Size-exclusion chromatography in multidimensional separation schemes for proteome analysis

被引:32
作者
Lecchi, P [1 ]
Gupte, AR [1 ]
Perez, RE [1 ]
Stockert, LV [1 ]
Abramson, FP [1 ]
机构
[1] George Washington Univ, Sch Med & Hlth Sci, Dept Pharmacol, Washington, DC 20037 USA
来源
JOURNAL OF BIOCHEMICAL AND BIOPHYSICAL METHODS | 2003年 / 56卷 / 1-3期
关键词
size-exclusion chromatography; rotofor; proteomics; mass spectrometry; CRIMS; stable isotopes;
D O I
10.1016/S0165-022X(03)00055-1
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Size-exclusion chromatography (SEC) is a separation technique with a relatively low resolving power, compared to those usually utilized in proteomics. Therefore, it is often overlooked in experimental protocols, when the main goal is resolving complex biological mixtures. In this report, we introduce innovative multidimensional schemes for proteomics analysis, in which SEC plays a practical role. Liquid isoelectric focusing (IEF) was combined with SEC, and experimental results were compared to those obtained by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), well-established techniques relying upon similar criteria for separation. Additional experiments were performed to evaluate the practical contribution of SEC in multidimensional chromatographic separations. Specifically, we evaluated the combination of SEC and ion exchange chromatography in an analytical scheme for the mass spectrometric analysis of protein-extracts obtained from bacterial cultures grown in stable isotope enriched media. Experimental conditions and practical considerations are discussed. (C) 2003 Elsevier Science B.V. All rights reserved.
引用
收藏
页码:141 / 152
页数:12
相关论文
共 19 条
[1]   CRIMS - CHEMICAL-REACTION INTERFACE MASS-SPECTROMETRY [J].
ABRAMSON, FP .
MASS SPECTROMETRY REVIEWS, 1994, 13 (04) :341-356
[2]   Use of Rotofor preparative isoelectrofocusing cell in protein purification procedure [J].
Ayala, A ;
Parrado, J ;
Machado, A .
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY, 1998, 69 (01) :11-16
[3]  
Coligan JE., 1998, CURRENT PROTOCOLS PR
[4]   Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology [J].
Gygi, SP ;
Corthals, GL ;
Zhang, Y ;
Rochon, Y ;
Aebersold, R .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2000, 97 (17) :9390-9395
[5]  
Harris ELV, 1990, PROTEIN PURIFICATION, P175
[6]   Analysis of biopolymers by size-exclusion chromatography mass spectrometry [J].
Lecchi, P ;
Abramson, FP .
JOURNAL OF CHROMATOGRAPHY A, 1998, 828 (1-2) :509-513
[7]   Size exclusion chromatography-chemical reaction interface mass spectrometry: "A perfect match" [J].
Lecchi, P ;
Abramson, FP .
ANALYTICAL CHEMISTRY, 1999, 71 (14) :2951-2955
[8]  
Lederberg J, 2001, SCIENTIST, V15, P8
[9]   Two-dimensional gel electrophoresis: recent advances in sample preparation, detection and quantitation [J].
Lilley, KS ;
Razzaq, A ;
Dupree, P .
CURRENT OPINION IN CHEMICAL BIOLOGY, 2002, 6 (01) :46-50
[10]   Multidimensional separations for protein/peptide analysis in the post-genomic era [J].
Liu, HB ;
Lin, DY ;
Yates, JR .
BIOTECHNIQUES, 2002, 32 (04) :898-+