Toward chromatographic analysis of interacting protein networks.
Protein complexes, collectively referred to as the cellular interactome, appear to play a major role in cellular regulation. At present it is thought that the interactome could be composed of hundreds of protein assemblies. The objective of the work described here was to examine the prospect that chromatographic methods widely used in the preparative isolation of native proteins could be incorporated into global proteomics methods in such a way that the primary structure of protein complexes of sufficient stability to survive chromatography could be recognized along with their participation in protein complexes. Because wide differences in sizes are a unique feature of protein complexes, size-exclusion chromatography (SEC) was incorporated into all the fractionation strategies examined. Anion-exchange chromatography (AEC) and hydrophobic-interaction chromatography (HIC) were also examined because of the broad utility that these methods have shown in the preparation of proteins with native structure. Slightly more than a third of all proteins identified in yeast lysates were found to elute from SEC, AEC, and HIC columns with an apparent molecular weight much higher than that predicted from their parent gene. These results were interpreted to mean that these proteins were migrating through columns as components of protein complexes. Based on studies with multidimensional SEC-->RPLC (reversed-phase liquid chromatography), AEC-->SEC, and HIC-->SEC systems, it was concluded that recognition of proteins in complexes could be easily incorporated into multidimensional chromatographic methods for global proteomics when at least one of the fractionation dimensions included SEC of native proteins.
Liu
Xiuping
X
Department of Chemistry, Purdue University, West Lafayette IN 47907, USA.
Yang
Wen-chu
WC
Gao
Qiang
Q
Regnier
Fred
F
D002845:Chromatography; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D057927:Hydrophobic and Hydrophilic Interactions; D025941:Protein Interaction Mapping; D011506:Proteins / Q000032:analysis / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000302:isolation & purification*
D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins