Chromatographic fractionation of protein complexes

Xiuping Liu, Purdue University

Abstract

Protein complexes appear to play a major role in cellular regulation, being collectively referred to as the interactome. At present it is thought that the cellular 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 therapeutic proteins of native structure could be incorporated into global proteomics methods in such a way that the primary structure of proteins could be recognized along with their participation in these protein complexes. Because wide differences in size 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 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 predicted from their parent gene. These results were interpreted to mean that these proteins were migrating through columns as components of high molecular weight protein complexes. Based on studies with multidimensional SEC→RPC, AEC→SEC, and HIC→SEC systems it was concluded that recognition of proteins in complexes could be easy incorporated into multidimensional chromatographic methods for global proteomics when at least one of the fractionation dimensions included SEC of native proteins.

Degree

Ph.D.

Advisors

Regnier, Purdue University.

Subject Area

Analytical chemistry

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