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Detection and Identification of Heme c‑Modified Peptides by Histidine Affinity Chromatography, High-Performance Liquid Chromatography–Mass Spectrometry, and Database Searching

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posted on 2012-12-07, 00:00 authored by Eric D. Merkley, Brian J. Anderson, Jea Park, Sara M. Belchik, Liang Shi, Matthew E. Monroe, Richard D. Smith, Mary S. Lipton
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–tandem mass spectrometry (LC–MS/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–MS/MS analysis. Enriched bovine cytochrome c samples yielded 3- to 6-fold more confident peptide–spectrum 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–MS/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 × 10–4 was detected with good signal-to-noise after HAC and LC–MS/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–spectrum matches of 20–100-fold for bovine cytochrome c.

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