A single protein to multiple peptides: Investigation of protein-peptide relationship using targeted alpha-2-macroglobulin analysis

Recent advances in proteomics technologies have enabled analysis of thousands of proteins in a high-throughput manner. Mass Spectrometry (MS) based proteomics, uses a peptide centric approach where biological samples undergo a specific proteolytic digestion and then only unique peptides are used for protein identification and quantification. Considering the fact that a single protein may have multiple unique peptides and a number of different forms, it becomes essential to understand dynamic protein-peptide relationship to ensure robust and reliable peptide centric protein analysis. In this study, we investigated the correlation between protein concentration and corresponding unique peptide responses under conventional proteolytic digestion conditions. Protein-peptide correlation, digestion efficiency, matrix-effect, and concentration-effect were evaluated. Twelve unique alpha-2-macroglobulin (A2MG) peptides were monitored using a targeted MS approach to acquire insights into protein-peptide dynamics. Although the peptide responses were reproducible between replicates, protein-peptide correlation was moderate in protein standards and low in complex matrices. The results suggest that reproducible peptide signal could be misleading in clinical studies and a peptide selection could dramatically change the outcome at protein level. This is the first study investigating quantitative protein-peptide correlations in biological samples using all unique peptides representing the same protein and opens a discussion on peptide-based proteomics. ### Competing Interest Statement The authors have declared no competing interest. * A2MG : alpha-2-macroglobulin ABC : ammonium bicarbonate ACN : acetonitrile CPTAC : Clinical Proteomic Tumor Analysis Consortium CV : coefficient of variation DTT : dithiothreitol ESI : electrospray ionization IAA : iodoacetamide IS : internal standard LC : liquid-chromatography Lys-C : lysine-C MS : mass spectrometry MRM : multiple reaction monitoring m/z : mass-to-charge ratio PDB : Protein Database Bank Q : quadrupole QC : quality control QQQ : triple quadrupole.