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

Jen-Hung Wang,Ting-Yi Sung,Wai-Kok Choong,Ching-Tai Chen

doi:10.1038/s41598-022-06026-9

Abstract

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.

Highlights

Identifying peptides and proteins from mass spectrometry (MS) data, spectral library searching has emerged as a complementary approach to the conventional database searching
We first used dependent acquisition (DDA)-based spectral libraries to perform spectral library searching on pseudo MS2 spectra generated from data-independent acquisition (DIA) data sets
We have developed a command-line spectral library search tool, called Calibr, and a Windows-based friendly user interface application, called CalibrWizard, to seamlessly perform spectral library searches on pseudo MS2 spectra generated using DIA-Umpire, followed by Percolator validation for spectrum-centric analysis of DIA data

Summary

Introduction

Identifying peptides and proteins from mass spectrometry (MS) data, spectral library searching has emerged as a complementary approach to the conventional database searching. Several spectral library search tools have been developed in the past years, for example, S pectraST2,6,15, Pepitome16, COSS17, and Epsilon-Q18 These tools rely on the same premise that the MS2 spectrum of a peptide under fixed conditions is a reproducible fingerprint of that peptide such that unidentified spectra under the same condition can be identified by spectral matching. The other is spectrum-centric approach which de-convolves the acquired DIA data into pseudo MS2 spectra for untargeted identification by conventional database searching same as for DDA data[19]. The pseudo MS2 spectra generated from DIA data can have significantly different spectral characteristics and varying signal qualities, leading to lower reproducibility of fragmentation patterns for peptides[21,22] For such cases, existing spectral library search tools designed and optimized for comparing DDA spectra could suffer from decreased sensitivity.

Methods

Results

Conclusion