High-throughput mass spectrometric discovery of protein post-translational modifications.

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.

[1]  B. Barrell,et al.  Life with 6000 Genes , 1996, Science.

[2]  M. Karas,et al.  Suppression effects in enzymatic peptide ladder sequencing using ultraviolet ‐ matrix assisted laser desorption/ionization ‐ mass spectrometry , 1998, Electrophoresis.

[3]  J. Berg.  Genome sequence of the nematode C. elegans: a platform for investigating biology. , 1998, Science.

[4]  Rolf Apweiler,et al.  The SWISS-PROT protein sequence data bank and its supplement TrEMBL in 1998 , 1998, Nucleic Acids Res..

[5]  K. Kinzler,et al.  Serial Analysis of Gene Expression , 1995, Science.

[6]  D. Hochstrasser,et al.  Extraction of membrane proteins by differential solubilization for separation using two‐dimensional gel electrophoresis , 1998, Electrophoresis.

[7]  C. Watanabe,et al.  Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[8]  A. Burlingame,et al.  Rapid mass spectrometric peptide sequencing and mass matching for characterization of human melanoma proteins isolated by two-dimensional PAGE. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[9]  T. Hunkapiller,et al.  Peptide mass maps: a highly informative approach to protein identification. , 1993, Analytical biochemistry.

[10]  S. Henikoff,et al.  Amino acid substitution matrices from protein blocks. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[11]  M. Mann,et al.  Developments in matrix-assisted laser desorption/ionization peptide mass spectrometry. , 1996, Current opinion in biotechnology.

[12]  P Ferrara,et al.  In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. , 1992, Analytical biochemistry.

[13]  Rolf Apweiler,et al.  The SWISS-PROT protein sequence database and its supplement TrEMBL in 2000 , 2000, Nucleic Acids Res..

[14]  R. Fleischmann,et al.  Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. , 1995, Science.

[15]  C. Gray,et al.  From genome to proteome: Protein map of Haemophilus influenzae , 1997, Electrophoresis.

[16]  Wei Zhou,et al.  Characterization of the Yeast Transcriptome , 1997, Cell.

[17]  D. Hochstrasser,et al.  Towards an automated approach for protein identification in proteome projects , 1998, Electrophoresis.

[18]  A. Podtelejnikov,et al.  Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[19]  A Bairoch,et al.  Two‐dimensional gel electrophoresis of Escherichia coli homogenates: The Escherichia coli SWISS‐2DPAGE database , 1996, Electrophoresis.

[20]  G. Gonnet,et al.  Protein identification by mass profile fingerprinting. , 1993, Biochemical and biophysical research communications.

[21]  D. Hochstrasser,et al.  Improved and simplified in‐gel sample application using reswelling of dry immobilized pH gradients , 1997, Electrophoresis.

[22]  R. Aebersold,et al.  A microfabricated device for rapid protein identification by microelectrospray ion trap mass spectrometry. , 1997, Analytical chemistry.

[23]  P. Højrup,et al.  Rapid identification of proteins by peptide-mass fingerprinting , 1993, Current Biology.

[24]  Rolf Apweiler,et al.  The SWISS-PROT protein sequence data bank and its supplement TrEMBL , 1997, Nucleic Acids Res..

[25]  Amos Bairoch,et al.  The PROSITE database, its status in 1997 , 1997, Nucleic Acids Res..

[26]  D. Hochstrasser,et al.  Progress with proteome projects: why all proteins expressed by a genome should be identified and how to do it. , 1996, Biotechnology & genetic engineering reviews.

[27]  P. Højrup,et al.  Use of mass spectrometric molecular weight information to identify proteins in sequence databases. , 1993, Biological mass spectrometry.

[28]  P. Roepstorff,et al.  Mass spectrometry in protein studies from genome to function. , 1997, Current opinion in biotechnology.

[29]  Amos Bairoch,et al.  Detailed peptide characterization using PEPTIDEMASS – a World‐Wide‐Web‐accessible tool , 1997, Electrophoresis.

[30]  W. G. Bryson,et al.  Characterisation of wool intermediate filament proteins separated by micropreparative two‐dimensional electrophoresis , 1997, Electrophoresis.