Application of displacement chromatography for the proteome analysis of a human plasma protein fraction.
It was the aim of this study to compare the performance of displacement chromatography with gradient elution chromatography both applied as the cation-exchange separation step for a proteome analysis in a bottom-up approach using multidimensional chromatography for the separation of tryptic peptides prior to their mass spectrometric analysis. The tryptic digest of the human Cohn fraction IV-4 served as a sample. For both chromatography modes commonly used operating parameters were chosen thus ensuring optimal separation results of equal sample amounts for each mode. All resulting fractions were analyzed with an HPLC-chip-LC-MS system. The eluate of the HPLC-chip column was ionized by electrospray ionization (ESI) and analyzed with an ion-trap mass spectrometer. For guaranteeing high confidence concerning the identity of the peptides, the mass spectrometric data were processed by different bioinformatic tools applying stringent criteria. By the displacement approach the total amount of identified proteins (78) was significantly higher than in the gradient mode (58). The results showed that displacement chromatography is a well suited alternative in comparison to gradient elution separation for analysis of proteomes via the bottom-up approach applying multidimensional chromatography, especially in those cases when larger quantities of proteins are available.
University Medical Center Hamburg-Eppendorf, Department of Clinical Chemistry, Mass Spectrometric Proteomics, Campus Forschung, N27 Room 00.008, Martinistr 52, 20246 Hamburg, Germany.
Robert
R
Ahrends
Björn
B
Lichtner
Andreas
A
Bertsch
Oliver
O
Kohlbacher
Diana
D
Hildebrand
Maria
M
Trusch
Hartmut
H
Schlüter
D001798:Blood Proteins / Q000032:analysis* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D006801:Humans; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D014357:Trypsin / Q000378:metabolism
D001798:Blood Proteins; D010446:Peptide Fragments; D020543:Proteome; D014357:Trypsin