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

Abstract

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.

Targeted On‐line SPE‐LC‐MS/MS Assay for the Quantitation of 12 Apolipoproteins from Human Blood

“…Depending on the modification, origin and degree of purity of trypsin, prices vary by up to a factor of 1000. Therefore, we tested bovine and porcine trypsin of different price categories with regards to their digestion efficiency in digestion time courses established in a human serum pool as recommended by Arnold et al . The digestion of apoA‐II and apoC‐I was significantly slower applying the low‐price bovine trypsin, whereas other apos were digested equally regardless of the trypsin's origin.…”

Critical Issues and Optimized Practices in Quantification of Protein Abundance Level to Determine Interindividual Variability in DMET Proteins by LC‐MS/MS Proteomics

“…While maximum digestion efficiency can be reached by changing the digestion time or protease concentration, applying microwave‐assisted digestion, and adding a different surfactant and solubilizing agents or protease type (Lyc‐C plus trypsin), it is usually difficult to prove whether digestion is complete (100% efficiency). Peptide stability is an important factor determining apparent digestion efficiency, as some peptides degrade faster than others …”

“…Arnold et al . demonstrated a matrix‐dependent effect on trypsin digestion and absolute quantification of ALDH1A1 . A comprehensive study by Walmsley et al .…”

“…Peptide stability is an important factor determining apparent digestion efficiency, as some peptides degrade faster than others. 73 It is a common apprehension in the PBPK field that the protein quantification data, which are generated using a peptide calibrator, may not be applicable to IVIVE or to predict interindividual variability, as these are not absolute values due to incomplete digestion inefficiency. 74 However, in our opinion, irrespective of digestion efficiency, intersystem or interindividual variability (relative fold difference) in DMET protein abundance can be accurately measured if the extraction and digestion efficiency remain reproducible across samples.…”

“…Arnold et al demonstrated a matrix-dependent effect on trypsin digestion and absolute quantification of ALDH1A1. 73 A comprehensive study by Walmsley et al 77 revealed the origin of trypsin as a significant source of variability in proteomics. Notably, bovine trypsin produced a significantly higher number of missed cleavage peptides, whereas porcine trypsin resulted in more semitryptic peptides.…”