Absolute quantitation of peptides and proteins by coulometric mass spectrometry after derivatization

Praneeth Ivan Joel Fnu, Md Tanim Al Hassan, Timothy Yaroshuk, Yongling Ai, Hao Chen

Research output: Contribution to journalArticlepeer-review

1 Scopus citations


Peptide/protein quantitation using mass spectrometry (MS) is advantageous due to its high sensitivity. Traditional absolute peptide quantitation methods rely on making calibration curves using peptide standards or isotope-labelled peptide standards, which are expensive and take time to synthesize. A method which can eliminate the need for using standards would be beneficial. Recently, we developed coulometric mass spectrometry (CMS) which can be used to quantify peptides that are oxidizable (e.g., those containing tyrosine or tryptophan), without using peptide standard. The method is based on electrochemical oxidation of peptides followed by MS measurement of the oxidation yield. However, it cannot be directly used to quantify peptides without oxidizable residues. To extend this method for quantifying peptides/proteins in general, in this study, we adopted a derivatization strategy, in which a target peptide is first tagged with an electroactive reagent such as monocarboxymethylene blue NHS ester (MCMB-NHS ester), followed with quantitation by CMS. To illustrate the power of this method, we have analyzed peptides MG and RPPGFSPFR. The quantification error was less than 5%. Using RPPGFSPFR as an example, the quantitation sensitivity of the technique was found to be 0.25 pmol. Furthermore, we also used the strategy to quantify proteins cytochrome C and β-casein with an error of 2–26 %.

Original languageEnglish (US)
Article number117153
JournalInternational Journal of Mass Spectrometry
StatePublished - Jan 2024

All Science Journal Classification (ASJC) codes

  • Instrumentation
  • Condensed Matter Physics
  • Spectroscopy
  • Physical and Theoretical Chemistry


  • Absolute quantitation
  • Derivatization
  • Electrochemical tag
  • Mass spectrometry
  • Peptide reduction


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