling.

Fig. 6.

Ratios of changes in tyrosine phosphorylation 10 (A) and 30 min (B) after EGF stimulation detected in present study (Boersema et al.) plotted against ratios of overlapping phosphotyrosine sites found in Zhang et al.(5).