Protein quantification at sub-nanogram loads using Zeno SWATH data-independent analysis (DIA) and nanoflow chromatography

The combination of the ZenoTOF 7600 system with nanoflow chromatography enables reproducible protein and peptide identifications using very low amounts of sample. Here, a 300 nL/min flow rate was used with a 45-minute gradient to identify hundreds to thousands of proteins at loads of 5ng and below. Loads down to 250 pg on column were investigated to simulate protein amounts obtained when analyzing low numbers of cells. This workflow highlights the high sensitivity of the ZenoTOF 7600 system for Zeno SWATH DIA proteomics workflows.

Introduction
 

Data independent acquisition (DIA or SWATH DIA) has emerged as a comprehensive workflow for label-free quantitative proteomics, allowing for the acquisition of MS/MS spectra on all detectable peptides in an analysis. The ZenoTOF 7600 system is equipped to run Zeno SWATH DIA, which leverages Zeno trap activation on the platform for 5-6x increases in peptide MS/MS sensitivity in SWATH DIA workflows.¹ Zeno SWATH DIA data can be interrogated using experiment-specific spectral libraries or libraries generated using in silico digestion approaches to identify and quantify proteins and peptides in the sample.^2,3

The sensitivity gains provided by Zeno trap activation enable the use of nanogram-scale sample amounts on the platform for proteomics workflows. Recently, Zeno SWATH DIA was demonstrated to identify 3927 precursors at a 0.98 ng load of K562 cell digest and 7730 precursors at a 1.95 ng load of K562 cell digest with a 20 min microflow gradient (5 µL/min) and library-free data processing with DIA-NN.⁴

This work expands upon previous low-load experiments by leveraging nanoflow chromatography⁵ for enhanced protein and peptide detection. A 300 nL/min flow rate was used with 45 min gradients and a 15 cm nanoflow column for the analysis of sub-nanogram and nanogram amounts of K562 digest. Both library-based and library-free data processing strategies in DIA-NN were assessed for this workflow.

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