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Microflow SWATH data analysed with DIA-NN

ABSTRACT: We have developed DIA-NN, an integrated software suite for fully-automated processing of raw SWATH/DIA data. DIA-NN enables deep and confident proteome coverage with fast chromatographic methods, paving the way for a new generation of high-throughput proteomics. This data set illustrates the capabilities of DIA-NN when analysing microflow SWATH data acquired with 19 - 23 minute gradients as well as the use of DIA-NN to generate spectral libraries directly from SWATH/DIA data.

INSTRUMENT(S): TripleTOF 6600

ORGANISM(S): Homo Sapiens (human) Saccharomyces Cerevisiae (baker's Yeast)

TISSUE(S): Blood Plasma, Cell Culture

SUBMITTER: Vadim Demichev

LAB HEAD: Markus Ralser

PROVIDER: PXD014690 | Pride | 2019-11-06

REPOSITORIES: Pride

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			Action	DRS
	20181206_k562_2ug_20min_3.wiff	Wiff
	20181206_k562_2ug_20min_3.wiff.scan	Wiff
	20181206_k562_2ug_20min_4.wiff	Wiff
	20181206_k562_2ug_20min_4.wiff.scan	Wiff
	20181206_k562_2ug_20min_5.wiff	Wiff

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Publications

DIA-NN: neural networks and interference correction enable deep proteome coverage in high throughput.

Demichev Vadim V Messner Christoph B CB Vernardis Spyros I SI Lilley Kathryn S KS Ralser Markus M

Nature methods 20191125 1

We present an easy-to-use integrated software suite, DIA-NN, that exploits deep neural networks and new quantification and signal correction strategies for the processing of data-independent acquisition (DIA) proteomics experiments. DIA-NN improves the identification and quantification performance in conventional DIA proteomic applications, and is particularly beneficial for high-throughput applications, as it is fast and enables deep and confident proteome coverage when used in combination with ...[more]

PMID: 31768060

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Minute-scale proteomics with Scanning SWATH and high-flow chromatography

Project description:Great advances have been made to quantify proteomes at depth and high precision. However, the inherent complexity of the proteome still creates huge challenges to deal with large sample series and longitudinal experiments. Here we report a data-independent acquisition (DIA) method, Scanning SWATH, and new software algorithms, that combine the advantages of data-dependent and DIA acquisition. Exploiting a continuous movement of the precursor isolation window to assign precursor mass to the MS2 fragment traces, Scanning SWATH increases the precursor identifications of up to 70% compared to conventional DIA methods on 1-5 minute chromatographic gradients. Moreover, we achieve significant acceleration of the mass spectrometric duty cycles, facilitating the generation of quantitative precise proteomes with high-flow (800 µL/min) chromatography and gradients that can be as fast as 30 seconds. We demonstrate the application of ultra-fast proteomics in drug mode-of-action screening. Scanning SWATH proteomes capture the mode-of-action of azoles and statins acting as fungistatic drugs.

2021-03-02 | PXD023613 | Pride

Robust and High-Throughput Analytical Flow Proteomics Analysis of Cynomolgus Monkey and Human Matrices with Zeno SWATH Data Independent Acquisition

Project description:Modern mass spectrometers routinely allow deep proteome coverage in a single experiment. These methods are typically operated at nano and micro flow regimes, but they often lack throughput and chromatographic robustness, which is critical for large-scale studies. In this context, we have developed, optimized and benchmarked LC-MS methods combining the robustness and throughput of analytical flow chromatography with the added sensitivity provided by the Zeno trap across a wide range of cynomolgus monkey and human matrices of interest for toxicological studies and clinical biomarker discovery. SWATH data independent acquisition (DIA) experiments with Zeno trap activated (Zeno SWATH DIA) provided a clear advantage over conventional SWATH DIA in all sample types tested with improved sensitivity, quantitative robustness and signal linearity as well as increased protein coverage by up to 9-fold. Using a 10-min gradient chromatography, up to 3,300 proteins were identified in tissues at 2 µg peptide load. Importantly, the performance gains with Zeno SWATH translated into better biological pathway representation and improved the ability to identify dysregulated proteins and pathways associated with two metabolic diseases in human plasma. Finally, we demonstrate that this method is highly stable over time with the acquisition of reliable data over the injection of 1,000+ samples (14.2 days of uninterrupted acquisition) without the need for human intervention or normalization. Altogether, Zeno SWATH DIA methodology allows fast, sensitive and robust proteomic workflows using analytical flow and is amenable to large-scale studies. This work provides detailed method performance assessment on a variety of relevant biological matrices and serves as a valuable resource for the proteomics community.

