Sensitive, specific, and quantitatively precise detection of peptides and proteins is the cornerstone of proteomics, helping to drive biological insight for a wide range of disease research interests. Data-independent acquisition (DIA) has come to the forefront of MS-based proteomics research. With ZT Scan DIA, a Zeno trap-enabled scanning DIA workflow, the quadrupole moves continuously along the precursor mass range while MS/MS spectra are acquired. Fragment ion signals appear when the leading edge of the quadrupole window passes the precursor m/z and disappear when it falls out of the quadrupole window. A fragment ion signal detected in a TOF pulse is assigned to all the precursors associated with this TOF pulse. Data from each TOF pulse are summed and sectioned into narrow precursor bins (1–2 Da), resulting in a triangular shape of the quadrupole ion trace (QIT) of the fragment. The highest signal of a fragment ion (tip of the triangle) is in the precursor bin that contains the respective precursor ion. Zeno trap pulsing significantly improves fragment sensitivity and has been shown to be beneficial in quantitative proteomics.

In this work, with analytical-flow chromatography, ZT Scan DIA with Zeno trap pulsing was used for the analysis of commercial K562 digest using a research modified Zeno trap-enabled QTOF system. The number of proteins and peptides identified and quantified were compared between variable window Zeno SWATH DIA and ZT Scan DIA methods with equivalent accumulation times and cycle times. In addition, the quantitation performance of ZT Scan DIA in terms of lower limits of quantitation (LLOQs), upper limits of quantitation (ULOQs) and linear dynamic ranges (LDRs) were evaluated by spiking various concentrations of isotopically labeled synthetic peptides into the K562 matrix.