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

作者：Merkley Eric D; Anderson Brian J; Park Jea; Belchik Sara M; Shi Liang; Monroe Matthew E; Smith Richard D; Lipton Mary S^*

来源：Journal of Proteome Research, 2012, 11(12): 6147-6158.

DOI：10.1021/pr3007914

• C-type cytochromes
• heme c
• peptides
• mass spectrometry
• histidine affinity chromatography
• enrichment
• database searching
• reporter ion
• isotopic envelope

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 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 c-ytochrome 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 X 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.

• 出版日期2012-12