Automated LC-LC-MS-MS platform using binary ion-exchange and gradient reversed-phase chromatography for improved proteomic analyses.
A simple multidimensional liquid chromatography system utilizing an isocratic pump and a HPLC system is described for the comprehensive proteomic analysis of complex peptide digest mixtures by coupled LC-LC-MS-MS techniques. A binary ion-exchange separation was achieved through the use of a strong cation-exchange column followed by a reversed-phase column for data-dependent LC-MS-MS analysis of the unbound analytes, and following salt elution (and concomitant column reequilibration), the bound analytes. Off-line validation of the platform showed near quantitative recovery of fractionated peptides and essentially complete ion-exchange partitioning. In comparative analyses of a highly complex peptide digest mixture a >40% increase in the number of peptide and protein identifications was achieved using this multidimensional platform compared to an unfractionated control.
Department of Biochemistry, Amgen, Inc., Thousand Oaks, CA 91320, USA.
M T
MT
Davis
J
J
Beierle
E T
ET
Bures
M D
MD
McGinley
J
J
Mort
J H
JH
Robinson
C S
CS
Spahr
W
W
Yu
R
R
Luethy
S D
SD
Patterson
D000595:Amino Acid Sequence; D001331:Automation; D002852:Chromatography, Ion Exchange / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D020543:Proteome / Q000737:chemistry*; D015203:Reproducibility of Results
D020543:Proteome