title: Towards defining the urinary proteome using liquid chromatography-tandem mass spectrometry. I. Profiling an unfractionated tryptic digest.
abstract: The proteome of normal male urine from a commercial pooled source has been examined using direct liquid chromatography-tandem mass spectrometry (LC-MS/MS). The entire urinary protein mixture was denatured, reduced and enzymatically digested prior to LC-MS/MS analysis using a hybrid-quadrupole time-of-flight mass spectrometer (Q-TOF) to perform data-dependent ion selection and fragmentation. To fragment as many peptides as possible, the mixture was analyzed four separate times, with the mass spectrometer selecting ions for fragmentation from a subset of the entire mass range for each run. This approach requires only an autosampler on the HPLC for automation (i.e, unattended operation). Across these four analyses, 1.450 peptide MS/MS spectra were matched to 751 sequences to identify 124 gene products (proteins and translations of expressed sequence tags). Interestingly, the experimental time for these analyses was less than that required to run a single two-dimensional gel.
authors.affiliation: Departments of Biochemistry and Genetics, Amgen, Thousand Oaks, CA, USA.
authors.forename: C S
authors.initials: CS
authors.lastname: Spahr
authors.forename: M T
authors.initials: MT
authors.lastname: Davis
authors.forename: M D
authors.initials: MD
authors.lastname: McGinley
authors.forename: J H
authors.initials: JH
authors.lastname: Robinson
authors.forename: E J
authors.initials: EJ
authors.lastname: Bures
authors.forename: J
authors.initials: J
authors.lastname: Beierle
authors.forename: J
authors.initials: J
authors.lastname: Mort
authors.forename: P L
authors.initials: PL
authors.lastname: Courchesne
authors.forename: K
authors.initials: K
authors.lastname: Chen
authors.forename: R C
authors.initials: RC
authors.lastname: Wahl
authors.forename: W
authors.initials: W
authors.lastname: Yu
authors.forename: R
authors.initials: R
authors.lastname: Luethy
authors.forename: S D
authors.initials: SD
authors.lastname: Patterson
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D002853:Chromatography, Liquid / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional; D015894:Genome, Human; D006801:Humans; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification; D020543:Proteome / Q000235:genetics / Q000302:isolation & purification*; D014357:Trypsin; D014556:Urine / Q000737:chemistry
chemical_list: D010446:Peptide Fragments; D020543:Proteome; D014357:Trypsin