Abstract Efficient peptide and protein identification from data-independent acquisition mass spectrometric (DIA-MS) data typically rely on an experiment-specific spectral library with a suitable size. Here, we report a computational strategy for optimizing the spectral library for a specific DIA dataset based on a comprehensive spectral library, which is accomplished by a priori analysis of the DIA dataset. This strategy achieved up to 44.7% increase in peptide identification and 38.1% increase in protein identification in the test dataset of six colorectal tumor samples compared with the comprehensive pan-human library strategy. We further applied this strategy to 389 carcinoma samples from 15 tumor datasets and observed up to 39.2% increase in peptide identification and 19.0% increase in protein identification. In summary, we present a computational strategy for spectral library size optimization to achieve deeper proteome coverage of DIA-MS data.

Optimization of Spectral Library Size Improves DIA-MS Proteome Coverage