title: High-throughput mass spectrometric discovery of protein post-translational modifications.
abstract: The availability of genome sequences, affordable mass spectrometers and high-resolution two-dimensional gels has made possible the identification of hundreds of proteins from many organisms by peptide mass fingerprinting. However, little attention has been paid to how information generated by these means can be utilised for detailed protein characterisation. Here we present an approach for the systematic characterisation of proteins using mass spectrometry and a software tool FindMod. This tool, available on the internet at http://www.expasy.ch/sprot/findmod.html , examines peptide mass fingerprinting data for mass differences between empirical and theoretical peptides. 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 currently considered (acetylation, amidation, biotinylation, C-mannosylation, deamidation, flavinylation, farnesylation, formylation, geranyl-geranylation, gamma-carboxyglutamic acids, hydroxylation, lipoylation, methylation, myristoylation, N -acyl diglyceride (tripalmitate), O-GlcNAc, palmitoylation, phosphorylation, pyridoxal phosphate, phospho-pantetheine, pyrrolidone carboxylic acid, sulphation) a total of 29 different rules were made. These consider which amino acids can carry a modification, whether the modification 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 utility of the approach with proteins from 2-D gels of Escherichia coli and sheep wool, where post-translational modifications predicted by FindMod were confirmed by MALDI post-source decay peptide fragmentation. As the approach is amenable to automation, it presents a potentially large-scale means of protein characterisation in proteome projects.
authors.affiliation: Macquarie University, Sydney, NSW 2109, Australia. m.wilkins@rna.bio.mq.edu.au
authors.forename: M R
authors.initials: MR
authors.lastname: Wilkins
authors.forename: E
authors.initials: E
authors.lastname: Gasteiger
authors.forename: A A
authors.initials: AA
authors.lastname: Gooley
authors.forename: B R
authors.initials: BR
authors.lastname: Herbert
authors.forename: M P
authors.initials: MP
authors.lastname: Molloy
authors.forename: P A
authors.initials: PA
authors.lastname: Binz
authors.forename: K
authors.initials: K
authors.lastname: Ou
authors.forename: J C
authors.initials: JC
authors.lastname: Sanchez
authors.forename: A
authors.initials: A
authors.lastname: Bairoch
authors.forename: K L
authors.initials: KL
authors.lastname: Williams
authors.forename: D F
authors.initials: DF
authors.lastname: Hochstrasser
mesh_terms: D000107:Acetylation; D000577:Amides / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D003545:Cysteine / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry; D007091:Image Processing, Computer-Assisted; D007633:Keratins / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D008715:Methionine / Q000031:analogs & derivatives / Q000378:metabolism; D008745:Methylation; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000378:metabolism; D010444:Peptide Elongation Factor Tu / Q000378:metabolism; D010449:Peptide Mapping; D010544:Peroxidases; D054464:Peroxiredoxins; D010649:Phenylalanine; D011499:Protein Processing, Post-Translational; D012984:Software; D013045:Species Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014443:Tyrosine
chemical_list: D000577:Amides; D014443:Tyrosine; D010649:Phenylalanine; D007633:Keratins; D008715:Methionine; D010088:Oxidoreductases; D010544:Peroxidases; D054464:Peroxiredoxins; D010444:Peptide Elongation Factor Tu; D008239:Lysine; D003545:Cysteine; C034666:propionamide; C013111:methionine sulfoxide