배나현 (성신여자대학교)

고병준 (성신여자대학교)

이재범 (성신여자대학교)

배나현 (성신여자대학교)

고병준 (성신여자대학교)

이재범 (성신여자대학교)

Relative quantification enables comparison of protein expression levels among samples, whereas absolute quantification provides direct measurements of target protein abundance, facilitating a better understanding of biological functions. Stable isotope-labeled peptides are widely used as internal standards for mass spectrometry-based absolute quantification; however, their high cost limits their widespread application. In this study, we developed a cost-effective absolute quantification method for target proteins, including PykF, Glk, and GapA, using a standard addition strategy with purified target proteins as internal standards. The target proteins were expressed in Escherichia coli, purified by His-tag affinity chromatography, and characterized and quantified using SDS-PAGE and bicinchoninic acid (BCA) assay. For absolute quantification, increasing amounts of the purified target proteins were added stepwise to cell lysates, followed by tryptic digestion, C18 solid-phase extraction, and parallel reaction monitoring (PRM) analysis. Each sample was analyzed in triplicate, and standard addition curves were generated to determine the absolute abundance of the target proteins.