1. Absolute quantitation of peptides and proteins by coulometric mass spectrometry after derivatization.
- Author
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Fnu, Praneeth Ivan Joel, Hassan, Md Tanim-Al, Yaroshuk, Timothy, Ai, Yongling, and Chen, Hao
- Subjects
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MASS spectrometry , *PEPTIDES , *DERIVATIZATION , *PROTEINS , *METHYLENE blue - Abstract
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 %. [Display omitted] • Coulometric mass spectrometry (CMS) in combination with derivatization strategy can be used for absolute quantitation of peptides and proteins in general. • Our quantitation method reported in this paper does not require the use of standards or isotope-labelled standards. • Our quantitation method reported in this paper is applicable to peptides that are not electrochemically active. • The derivatization of peptide with methylene blue electrochemical tag allows the oxidation of peptides at a very low potential (0.2 V vs. Ag/AgCl), thus providing the method selectivity. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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