High-throughput mass spectrometric discovery of protein post-translationalmodifications

The availability of genome sequences, affordable mass spectrometers and hig h-resolution two-dimensional gels has made possible the identification of h undreds of proteins from many organisms by peptide mass fingerprinting. How ever, Little attention has been paid to how information generated by these means can be utilised for detailed protein characterisation. Here we presen t an approach for the systematic characterisation of proteins using mass sp ectrometry and a software tool FindMod. This tool, available on the interne t at http://wwM.expasy.ch/sprot/findmod.html, examines peptide mass fingerp rinting data for mass differences between empirical and theoretical peptide s. Where mass differences correspond to a post-translational modification, intelligent rules are applied to predict the amino acids in the peptide, if any, that might carry the modification. FindMod rules were constructed by examining 5153 incidences of post-translational modifications documented in the SWISS-PROT database, and for the 22 post-translational modifications c urrently considered (acetylation, amidation, biotinylation, C-mannosylation , deamidation, flavinylation, farnesylation, formylation, geranyl-geranylat ion, gamma-carboxyglutamic acids, hydroxylation, lipoylation, methylation, myristoylation, N-acyl diglyceride (tripalmitate), O-GlcNAc, palmitoylation , phosphorylation, pyridoxal phosphate, phospho-pantetheine, pyrrolidone ca rboxylic acid, sulphation) a total of 29 different rules were made. These c onsider which amino acids can carry a modification, whether the modificatio n occurs on N-terminal, C-terminal or internal amino acids, and the type of organisms on which the modification can be found. We illustrate the utilit y of the approach with proteins from 2-D gels of Escherichia coil and sheer wool, where post-translational modifications predicted by FindMod were con firmed by MALDI post-source decay peptide fragmentation. As the approach is amenable to automation, it presents a potentially large-scale means of pro tein characterisation in proteome projects. (C) 1999 Academic Press.