High-throughput proteomics of nanogram-scale samples with Zeno SWATH DIA

Abstract

The ability to conduct high-quality proteomic experiments in high throughput has opened new avenues in clinical research, drug discovery, and systems biology. Next to an increase in quantitative precision, recent developments in high-throughput proteomics have also gained proteomic depth, to the extent that earlier gaps between classic and high-throughput experiments have significantly narrowed. Here we introduce and benchmark Zeno SWATH, a data-independent acquisition technique that employs a linear ion trap pulsing (Zeno trap pulsing) in order to increase proteomic depth and dynamic range in proteomic experiments. Combined with the high acquisition speed, these gains in sensitivity are particularly attractive for conducting high-throughput proteomics experiments with high chromatographic flow rates and fast gradients. We demonstrate that when combined with either micro-flow- or analytical-flow-rate chromatography, Zeno SWATH increases protein identification in complex samples 5- to 10-fold when compared to current SWATH acquisition methods on the same instrument. Using 20-min micro-flow chromatography, Zeno SWATH identified > 6,000 proteins from a 62.5 ng load of human cell lysate with more than 5,000 proteins consistently quantified in triplicate injections with a median CV of 6%. Using 5-min analytical-flow-rate chromatography (800 μl/min), Zeno SWATH identified 4,907 proteins from a triplicate injection of 2 μg of a human cell lysate; or more than 3,000 proteins from 250 ng tryptic digest. Zeno SWATH hence facilitates precise proteomic experiments with small sample amounts using a fast and robust high flow-rate chromatographic method, broadening the application space that requires precise proteomic experiments on a large scale.

The human host response to monkeypox infection: a proteomic case series study

“…To our knowledge, the repurposing of panel assays for assessing disease severity, for clinical trial monitoring, or for predicting future disease courses has not been explored previously but could prove a helpful tool in mounting a rapid response to emerging diseases. [28]) in parallel to a targeted proteomic assay that quantifies COVID-19 severity biomarkers ( [22]) to characterize the plasma proteome in an MPX case series, and comparison the proteomes to those of healthy volunteers and COVID-19 patients. b) Volcano plot of contrast MPX vs healthy controls; α <= 0.015 and |logFC| >= 1.35 were used for selection of regulated proteins.…”

“…Moreover, we included a broad panel of stable-isotope-labeled internal standards (PQ500, Biognosys). The tryptic digests obtained were then recorded using an online coupling of microflow chromatography and Zeno SWATH DIA, the latest generation of DIA proteomic technology [28]. Indeed, to our knowledge, the present study represents the first biomedical application of Zeno SWATH MS. After data were recorded as a single batch, raw data were processed with DIA-NN [40], and data were post-processed to detect differentially concentrated proteins as well as the enrichment of pathway terms using pathway definitions from REACTOME [41].…”

“…2 µl of each sample (360 ng sample + 0.02 µl PQ500, Biognosys) were loaded on a HSS T3 column (300 µm × 150 mm, 1.8 µm, Waters) heated to 35 °C, then chromatographically separated with a 20-min gradient using a flow rate of 5 µl/min [27]. A Zeno SWATH acquisition scheme with 85 variable-size windows and 11-ms accumulation time was used [28] for MS detection.…”

“…The human host response to monkeypox virus infection determined at the level of the plasma proteome. a) Schematic overview of the workflow using discovery proteomics (Zeno SWATH MS [28]) in parallel to a targeted proteomic assay that quantifies COVID-19 severity biomarkers ( [22]) to characterize the plasma proteome in an MPX case series, and comparison the proteomes to those of healthy volunteers and COVID-19 patients. b) Volcano plot of contrast MPX vs healthy controls; α <= 0.015 and |logFC| >= 1.35 were used for selection of regulated proteins.…”

Increasing proteome depth while maintaining quantitative precision in short gradient data-independent acquisition proteomics

“…Research in improving DIA-MS has focused on the implementation of the DIA acquisition strategy on new MS hardware 2,4 , the development of novel acquisition schemes 3,[5][6][7][8] and the improvement of data analysis [9][10] as well as library generation [10][11][12][13] . However, one of the key aspects of setting up of DIA acquisition methods is largely understudied, namely determining the optimal number of data points per peak (DPPP) 14 .…”