Addressing the Protease Bias in Quantitative Proteomics

Protein quantification strategies using multiple proteases have been shown to deliver poor inter-protease accuracy in label-free mass spectrometry experiments. By utilizing six different proteases with different cleavage sites, this study explores the protease bias and its effect on accuracy and precision by using recombinant protein standards. We established 557 SRM assays, using a recombinant protein standard resource, towards ten proteins in human plasma and determined their concentration with multiple proteases. The quantified peptides of these plasma proteins spanned three orders of magnitude (0.02-70 µM). In total, 60 peptides were used for absolute quantification and the majority of the peptides showed high robustness. The retained reproducibility was achieved by quantifying plasma proteins using spiked stable isotope standard recombinant proteins in a targeted proteomics workflow.