Coupling a Detergent Lysis/Cleanup Methodology with Intact Protein Fractionation for Enhanced Proteome Characterization
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- 作者：Ritin Sharma ; Brian D. Dill ; Karuna Chourey ; Manesh Shah ; Nathan C. VerBerkmoes ; Robert L. Hettich
- 刊名：Journal of Proteome Research
- 出版年：2012
- 出版时间：December 7, 2012
- 年：2012
- 卷：11
- 期：12
- 页码：6008-6018
- 全文大小：518K
- 年卷期：v.11,no.12(December 7, 2012)
- ISSN：1535-3907
文摘
The expanding use of surfactants for proteome sample preparations has prompted the need to systematically optimize the application and removal of these MS-deleterious agents prior to proteome measurements. Here we compare four detergent cleanup methods (trichloroacetic acid (TCA) precipitation, chloroform/methanol/water (CMW) extraction, a commercial detergent removal spin column method (DRS) and filter-aided sample preparation (FASP)) to provide efficiency benchmarks with respect to protein, peptide, and spectral identifications in each case. Our results show that for protein-limited samples, FASP outperforms the other three cleanup methods, while at high protein amounts, all the methods are comparable. This information was used to investigate and contrast molecular weight-based fractionated with unfractionated lysates from three increasingly complex samples (Escherichia coli K-12, a five microbial isolate mixture, and a natural microbial community groundwater sample), all of which were prepared with an SDS-FASP approach. The additional fractionation step enhanced the number of protein identifications by 8% to 25% over the unfractionated approach across the three samples.