Detection and identification of heme c-modified peptides by histidine affinity chromatography, high-performance liquid chromatography-mass spectrometry, and database searching.
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.
Pacific Northwest National Laboratories, Richland, Washington 99352, United States.
Eric D
ED
Merkley
Brian J
BJ
Anderson
Jea
J
Park
Sara M
SM
Belchik
Liang
L
Shi
Matthew E
ME
Monroe
Richard D
RD
Smith
Mary S
MS
Lipton
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry; D020562:Betaproteobacteria / Q000201:enzymology; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D045304:Cytochromes c / Q000032:analysis / Q000737:chemistry; D030562:Databases, Protein; D004926:Escherichia coli / Q000737:chemistry; D006418:Heme / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D007477:Ions / Q000737:chemistry; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods; D010455:Peptides / Q000737:chemistry; D057386:Search Engine / Q000379:methods*; D059629:Signal-To-Noise Ratio; D053719:Tandem Mass Spectrometry / Q000379:methods*
D001426:Bacterial Proteins; D007477:Ions; D010455:Peptides; C031690:heme C; D006418:Heme; D006639:Histidine; D045304:Cytochromes c