Multiheme c-type cytochrome
s (protein
s with covalently attached heme
c moietie
s) play important role
s in extracellular metal re
spiration in di
ssimilatory metal-reducing bacteria. Liquid chromatography鈥搕andem ma
ss spectrometry (LC鈥揗S/MS) characterization of c-type cytochrome
s i
s hindered by the pre
sence of multiple heme group
s,
since the heme
c modified peptide
s are typically not ob
served or, if ob
served, not identified. U
sing a recently reported hi
stidine affinity chromatography (HAC) procedure, we enriched heme
c tryptic peptide
s from purified bovine heart cytochrome
c, two bacterial decaheme cytochrome
s, and
subjected the
se
sample
s to LC鈥揗S/MS analy
si
s. Enriched bovine cytochrome
c sample
s yielded 3- to 6-fold more confident peptide鈥搒pectrum matche
s to heme
c containing peptide
s than unenriched dige
st
s. In unenriched dige
st
s of the decaheme cytochrome MtoA from
Sideroxydans lithotrophicus ES-1, heme
c peptide
s for 4 of the 10 expected
site
s were ob
served by LC鈥揗S/MS; following HAC fractionation, peptide
s covering 9 out of 10
site
s were obtained. Heme
c peptide
spiked into
E. coli ly
sate
s at ma
ss ratio
s a
s low a
s 1 脳 10<
sup>鈥?
sup> wa
s detected with good
signal-to-noi
se after HAC and LC鈥揗S/MS analy
si
s. In addition to HAC, we have developed a proteomic
s databa
se
search
strategy that take
s into account the unique phy
sicochemical propertie
s of heme
c peptide
s. The re
sult
s sugge
st that accounting for the double thioether link between heme
c and peptide, and the u
se of the labile heme fragment a
s a reporter ion, can improve databa
se
searching re
sult
s. The combination of affinity chromatography and heme-
specific informatic
s yielded increa
se
s in the number of peptide鈥搒pectrum matche
s of 20鈥?00-fold for bovine cytochrome
c.
Keywords:
s" href="http://pubs.acs.org/action/doSearch?action=search&searchText=C%5C-type+cytochromes&qsSearchArea=searchText">C-type cytochromes; s" href="http://pubs.acs.org/action/doSearch?action=search&searchText=heme+c&qsSearchArea=searchText">heme c; s" href="http://pubs.acs.org/action/doSearch?action=search&searchText=peptides&qsSearchArea=searchText">peptides; s" href="http://pubs.acs.org/action/doSearch?action=search&searchText=mass+spectrometry&qsSearchArea=searchText">mass spectrometry; s" href="http://pubs.acs.org/action/doSearch?action=search&searchText=histidine+affinity+chromatography&qsSearchArea=searchText">histidine affinity chromatography; s" href="http://pubs.acs.org/action/doSearch?action=search&searchText=enrichment&qsSearchArea=searchText">enrichment; s" href="http://pubs.acs.org/action/doSearch?action=search&searchText=database+searching&qsSearchArea=searchText">database searching; s" href="http://pubs.acs.org/action/doSearch?action=search&searchText=reporter+ion&qsSearchArea=searchText">reporter ion; s" href="http://pubs.acs.org/action/doSearch?action=search&searchText=isotopic+envelope&qsSearchArea=searchText">isotopic envelope