Improving protein and proteome coverage through data-independent multiplexed peptide fragmentation

Area of Science:

• Proteomics
• Mass Spectrometry
• Biochemistry

Background:

• Data-dependent acquisition (DDA) LC/MS/MS typically interrogates only the most abundant precursor ions, leading to incomplete proteome coverage.
• Data-independent acquisition (DIA) LC/MS(E) fragments all coeluting precursors in parallel, maximizing data acquisition efficiency.
• Understanding the performance differences between DIA and DDA is crucial for advancing protein characterization.

Purpose of the Study:

• To investigate and compare the protein and proteome characterization performance of DIA LC/MS(E) and DDA LC/MS/MS.
• To evaluate the impact of acquisition strategy on protein sequence coverage and identification of low-abundance components.
• To highlight the advantages of parallel fragmentation in DIA for comprehensive proteomic analysis.

Main Methods:

• Applied both DIA LC/MS(E) and DDA LC/MS/MS to a four-protein standard mixture, an immunoprecipitated protein (Arabidopsis thaliana FLS2), and a tomato leaf proteome sample.
• Analyzed raw data, focusing on precursor detection, fragmentation timing, and resulting product ion spectra quality.
• Quantified protein sequence coverage and identified differences in the interrogation of coeluting precursors.

Main Results:

• DIA demonstrated dramatic improvements in individual protein sequence coverage across all tested samples, particularly for low-abundance proteins.
• DDA often failed to interrogate crucial precursors or did so at suboptimal chromatographic points, yielding poor spectra.
• DIA's parallel fragmentation approach captured precursors missed by DDA's serial, intensity-driven selection.

Conclusions:

• DIA LC/MS(E) offers significant advantages over DDA LC/MS/MS for comprehensive protein identification and characterization.
• The parallel fragmentation strategy of DIA overcomes fundamental limitations of serial MS/MS interrogation.
• DIA holds promise for enhanced analysis of protein isoforms and post-translational modifications due to its superior data acquisition.