Value of using multiple proteases for large-scale mass spectrometry-based proteomics.

Large-scale protein sequencing methods rely on enzymatic digestion of complex protein mixtures to generate a collection of peptides for mass spectrometric analysis. Here we examine the use of multiple proteases (trypsin, LysC, ArgC, AspN, and GluC) to improve both protein identification and characterization in the model organism Saccharomyces cerevisiae. Using a data-dependent, decision tree-based algorithm to tailor MS(2) fragmentation method to peptide precursor, we identified 92 095 unique peptides (609 665 total) mapping to 3908 proteins at a 1% false discovery rate (FDR). These results were a significant improvement upon data from a single protease digest (trypsin) - 27 822 unique peptides corresponding to 3313 proteins. The additional 595 protein identifications were mainly from those at low abundances (i.e., < 1000 copies/cell); sequence coverage for these proteins was likewise improved nearly 3-fold. We demonstrate that large portions of the proteome are simply inaccessible following digestion with a single protease and that multiple proteases, rather than technical replicates, provide a direct route to increase both protein identifications and proteome sequence coverage.

[1]  Jeroen Krijgsveld,et al.  Lys-N and trypsin cover complementary parts of the phosphoproteome in a refined SCX-based approach. , 2009, Analytical chemistry.

[2]  S. Ficarro,et al.  Improved electrospray ionization efficiency compensates for diminished chromatographic resolution and enables proteomics analysis of tyrosine signaling in embryonic stem cells. , 2009, Analytical chemistry.

[3]  Joshua J. Coon,et al.  Post-acquisition ETD spectral processing for increased peptide identifications , 2009, Journal of the American Society for Mass Spectrometry.

[4]  Rainer Malik,et al.  Evaluation of the low-specificity protease elastase for large-scale phosphoproteome analysis. , 2008, Analytical chemistry.

[5]  G. McAlister,et al.  Decision tree–driven tandem mass spectrometry for shotgun proteomics , 2008, Nature Methods.

[6]  M. Mann,et al.  Comprehensive mass-spectrometry-based proteome quantification of haploid versus diploid yeast , 2008, Nature.

[7]  G. McAlister,et al.  A proteomics grade electron transfer dissociation-enabled hybrid linear ion trap-orbitrap mass spectrometer. , 2008, Journal of proteome research.

[8]  Richard D. Smith,et al.  The influence of sample preparation and replicate analyses on HeLa Cell phosphoproteome coverage. , 2008, Journal of proteome research.

[9]  G. McAlister,et al.  Performance Characteristics of Electron Transfer Dissociation Mass Spectrometry*S , 2007, Molecular & Cellular Proteomics.

[10]  G. McAlister,et al.  Implementation of electron-transfer dissociation on a hybrid linear ion trap-orbitrap mass spectrometer. , 2007, Analytical chemistry.

[11]  Steven P Gygi,et al.  Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry , 2007, Nature Methods.

[12]  Suresh Mathivanan,et al.  Global proteomic profiling of phosphopeptides using electron transfer dissociation tandem mass spectrometry , 2007, Proceedings of the National Academy of Sciences.

[13]  Steven P. Gygi,et al.  Large-scale phosphorylation analysis of mouse liver , 2007, Proceedings of the National Academy of Sciences.

[14]  Roger G Biringer,et al.  Enhanced sequence coverage of proteins in human cerebrospinal fluid using multiple enzymatic digestion and linear ion trap LC-MS/MS. , 2006, Briefings in functional genomics & proteomics.

[15]  Alan L Rockwood,et al.  Proteomic identification of oncogenic chromosomal translocation partners encoding chimeric anaplastic lymphoma kinase fusion proteins. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[16]  M. Mann,et al.  Status of complete proteome analysis by mass spectrometry: SILAC labeled yeast as a model system , 2006, Genome Biology.

[17]  George C Tseng,et al.  Statistical characterization of the charge state and residue dependence of low-energy CID peptide dissociation patterns. , 2005, Analytical chemistry.

[18]  Achim Kramer,et al.  Mapping of phosphorylation sites by a multi-protease approach with specific phosphopeptide enrichment and NanoLC-MS/MS analysis. , 2005, Analytical chemistry.

[19]  J. Shabanowitz,et al.  Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[20]  S. Bryant,et al.  Open mass spectrometry search algorithm. , 2004, Journal of proteome research.

[21]  Jennifer N. Sutton,et al.  Low‐molecular‐weight human serum proteome using ultrafiltration, isoelectric focusing, and mass spectrometry , 2004, Electrophoresis.

[22]  E. O’Shea,et al.  Global analysis of protein expression in yeast , 2003, Nature.

[23]  E. O’Shea,et al.  Global analysis of protein localization in budding yeast , 2003, Nature.

[24]  Eugene A. Kapp,et al.  Mining a tandem mass spectrometry database to determine the trends and global factors influencing peptide fragmentation. , 2003, Analytical chemistry.

[25]  William S Hancock,et al.  Multiple enzymatic digestion for enhanced sequence coverage of proteins in complex proteomic mixtures using capillary LC with ion trap MS/MS. , 2003, Journal of proteome research.

[26]  Joshua E. Elias,et al.  Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome. , 2003, Journal of proteome research.

[27]  John I. Clark,et al.  Shotgun identification of protein modifications from protein complexes and lens tissue , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[28]  J. Yates,et al.  Large-scale analysis of the yeast proteome by multidimensional protein identification technology , 2001, Nature Biotechnology.

[29]  F. McLafferty,et al.  Electron Capture Dissociation of Multiply Charged Protein Cations. A Nonergodic Process , 1998 .

[30]  Vicki H. Wysocki,et al.  Influence of Peptide Composition, Gas-Phase Basicity, and Chemical Modification on Fragmentation Efficiency: Evidence for the Mobile Proton Model , 1996 .

[31]  L. Hood,et al.  Internal amino acid sequence analysis of proteins separated by one- or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose. , 1987, Proceedings of the National Academy of Sciences of the United States of America.