PURIFICATION AND IDENTIFICATION OF TYROSINE-PHOSPHORYLATED PROTEINS FROM B-LYMPHOCYTES STIMULATED THROUGH THE ANTIGEN RECEPTOR

The activation of protein tyrosine kinases (PTKs) and subsequent tyros ine phosphorylation of cellular proteins is a critical initial signal in the response of eukaryotic cells to mitogens, differentiative signa ls, 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 ty rosine-phosphorylated in response to receptor signaling remain unident ified. As some of these unidentified PTK substrates may also be signal -transducing proteins, their identification and functional characteriz ation is an important objective towards understanding receptor signali ng. We describe the development of a comprehensive and general process for the isolation and structural characterization of tyrosine-phospho rylated proteins. The method involves enrichment by anti-phosphotyrosi ne affinity chromatography, electrophoretic concentration and separati on, and proteolytic fragmentation of individual purified phosphoprotei ns. Resulting peptide fragments are separated by microbore reverse-pha se high performance liquid chromatography (RP-HPLC) and a portion of t he eluted peptides are subjected to electrospray-mass spectrometry (ES /MS) for accurate determination of peptide masses. Proteolytic fragmen tation of a protein produces a characteristic set of peptide masses th at can be used to rapidly identify the protein by searching databases containing the peptide mass ''fingerprints'' for all known proteins. T he identity of the protein established by this method can be confirmed by sequence analysis of selected peptides. We have applied this proce dure to the analysis of PTK substrates from B lymphocytes that have be en stimulated through the B cell antigen receptor (BCR). Signaling by this receptor is involved in the generation of antibodies against fore ign molecules (antigens). The BCR activates multiple PTKs which phosph orylate at least 30 different proteins. We have identified several of these tyrosine-phosphorylated proteins, including Syk, a PTK that is k nown to be tyrosine-phosphorylated in activated B cells. Thus, the pro cedure described here can be used to identify regulatory proteins of l ow abundance. The process consists of a logical succession of compatib le steps that avoids pitfalls inherent to prior attempts to characteri ze low abundance phosphoproteins and should find wide use for the iden tification of tyrosine-phosphorylated proteins in other cell types.