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Protein quantification based on peptides using LC-MS/MS has emerged as a promising method to measure biomarkers, protein drugs, and endogenous proteins. However, the best practices for selection, optimization, and validation of the quantification peptides are not well established, and the influence of different matrices on protein digestion, peptide stability, and MS detection has not been systematically addressed. The aim of this study was to determine how biological matrices affect digestion, detection, and stability of peptides. The microsomal retinol dehydrogenase (RDH11) and cytosolic soluble aldehyde dehydrogenases (ALDH1As) involved in the synthesis of retinoic acid (RA) were chosen as model proteins. Considerable differences in the digestion efficiency, sensitivity, and matrix effects between peptides were observed regardless of the target protein's subcellular localization. The precision and accuracy of the quantification of RDH11 and ALDH1A were affected by the choice of calibration and internal standards. The final method using recombinant protein calibrators and stable isotope labeled (SIL) peptide internal standards was validated for human liver. The results demonstrate that different sample matrices have peptide, time, and matrix specific effects on protein digestion and absolute quantification.

Impact of Sample Matrix on Accuracy of Peptide Quantification: Assessment of Calibrator and Internal Standard Selection and Method Validation