posted on 2021-06-08, 19:09authored byYongling Ai, Pengyi Zhao, Praneeth Ivan Joel FNU, Hao Chen
Isotope-labeled internal standards
are routinely used for mass
spectrometry (MS)-based absolute quantitation. However, syntheses
of isotope-labeled peptides are time-consuming and costly. To tackle
this issue, we recently developed a coulometric mass spectrometric
(CMS) approach for absolute quantitation without the use of standards,
based on the electrochemical oxidation of cysteine or tyrosine-containing
peptides followed by mass spectrometric measurement of the oxidation
yield. To further expand the utility of this method, herein we present
the CMS method for absolute quantitation of peptides based on tryptophan
electrochemical oxidation. Several tryptophan-containing peptides,
such as WGG, WQPPRARI, WAGGDASGE, RTRPLWVRME, and
KVPRNQDWL, were successfully quantified with a quantification
error ranging from −4.5 to +4.3%. Furthermore, this quantitation
approach is also applicable to protein, in which protein can be digested
and a surrogate peptide can be selected for quantification to reflect
the amount of the parent protein, as exemplified by CMS analysis of
peptide GITWK from cytochrome c. The CMS result agreed well with the
traditional isotope dilution method, with only a small difference
of 3.5%. In addition, CMS was used to successfully quantify amyloid
beta (Aβ) peptide fragments (up to 28 amino acid residues) based
on tyrosine oxidation. The validity of the CMS method for peptide
and protein absolute quantitation without using isotope-labeled peptide
standards would greatly facilitate proteomics research.