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

Recent advances in proteomics technologies have enabled the analysis of thousands of proteins in a high-throughput manner. Mass spectrometry (MS) based proteomics uses a peptide-centric approach where biological samples undergo 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 relationships 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 a conventional proteolytic digestion condition. Protein-peptide correlation, digestion efficiency, matrix-effect, and concentration-effect were evaluated. Twelve unique peptides of alpha-2-macroglobulin (A2MG) 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.