Size-exclusion chromatography in multidimensional separation schemes for proteome analysis.
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.
Lecchi
Paolo
P
Department of Pharmacology, The George Washington University School of Medicine and Health Sciences, 2300 I St. N.W., Washington, DC, 20037, USA. phmpxl@gwumc.edu
Gupte
Anand R
AR
Perez
Ricardo E
RE
Stockert
Lyubov V
LV
Abramson
Fred P
FP
D001426:Bacterial Proteins / Q000032:analysis*; D002850:Chromatography, Gel / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry*; D007525:Isoelectric Focusing / Q000379:methods*; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D001426:Bacterial Proteins; D020543:Proteome