Tandem mass spectrometry methods for definitive protein identification in proteomics research

Optimized procedures have been developed for the addition of sulfonic acid groups to the N‐termini of low‐level peptides. These procedures have been applied to peptides produced by tryptic digestion of proteins that have been separated by two‐dimensional (2‐D) gel electrophoresis. The derivatized peptides were sequenced using matrix‐assisted laser desorption/ionization (MALDI) post‐source decay (PSD) and electrospray ionization‐tandem mass spectrometry methods. Reliable PSD sequencing results have been obtained starting with sub‐picomole quantities of protein. We estimate that the current PSD sequencing limit is about 300 fmol of protein in the gel. The PSD mass spectra of the derivatized peptides usually allow much more specific protein sequence database searches than those obtained without derivatization. We also report initial automated electrospray ionization‐tandem mass spectrometry sequencing of these novel peptide derivatives. Both types of tandem mass spectra provide predictable fragmentation patterns for arginine‐terminated peptides. The spectra are easily interpreted de novo, and they facilitate error‐tolerant identification of proteins whose sequences have been entered into databases.

[1]  M. M. Bursey,et al.  Some proline substituent effects in the tandem mass spectrum of protonated pentaalanine. , 1992, Biological mass spectrometry.

[2]  J. Watson,et al.  Charge derivatization of peptides for analysis by mass spectrometry. , 1998, Mass spectrometry reviews.

[3]  M. Mann,et al.  Automation of micro‐preparation and enzymatic cleavage of gel electrophoretically separated proteins , 1995, FEBS letters.

[4]  A. Dell,et al.  High sensitivity collisionally-activated decomposition tandem mass spectrometry on a novel quadrupole/orthogonal-acceleration time-of-flight mass spectrometer. , 1996, Rapid communications in mass spectrometry : RCM.

[5]  M. Karas,et al.  Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. , 1988, Analytical chemistry.

[6]  D. Arnott,et al.  An integrated approach to proteome analysis: identification of proteins associated with cardiac hypertrophy. , 1998, Analytical biochemistry.

[7]  D. Kirsch,et al.  Sequenching of peptides in a time-of-flight mass spectrometer: evaluation of postsource decay following matrix-assisted laser desorption ionisation (MALDI) , 1994 .

[8]  M. Wilm,et al.  Error-tolerant identification of peptides in sequence databases by peptide sequence tags. , 1994, Analytical chemistry.

[9]  J. Yates,et al.  An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database , 1994, Journal of the American Society for Mass Spectrometry.

[10]  Waterfield,et al.  Enhancement of charge remote fragmentation in protonated peptides in high-energy CID MALDI-TOF-MS using 'cold' matrices , 1997 .

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

[12]  R Kaufmann,et al.  Mass spectrometric sequencing of linear peptides by product-ion analysis in a reflectron time-of-flight mass spectrometer using matrix-assisted laser desorption ionization. , 1993, Rapid communications in mass spectrometry : RCM.

[13]  B. Spengler.  Post-source decay analysis in matrix-assisted laser desorption/ionization mass spectrometry of biomolecules† , 1997 .

[14]  Marc R. Wilkins,et al.  Protein Identification in Proteome Projects , 1997 .

[15]  M. Mann,et al.  Electrospray ionization for mass spectrometry of large biomolecules. , 1989, Science.

[16]  J. Yates,et al.  The quadrupole ion trap mass spectrometer--a small solution to a big challenge. , 1997, Analytical biochemistry.

[17]  D. Hochstrasser,et al.  Introduction to the Proteome , 1997 .

[18]  A. Shevchenko,et al.  Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. , 1996, Analytical chemistry.

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

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

[21]  J R Yates,et al.  Protein sequencing by tandem mass spectrometry. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[22]  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.

[23]  Marc R. Wilkins,et al.  Proteome Research: New Frontiers in Functional Genomics , 1997, Principles and Practice.

[24]  T. Keough,et al.  A method for high-sensitivity peptide sequencing using postsource decay matrix-assisted laser desorption ionization mass spectrometry. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[25]  Peter R. Baker,et al.  Role of accurate mass measurement (+/- 10 ppm) in protein identification strategies employing MS or MS/MS and database searching. , 1999, Analytical chemistry.

[26]  A. Burlingame,et al.  Protein sequence and structural studies employing matrix-assisted laser desorption ionization-high energy collision-induced dissociation , 1997 .

[27]  R Kaufmann,et al.  Peptide sequencing by matrix-assisted laser-desorption mass spectrometry. , 1992, Rapid communications in mass spectrometry : RCM.

[28]  A. Podtelejnikov,et al.  Delayed extraction improves specificity in database searches by matrix-assisted laser desorption/ionization peptide maps. , 1996, Rapid communications in mass spectrometry : RCM.

[29]  D. Kirsch,et al.  Fundamental aspects of postsource decay in matrix-assisted laser desorption mass spectrometry. 1. Residual gas effects , 1992 .

[30]  R. Cotter,et al.  Sequencing peptides without scanning the reflectron: post-source decay with a curved-field reflectron time-of-flight mass spectrometer. , 1995, Rapid communications in mass spectrometry : RCM.

[31]  K. Biemann.  Contributions of mass spectrometry to peptide and protein structure. , 1988, Biomedical & environmental mass spectrometry.

[32]  V. Neuhoff,et al.  Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G‐250 and R‐250 , 1988, Electrophoresis.

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

[34]  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.

[35]  P. Chaurand,et al.  Post-source decay and delayed extraction in matrix-assisted laser desorption/ionization-reflectron time-of-flight mass spectrometry. Are there trade-offs? , 1996, Rapid communications in mass spectrometry : RCM.