Briefings in Functional Genomics and Proteomics Advance Access published online on May 26, 2006
Briefings in Functional Genomics and Proteomics, doi:10.1093/bfgp/ell026
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* To whom correspondence should be addressed. The cerebrospinal fluid (CSF) provides a ready access into the health state of the central nervous system, and alterations in some CSF proteins have been documented in brain disease. However, the complete variety of proteins is not known and methods to identify protein components are still being developed. The goal of this study was to examine the sequence coverage obtained from human CSF digests produced with different proteases. Enzymatic digests of CSF proteins were obtained with arginine-C endopeptidase (ArgC), glutamic acid endopeptidase (GluC), chymotrypsin, trypsin and their combinations, and then examined using reverse phase chromatography and a FinniganTM LTQTM linear ion trap mass spectrometer. Peptide sequences were identified with BioWorks 3.1 and sequence coverage calculated for the 38 most confidently identified proteins. Trypsin and GluC yielded greater coverage than chymotrypsin, while ArgC had the least sequence coverage. Protein sequence coverage was affected only slightly over four orders of magnitude dynamic range of abundance. Combining the peptides derived from different proteases further increased the coverage. Maximal sequence coverage was achieved by combining digest results from both GluC and trypsin. These results have implications for future studies to identify CSF proteins and their post-translational modifications. Dr Roger G. Biringer is a Senior Scientist in the Scientific Instruments Division of Thermo Electron Corporation. His research interests include proteomics of neurological disorders and the development of mass spectrometric methodologies to solve biological problems. Heidi Amato received her Master's Degree in Chemistry from San Jose State University, San Jose, CA. She is currently an entrepreneur in Oregon. Michael G. Harrington is the Clan Chief of the Molecular Neurology Program at Huntington Medical Research Institutes where his main focus is to elucidate the biochemistry of migraine and neurodegenerative diseases. Alfred N. Fonteh is a Principal Investigator/Science Director of the Molecular Neurology Program at the Huntington Medical Research Institutes. His major interest is in understanding the biochemical mechanisms of neurological diseases. James N. Riggins is a scientist in the Molecular Neurology Program and in the Liver Program at Huntington Medical Research Institutes. Andreas F.R. Hühmer is the Program Manager for the Scientific Instruments Division at Thermo Electron in San Jose, California. His research interests include the development of mass spectrometry-based techniques and software tools to address scientific problems in Biology and Medicine.
Original Article
Enhanced sequence coverage of proteins in human cerebrospinal fluid using multiple enzymatic digestion and linear ion trap LC-MS/MS
Roger G. Biringer *,
Heidi Amato,
Michael G. Harrington,
Alfred N. Fonteh,
James N. Riggins,
and
Andreas F. R. Hühmer
Roger G. Biringer, E-mail: roger.biringer{at}thermo.com
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