Automated immobilized metal affinity chromatography system for enrichment of Escherichia coli phosphoproteome

• DOI: 10.1002/elps.201200628
• Publication Date: 2013-03-14T10:54:56Z
• Authors: Yi Qu; Si Wu; Rui Zhao; Erika Zink; Daniel J. Orton; Ronald J. Moore; Da Meng; Therese R. W. Clauss; Joshua T. Aldrich; Mary S. Lipton; Ljiljana Paša‐Tolić
• Keywords: Proteome

Enrichment of bacterial phosphopeptides is an essential step prior to bottom-up mass spectrometry-based analysis the phosphoproteome, which fundamental understanding role phosphoproteins in cell signaling and regulation protein activity. We developed automated immobilized metal affinity chromatography (IMAC) system enrich strong cation exchange-fractionated from soluble proteome Escherichia coli MG1655 grown on minimal medium. Initial demonstration resulted identification 75 covering 52 phosphoproteins. Consistent with previous studies, many these are involved carbohydrate portion central metabolism. The utilizes a large capacity IMAC column that can effectively sample by increasing peptide loading reducing wash time. An additional benefit reduced labor associated costs.