Challenges in quantitative accuracy for high-throughput mass spectrometry remain a concern as demands continue to increase for proteomics-based precision medicine and clinical studies. With a new wave of research shifting towards DIA strategies to increase proteomic coverage, depth, and reproducibility, larger amounts of data are generated as more information is acquired. Furthermore, successful quantitative measurements in complex samples require a great deal of method optimization to ensure critical factors, such as the number of experimentally measured points across an LC peak, are met. Ultimately, the throughput of current quantitative proteomics workflows is negatively affected by these factors to accommodate the longer times needed to collect and process large sets of data, or the quality and sensitivity of the data is sacrificed to maintain high throughput.Here, we present a full workflow solution to overcome these barriers and optimize the throughput of large scale, LC-MS/MS proteomics from data acquisition to analysis. First, we utilize an extremely fast, novel acquisition method (ZT Scan DIA), that requires minimal method development compared to existing DIA workflows. In this work, ZT Scan DIA exploits a continuous sliding quadrupole (Q1), at a rate of 640Hz, in combination with a research-grade Zeno trap enabled QTOF to simultaneously assign precursor masses to the MS/MS fragments. Furthermore, 20x more fragment ions can be detected with Zeno trap activation. Then, with the varying quadrupole scanning rate, a generally accepted range in m/z of 500 amu is used to cover the peptide parent masses, along with having the smallest quadrupole isolation window to maximize selectivity.
In the second part of the workflow, we introduce a software platform to automate the analysis of ZT Scan DIA data. The software utilizes a new algorithm integrating DDA and DIA data analysis approaches to increase sensitivity and accuracy of identification and quantitation results. Specifically, a DDA approach is used for associating precursor masses to the MS/MS fragments generated by the Q1. Using data acquired by ZT Scan DIA for 50ng of K562 on a 5-min gradient, the software identified 29886 peptides and 3716 protein groups at 1% FDR with direct database search. Compared with data from the same sample acquired by Zeno SWATH DIA, ZT Scan DIA demonstrates improved identification rates by >15% on both peptide- and protein-levels contributing to the added Q1 dimension. In addition, the software displays the Q1 profile of each identified peptide in an interactive GUI so that results can be easily interpreted and validated. Visualization of the Q1 profile demonstrates the added ability to deconvolve co-eluting fragment ion signals which originate from native and modified peptides. Overall, ZT Scan DIA together with the customized software platform, provide an extremely sensitive, superior-throughput workflow for large-scale quantitative proteomics.