Detection and Identification of Heme c-Modified Peptides by Histidine Affinity Chromatography, High-Performance Liquid Chromatography鈥揗ass Spectrometry, and Database Searching
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- 作者：Eric D. Merkley ; Brian J. Anderson ; Jea Park ; Sara M. Belchik ; Liang Shi ; Matthew E. Monroe ; Richard D. Smith ; Mary S. Lipton
- 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：6147-6158
- 全文大小：461K
- 年卷期：v.11,no.12(December 7, 2012)
- ISSN：1535-3907
文摘
Multiheme c-type cytochromes (proteins with covalently attached heme c moieties) play important roles in extracellular metal respiration in dissimilatory metal-reducing bacteria. Liquid chromatography鈥搕andem mass spectrometry (LC鈥揗S/MS) characterization of c-type cytochromes is hindered by the presence of multiple heme groups, since the heme c modified peptides are typically not observed or, if observed, not identified. Using a recently reported histidine affinity chromatography (HAC) procedure, we enriched heme c tryptic peptides from purified bovine heart cytochrome c, two bacterial decaheme cytochromes, and subjected these samples to LC鈥揗S/MS analysis. Enriched bovine cytochrome c samples yielded 3- to 6-fold more confident peptide鈥搒pectrum matches to heme c containing peptides than unenriched digests. In unenriched digests of the decaheme cytochrome MtoA from Sideroxydans lithotrophicus ES-1, heme c peptides for 4 of the 10 expected sites were observed by LC鈥揗S/MS; following HAC fractionation, peptides covering 9 out of 10 sites were obtained. Heme c peptide spiked into E. coli lysates at mass ratios as low as 1 脳 10鈥? was detected with good signal-to-noise after HAC and LC鈥揗S/MS analysis. In addition to HAC, we have developed a proteomics database search strategy that takes into account the unique physicochemical properties of heme c peptides. The results suggest that accounting for the double thioether link between heme c and peptide, and the use of the labile heme fragment as a reporter ion, can improve database searching results. The combination of affinity chromatography and heme-specific informatics yielded increases in the number of peptide鈥搒pectrum matches of 20鈥?00-fold for bovine cytochrome c.