Purification and identification of tyrosine-phosphorylated proteins from B lymphocytes stimulated through the antigen...

The activation of protein tyrosine kinase (PTKs) and subsequent tyrosine phosphorylation of cellular proteins is a critical initial signal in the response of eukaryotic cells to mitogens, differentiative signals, and other stimuli. A number of PTK substrates have been identified and many of these are components of signal transduction pathways that regulate cell function. However, the majority of proteins that are tyrosine-phosphorylated in response to receptor signaling remain unidentified. As some of these unidentified PTK substrates may also be signal-transducing proteins, their identification and functional characterization is an important objective towards understanding receptor signaling. We describe the development of a comprehensive and general process for the isolation and structural characterization of tyrosine-phosphorylated proteins. The method involves enrichment by anti-phosphotyrosine affinity chromatography, electrophoretic concentration and separation, and proteolytic fragmentation of individual purified phosphoproteins. Resulting peptide fragments are separated by microbore reverse-phase high performance liquid chromatography (RP-HPLC) and a portion of the eluted peptides are subjected to electrospray-mass spectrometry (ES/MS) for accurate determination of peptide masses. Proteolytic fragmentation of a protein produces a characteristic set of peptide masses that can be used to rapidly identify the protein by searching databases containing the peptide mass "fingerprints" for all known proteins. The identity of the protein established by this method can be confirmed by sequence analysis of selected peptides. We have applied this procedure to the analysis of PTK substrates from B lymphocytes that have been stimulated through the B cell antigen receptor (BCR). Signaling by this receptor is involved in the generation of antibodies against foreign molecules (antigens). The BCR activates multiple PTKs which phosphorylate at least 30 different proteins. We have identified several of these tyrosine-phosphorylated proteins, including Syk, a PTK that is known to be tyrosine-phosphorylated in activated B cells. Thus, the procedure described here can be used to identify regulatory proteins of low abundance. The process consists of a logical succession of compatible steps that avoids pitfalls inherent to prior attempts to characterize low abundance phosphoproteins and should find wide use for the identification of tyrosine-phosphorylated proteins in other cell types.