A Tyrosine Phosphoproteome Analysis Approach Enabled by Selective Dephosphorylation with Protein Tyrosine Phosphatase.

Protein tyrosine phosphorylation (pTyr) plays a prominent role in signal transduction and regulation in all eukaryotic cells. In conventional immunoaffinity purification (IP) methods, phosphotyrosine peptides are isolated from the digest of cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. However, low sensitivity, poor reproducibility, and high cost are universal concerns for IP approaches. In this study, we presented an antibody-free approach to identify phosphotyrosine peptides by using protein tyrosine phosphatase (PTP). It was found that most of the PTPs including PTP1B, TCPTP, and SHP1 can efficiently and selectively dephosphorylate phosphotyrosine peptides. We then designed a workflow by combining two Ti4+-IMAC-based phosphopeptide enrichment steps with PTP-catalyzed dephosphorylation for tyrosine phosphoproteomics analysis. This workflow was first validated by selective detection of phosphotyrosine peptides from semicomplex samples and then applied to analyze the tyrosine phosphoproteome of Jurkat T cells. Around 1000 putative former phosphotyrosine peptides were identified from less than 500 μg of cell lysate. The tyrosine phosphosites on the majority of these peptides could be unambiguously determined for over 70% of them possessing only one tyrosine residue. It was also found that the tyrosine sites identified by this method were highly complementary to those identified by the SH2 superbinder-based method. Therefore, the combination of Ti4+-IMAC enrichment with PTP dephosphorylation provides an alternative and cost-effective approach for tyrosine phosphoproteomics analysis.