Introduction:

Obtaining insight into biological systems requires the ability to detect and reproducibly quantitate peptides of interest. ZT Scan DIA is a Zeno trap-enabled scanning DIA strategy currently deployed on a research-modified Zeno trap-enabled QTOF instrument with a sliding quadrupole (Q1) through the precursor m/z to select precursors for CID (collision-induced dissociation) fragmentation in a collision cell. Continuous quadrupole movement across the precursor masses can be directly correlated to the continuously changing intensity of fragment ions arising from those masses, thereby enabling the direct assignment of those precursors to their respective MS/MS spectra in a time-dependent manner. Since each fragment ion must have an identical pattern of increasing and decreasing intensity mapping to the precursor being scanned by the quadrupole, fragments arising from different precursors can be distinguished enabling improved spectral deconvolution. Thereby, we have used varying levels of commercial K562 tryptic digest loads (low to 200 ng) across gradients as short as 1 min up to 30 min with nanoflow and microflow with ZT Scan DIA and Zeno SWATH DIA to assess protein detections and quantitation.

Methods:

A research modified Zeno trap-enabled QTOF with an OptiFlow Turbo V ion source in line with a Waters M-Class LC system with nano- and microflow columns was used to acquire data with ZT Scan DIA and Zeno SWATH DIA. ZT Scan DIA with nanoflow (150-500 nl/min) active gradient lengths (water to 5-35% acetonitrile with 0.01 % formic acid) from 5-30 min in length and microflow (5 µL/min) gradients of 1-15 min in length were evaluated across varying K562 cell digest (SCIEX) loads with acquisition speeds up to 640 Hz. Data was processed with DIA-NN (v. 1.8.1) and PEAKS Studio (v. 12 beta) using a spectral-library or library-free (FASTA) mode.

Results:

A novel acquisition strategy, ZT Scan DIA, delivers over 2x gains in quantitative performance at low loads using nanoflow relative to Zeno SWATH DIA using similar acquisition time and identical gradient length. At higher loads, ZT Scan DIA offers similar quantitative performance with an active LC gradient that is 3-5x shorter than with Zeno SWATH DIA. For a 200 ng K562 load with a 3 minute LC gradient at microflow, we can detect nearly 5300 protein groups with over 4600 having a CV under 20%.

Conclusions:

ZT Scan DIA improves qualitative and quantitative performance at higher throughput relative to Zeno SWATH DIA.