Application of displacement chromatography for the proteome analysis of a human plasma protein fraction

TY - JOUR

T1 - Application of displacement chromatography for the proteome analysis of a human plasma protein fraction

AU - Ahrends, Robert

AU - Lichtner, Björn

AU - Bertsch, Andreas

AU - Kohlbacher, Oliver

AU - Hildebrand, Diana

AU - Trusch, Maria

AU - Schlüter, Hartmut

N1 - Copyright (c) 2009 Elsevier B.V. All rights reserved.

PY - 2010/5/7

Y1 - 2010/5/7

N2 - 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.

AB - 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.

KW - Blood Proteins

KW - Chromatography, High Pressure Liquid

KW - Chromatography, Ion Exchange

KW - Humans

KW - Peptide Fragments

KW - Proteome

KW - Proteomics

KW - Sensitivity and Specificity

KW - Spectrometry, Mass, Electrospray Ionization

KW - Trypsin

U2 - 10.1016/j.chroma.2009.10.028

DO - 10.1016/j.chroma.2009.10.028

M3 - SCORING: Journal article

C2 - 19854443

VL - 1217

SP - 3321

EP - 3329

JO - J CHROMATOGR A

JF - J CHROMATOGR A

SN - 0021-9673

IS - 19

ER -