ELECTROSPRAY-IONIZATION MASS-SPECTROMETRY OF PHOSPHOPEPTIDES ISOLATEDBY ONLINE IMMOBILIZED METAL-ION AFFINITY-CHROMATOGRAPHY

Electrospray ionization mass spectrometry (ESI/MS) affords a rapid and sensitive technique for determining peptides produced by the enzymati c digestion of phosphoproteins. When coupled with on-line immobilized metal-ion affinity chromatography (IMAC), the combination allows separ ation and mass spectrometric identification of phosphorylated and nonp hosphorylated peptides. In this study, the feasibility and general app licability of on-line IMAC/ESI/MS is investigated by using immobilized ferric ions for selective chelation of several phosphotyrosine and ph osphoserine peptides. The sensitivity and practicality of the techniqu e for phosphoproteins are demonstrated via the analysis of 30 pmol (ap proximately 0.7 mug) of bovine beta-casein purified by sodium dodecyls ulfate-polyacrylamide gel electrophoresis, electroblotted onto a polyv inylidene difluoride membrane, and digested in situ with trypsin. It i s observed that on-line IMAC/ESI/MS suffers less from sample losses th an experiments performed off-line, suggesting that the limiting factor s in sensitivity for this technique are the purification procedures an d sample handling rather than the IMAC and mass spectrometry. Thus, th e ability to inject the tryptic digest of an electroblotted protein di rectly onto the column without buffer exchange and to analyze the elue nt directly via on-line coupling of the IMAC column to the mass spectr ometer greatly reduces sample losses incurred through sample handling and provides a convenient method for analyzing phosphopeptides at low levels.