Plasma Extraction by Acetonitrile and Trichloroacetic Acid With Dithiothreitol and Combination for Low Molecular Weight Proteome Analysis

The low molecular weight (LMW) proteome fraction comprises small proteins and endogenous peptide fragments present in peripheral circulation that could serve as potential diagnostic markers; however, the coverage of the mass spectrometry-based plasma or serum LMW proteomic analysis is limited. We found that 60%, 70%, 80%, and 90% acetonitrile and 10% trichloroacetic acid (TCA) with or without dithiothreitol (DTT) extracted different LMW proteins (below 50 kD or smaller). Extraction by 60%, 80% acetonitrile, and 10% TCA with or without DTT in the plasma and pooling after tryptic digestion for in-depth proteomic analysis by high pH reverse phase fractionation has led to the identification of 5638 tryptic peptides from 805 proteins. This comprehensive plasma LMW proteomic analysis is expected to promote success in blood biomarker discoveries.