title: Use of an integrated MS--multiplexed MS/MS data acquisition strategy for high-coverage peptide mapping studies.
abstract: Liquid chromatography/mass spectrometry (LC/MS) peptide maps have become a basic tool for characterizing proteins of biological and pharmaceutical interest. The ability to generate reproducible maps with high protein sequence coverage is a central goal of methods development. We have applied a recently developed analytical approach (termed LC/MS(E)) to LC/MS peptide mapping. Using the LC/MS(E) approach, the mass detector alternates between a low-energy scanning mode (MS) for accurate mass peptide precursor identification, and an elevated-energy mode (MS(E)) for generation of accurate mass multiplex peptide fragmentation data. In this paper, we evaluate this analytical approach against a tryptic digest of yeast enolase. From the low-energy data, high peptide map coverage (98% of sequence from peptides >3 amino acids) was reproducibly obtained. The MS signal for essentially equimolar peptides varied over 2 orders of magnitude in intensity, and peptide intensities could be precisely and reproducibly measured. Using the temporal constraint that MS(E) peptide fragment ions exhibit chromatographic profiles that parallel the precursor ions that generated them, we were able to produce accurate mass time-resolved MS/MS information for all enolase peptides with sufficient abundance to produce a detectable fragment ion.
authors.affiliation: Life Sciences R&D, Waters Corporation, Milford, MA, USA.
authors.forename: Asish B
authors.initials: AB
authors.lastname: Chakraborty
authors.forename: Scott J
authors.initials: SJ
authors.lastname: Berger
authors.forename: John C
authors.initials: JC
authors.lastname: Gebler
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry*; D010449:Peptide Mapping / Q000379:methods*; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D014357:Trypsin / Q000378:metabolism; D015003:Yeasts / Q000201:enzymology
chemical_list: D010446:Peptide Fragments; D014357:Trypsin; D010751:Phosphopyruvate Hydratase