Thematic Institutional Excellence Programme(TKP2021-EGA-24) Támogató: Emberi Erőforrások
Minisztériuma
(TKP2021-NVA)
(TKP2021-NKTA)
(SNN 138407)
(UNKP-22-4-II-SE-26)
Mass spectrometry has become the most prominent yet evolving technology in quantitative
proteomics. Today, a number of label-free and label-based approaches are available
for the relative and absolute quantification of proteins and peptides. However, the
label-based methods rely solely on the employment of stable isotopes, which are expensive
and often limited in availability. Here we propose a label-based quantification strategy,
where the mass difference is identified by the differential alkylation of cysteines
using iodoacetamide and acrylamide. The alkylation reactions were performed under
identical experimental conditions; therefore, the method can be easily integrated
into standard proteomic workflows. Using high-resolution mass spectrometry, the feasibility
of this approach was assessed with a set of tryptic peptides of human serum albumin.
Several critical questions, such as the efficiency of labeling and the effect of the
differential alkylation on the peptide retention and fragmentation, were addressed.
The concentration of the quality control samples calculated against the calibration
curves were within the ±20% acceptance range. It was also demonstrated that heavy
labeled peptides exhibit a similar extraction recovery and matrix effect to light
ones. Consequently, the approach presented here may be a viable and cost-effective
alternative of stable isotope labeling strategies for the quantification of cysteine-containing
proteins.