Effect of Dynamic Exclusion Duration on Spectral Count Based Quantitative Proteomics

To increase proteome coverage, dynamic exclusion (DE) is a widely used tool. When DE is enabled, more proteins can be identified, although the total spectral counts will decrease. To investigate the effects of DE duration on spectral-counting based quantitative proteomics, we analyzed the same sample via multidimensional protein identification technology while enabling different DE durations (15, 60, 90, 300, 600 s) or turning DE off. Normalized spectral abundance factors (NSAFs) measured for abundant proteins varied little with or without DE, while enabling DE lead to higher peptide counts, higher NSAFs, and better reproducibility of detection for proteins of relatively lower abundance. The optimal DE duration, which generated the maximum number of peptides, proteins, and peptides per protein, was observed to be 90 s in our settings. We developed a mathematical model for analyzing the effects of DE duration on peptide spectral counts. We found that the optimal DE duration depends on the average chromatographic peak width at the base of eluting peptides and mass spectrometry parameters, leading us to calculate an optimized DE duration of 97.9 s, in excellent agreement with our observations. In this study, we provide a systematic approach for the optimization of spectral counts for improved quantitative proteomics analysis.