title: Enhanced sequence coverage of proteins in human cerebrospinal fluid using multiple enzymatic digestion and linear ion trap LC-MS/MS.
abstract: 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 Finnigan LTQ 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.
authors.affiliation: Scientific Instruments Division of Thermo Electron Corporation, 355 River Oaks Parkway, San Jose, CA 95134, USA. roger.biringer@thermo.com
authors.forename: Roger G
authors.initials: RG
authors.lastname: Biringer
authors.forename: Heidi
authors.initials: H
authors.lastname: Amato
authors.forename: Michael G
authors.initials: MG
authors.lastname: Harrington
authors.forename: Alfred N
authors.initials: AN
authors.lastname: Fonteh
authors.forename: James N
authors.initials: JN
authors.lastname: Riggins
authors.forename: Andreas F R
authors.initials: AF
authors.lastname: Hühmer
mesh_terms: D000595:Amino Acid Sequence; D002555:Cerebrospinal Fluid / Q000737:chemistry*; D002853:Chromatography, Liquid / Q000379:methods*; D002918:Chymotrypsin / Q000494:pharmacology; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000494:pharmacology*; D020543:Proteome / Q000032:analysis* / Q000235:genetics; D012697:Serine Endopeptidases / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
chemical_list: D020543:Proteome; D010447:Peptide Hydrolases; D012697:Serine Endopeptidases; C060325:arginine endopeptidase; D002918:Chymotrypsin; D014357:Trypsin