Metal Affinity Enrichment Increases the Range and Depth of Proteome Identification for Extracellular Microbial Proteins
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- 作者：Korin E. Wheeler ; Brian K. Erickson ; Ryan Mueller ; Steven W. Singer ; Nathan C. VerBerkmoes ; Mona Hwang ; Michael P. Thelen ; Robert L. Hettich
- 刊名：Journal of Proteome Research
- 出版年：2012
- 出版时间：February 3, 2012
- 年：2012
- 卷：11
- 期：2
- 页码：861-870
- 全文大小：390K
- 年卷期：v.11,no.2(February 3, 2012)
- ISSN：1535-3907
文摘
Many key proteins, such as those involved in cellular signaling or transcription, are difficult to measure in microbial proteomic experiments due to the interfering presence of more abundant, dominant proteins. In an effort to enhance the identification of previously undetected proteins, as well as provide a methodology for selective enrichment, we evaluated and optimized immobilized metal affinity chromatography (IMAC) coupled with mass spectrometric characterization of extracellular proteins from an extremophilic microbial community. Seven different metals were tested for IMAC enrichment. The combined results added 20% greater proteomic depth to the extracellular proteome. Although this IMAC enrichment could not be conducted at the physiological pH of the environmental system, this approach did yield a reproducible and specific enrichment of groups of proteins with functions potentially vital to the community, thereby providing a more extensive biochemical characterization. Notably, 40 unknown proteins previously annotated as 鈥渉ypothetical鈥?were enriched and identified for the first time. Examples of identified proteins includes a predicted TonB signal sensing protein homologous to other known TonB proteins and a protein with a COXG domain previously identified in many chemolithoautotrophic microbes as having a function in the oxidation of CO.