Multiparameter Optimization of Two Common Proteomics Quantification Methods for Quantifying Low-Abundance Proteins

Quantitative proteomics has been extensively applied in the screening of differentially regulated proteins in various research areas for decades, but its sensitivity and accuracy have been a bottleneck for many applications. Every step in the proteomics workflow can potentially affect the quantification of low-abundance proteins, but a systematic evaluation of their effects has not been done yet. In this work, to improve the sensitivity and accuracy of label-free quantification and tandem mass tags (TMT) labeling in quantifying low-abundance proteins, multiparameter optimization was carried out using a complex 2-proteome artificial sample mixture for a series of steps from sample preparation to data analysis, including the desalting of peptides, peptide injection amount for LC-MS/MS, MS1 resolution, the length of LC-MS/MS gradient, AGC targets, ion accumulation time, MS2 resolution, precursor coisolation threshold, data analysis software, statistical calculation methods, and protein fold changes, and the ...