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

Enrichment of bacterial phosphopeptides is an essential step prior to bottom‐up mass spectrometry‐based analysis of the phosphoproteome, which is fundamental to understanding the role of phosphoproteins in cell signaling and regulation of protein activity. We developed an automated immobilized metal...

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Vydané v:Electrophoresis Ročník 34; číslo 11; s. 1619 - 1626
Hlavní autori: Qu, Yi, Wu, Si, Zhao, Rui, Zink, Erika, Orton, Daniel J., Moore, Ronald J., Meng, Da, Clauss, Therese R. W., Aldrich, Joshua T., Lipton, Mary S., Paša-Tolić, Ljiljana
Médium: Journal Article
Jazyk:English
Vydavateľské údaje: Germany Blackwell Publishing Ltd 01.06.2013
Predmet:
Affinity
Amino Acid Sequence
Automated
Bacteria
Chromatography
Chromatography, Affinity - instrumentation
Electrophoresis
Enrichment
Equipment Design
Escherichia coli
Escherichia coli - chemistry
Escherichia coli Proteins - chemistry
Escherichia coli Proteins - isolation & purification
Immobilized metal affinity chromatography enrichment
Metals - chemistry
Molecular Sequence Data
Peptides
Phosphopeptides - chemistry
Phosphopeptides - isolation & purification
Phosphoproteins - chemistry
Phosphoproteins - isolation & purification
Phosphorylation
Proteomics
Proteomics - methods
ISSN:0173-0835, 1522-2683, 1522-2683
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Popis
Shrnutí:Enrichment of bacterial phosphopeptides is an essential step prior to bottom‐up mass spectrometry‐based analysis of the phosphoproteome, which is fundamental to understanding the role of phosphoproteins in cell signaling and regulation of protein activity. We developed an automated immobilized metal affinity chromatography (IMAC) system to enrich strong cation exchange‐fractionated phosphopeptides from the soluble proteome of Escherichia coli MG1655 grown on minimal medium. Initial demonstration of the system resulted in identification of 75 phosphopeptides covering 52 phosphoproteins. Consistent with previous studies, many of these phosphoproteins are involved in the carbohydrate portion of central metabolism. The automated system utilizes a large capacity IMAC column that can effectively enrich phosphopeptides from a bacterial sample by increasing peptide loading and reducing the wash time. An additional benefit of the automated IMAC system is reduced labor and associated costs.
Bibliografia:Battelle Memorial Institute under DOE - No. DE-AC05-76RL01830
EMSL intramural research projects and EMSL capability development projects
U.S. Department of Energy, located at Pacific Northwest National Laboratory (PNNL)
istex:57E3E6EF1B7FC9428863A9D0E59C338ABB55AC1B
ark:/67375/WNG-K1TKT91K-J
U. S. Department of Energy Office of Biological and Environmental Research (DOE/BER)
ArticleID:ELPS4667
These authors contributed equally to this work.
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ISSN:0173-0835
1522-2683
1522-2683
DOI:10.1002/elps.201200628