Introduction
Data-independent acquisition (DIA) workflows using high-resolution mass spectrometry (HRMS) have become highly utilized for proteomics research, providing qualitative and quantitative information on proteins and their impact on biological pathways. MS sensitivity and quantitative dynamic range often limit the quantitative analysis of low-abundance proteins and peptides. This work demonstrates the impact of improved MS sensitivity on label-free quantitation (LFQ) using Zeno SWATH DIA on a novel QTOF system.
Methods
Sample analysis was performed using a Waters M-Class HPLC system coupled to a novel quadrupole time-of-flight (QTOF) system. Commercial tryptic digests (human K562, yeast, and E. coli) were mixed in different ratios/dilutions and analyzed using microflow reverse-phase chromatography, with various separation gradient lengths. LFQ analysis was done using Zeno SWATH DIA, and the resulting data were processed using PEAKS Studio software (BSI Inc). The precision and accuracy of the LFQ analysis at the protein and peptide levels were measured for the varied species ratios and dilutions.
Results
The MS/MS sensitivity of the novel QTOF platform was approximately 5x higher than that of its predecessor platform. The impact of this sensitivity gain is demonstrated by the number of quantitative identifications of peptides in proteins in the various LFQ mixtures. The novel QTOF system demonstrated higher precision and accuracy for the quantitation of protein ratios in the different human/yeast/E. coli mixtures, particularly for injections of low levels of sample.
Conclusion
These results underscore the importance of sensitivity for Zeno SWATH DIA when used for quantitative proteomics research.