Peptide-Centric Proteome Analysis: An Alternative Strategy for the Analysis of Tandem Mass Spectrometry Data

Abstract

In mass spectrometry-based bottom-up proteomics, data-independent acquisition is an emerging technique because of its comprehensive and unbiased sampling of precursor ions. However, current data-independent acquisition methods use wide precursor isolation windows, resulting in cofragmentation and complex mixture spectra. Thus, conventional database searching tools that identify peptides by interpreting individual tandem MS spectra are inherently limited in analyzing data-independent acquisition data. Here we discuss an alternative approach, peptide-centric analysis, which tests directly for the presence and absence of query peptides. We discuss how peptide-centric analysis resolves some limitations of traditional spectrum-centric analysis, and we outline the unique characteristics of peptide-centric analysis in general.

Fig. 1.

Spectrum-centric analysis and peptide-centric analysis. In spectrum-centric analysis, each MS/MS spectrum from either a DDA or DIA experiment is queried against a protein sequence database. The peptides that yield the best scoring N statistically significant PSMs are assigned to the corresponding MS/MS spectrum. Typically N is one for a DDA spectrum and multiple for a DIA spectrum (showing N = 4 here). In peptide-centric analysis, every peptide of interest is queried against the acquired MS/MS data. The bottom-middle panel shows the extracted MS/MS signal of the query peptide over time in which the signal is extracted from any MS/MS spectrum generated from isolating the query precursor m/z. The extraction window width corresponds to the acquisition method, showing here 2 m/z for DDA and 10 m/z for DIA. The precursor m/z of the query peptide is sampled stochastically and sparsely in DDA but systematically in DIA. The MS/MS signal that provides the best scoring evidence of detection is assigned to the query peptide (indicated by the arrows).