Improved Accuracy of Proteome Quantification by MS/MS Fragment Intensity

Abstract

Quantitative proteomics facilitates the discovery of potential biomarkers. Development of novel algorithms to improve the accuracy of proteome quantification is of vital importance. MS/MS fragment intensity has arisen as a novel abundance feature with unique advantages.In isobarically labelled proteome samples, the increase of MS/MS spectra complexity has adverse effect on the identification and coverage of quantification. A paired ion based scoring algorithm (PISA) based on Morpheus, was proposed to address this issue by scoring the matches using the number plus the fraction of intensity of matched paired ions. It was applied to the identification of Lys‐C digest of protein samples isobarically labelled with two different strategies involving both in‐vivo and chemical labeling. Compared to commercially available and open source software Mascot and Morpheus, more peptide spectrum matches, distinct peptides, and proteins can be identified at a 1% false discovery rate with the increase ranging from 22 % to 60 %. Moreover, 100% quantification coverage was achieved.Summed MS/MS Total ion current (SMT) cumulates all fragment intensities of all MS/MS spectra assigned to a protein. Spectral count in Absolute Protein Expression profiling (APEX) algorithm was replaced by SMT to enhance the accuracy of absolute quantification of proteomes in label‐free manner. The combination of APEX and SMT (abbreviated as APEX‐SMT) was capable of improving the accuracy of absolute quantification by reducing the average relative deviation by ~55‐85% compared to that of APEX, through a series of tests on Universal Proteomics Standard sample with a dynamic range of 5 orders of magnitude (UPS2).