Calibr improves spectral library search for spectrum-centric analysis of data independent acquisition proteomics

Identifying peptides and proteins from mass spectrometry (MS) data, spectral library searching has emerged as a complementary approach to the conventional database searching. However, for the spectrum-centric analysis of data-independent acquisition (DIA) data, spectral library searching has not been widely exploited because existing spectral library search tools are mainly designed and optimized for the analysis of data-dependent acquisition (DDA) data. We present Calibr, a spectral library search tool for spectrum-centric DIA data analysis. Calibr optimizes spectrum preprocessing for pseudo MS2 spectra, generating an 8.11% increase in spectrum–spectrum match (SSM) number and a 7.49% increase in peptide number over the traditional preprocessing approach. When searching against the DDA-based spectral library, Calibr improves SSM number by 17.6–26.65% and peptide number by 18.45–37.31% over two state-of-the-art tools on three different data sets. Searching against the public spectral library from MassIVE, Calibr improves state-of-the-art tools in SSM and peptide numbers by more than 31.49% and 25.24%, respectively, for two data sets. Our analyses indicate higher sensitivity of Calibr results from the use of various spectral similarity measures and statistical scores, coupled with machine learning-based statistical validation for FDR control. Calibr executable files including a graphical user-interface application are available at https://ms.iis.sinica.edu.tw/COmics/Software_CalibrWizard.html and https://sourceforge.net/projects/comics-calibr.