2023-05-09 | PXD039414 | Pride

Proteomics coupled with in vitro model to study the early crosstalk occurring between newly excysted juveniles of Fasciola hepatica and host intestinal cells

Project description:Here, we develop a new in vitro model replicating the first interaction of FhNEJ with mouse primary small intestinal epithelial cells (MPSIEC). FhNEJ and MPSIEC were incubated for 3 hours and protein extracts (tegument and soma for FhNEJ and membrane and cytosol for MPSIEC) were subjected to quantitative SWATH-MS proteomics compared with respective controls to evaluate the changes in both the parasite and the host.

2022-09-20 | PXD033952 | Pride

Benchmarking DIA data analysis workflows

Project description:Data independent acquisition (DIA) has become a well-established method in LC-MS driven proteomics. Nonetheless, there are still a lot of possibilities at the data analysis level. By benchmarking different DIA analysis workflows through a ground truth sample mimicking real differential abundance samples, consisting of a differential spike-in of UPS2 in a constant yeast background, we provide a roadmap for DIA data analysis of shotgun samples based on whether sensitivity, precision or accuracy is of the essence. Three different commonly used DIA software tools (DIA-NN, EncyclopeDIA and SpectronautTM) were tested in both spectral library mode and spectral library free mode. In spectral library mode we used the independent spectral library prediction tools Prosit and MS2PIP together with DeepLC, next to the classical DDA-based spectral libraries. In total we benchmarked 12 DIA workflows. DIA-NN in library free mode or using in silico predicted libraries shows the highest sensitivity maintaining a high reproducibility and accuracy.

2024-05-06 | PXD044981 | Pride

Comparative study of the data analysis tools for data-independent acquisition mass spectrometry

Project description:In-house generated datasets of different gradient lengths in comparing the performance of five DIA-MS analysis tools (OpenSWATH, EncyclopeDIA, Skyline, DIA-NN, Spectronaut).

2023-08-10 | PXD039759 | Pride

Study of Potent inhibitors for CT5-3hTERT fibroblast cell line

Project description:Data independent acquisition (DIA) mass spectrometry was used to study the potent inhibitors for cancer derived fibroblast cell line, CT5-3hTERT. The fibroblast spheroids were treated with 21 different tumor inhibitors to compare their effect in proteome in comparison to treated with DMSO.

2024-08-10 | PXD050005 | Pride

Differential gene expression of cells located in the top and bottom of yeast colonies

Project description:Saccharomyces cerevisiae colonies were grown on synthetic minimal media containing 1% glucose. The top layer of cells was scraped off using a custom plastic guide and a cell scraper. The remaining bottom layer of cells was then washed off with water and proteomics samples were prepared from both layers separately. Label-free quantification by DIA-SWATH was used to assess differences in gene expression between top and bottom layers.

2023-02-13 | PXD033395 | Pride

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

Project description: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.

2022-12-01 | PXD036786 | Pride

Yeast proteomic analysis upon temperature shift

Project description:DIA/SWATH analysis of yeast upon temperature shift.

2021-06-28 | PXD016320 | Pride

Comparison of fractionation proteomics for local SWATH library building

Project description:For data-independent acquisition by means of sequential window acquisition of all theoretical fragment ion spectra (SWATH), a reference library of data-dependent acquisition (DDA) runs is typically used to correlate the quantitative data from the fragment ion spectra with peptide identifications. The quality and coverage of such a reference library is therefore essential when processing SWATH data. In general, library sizes can be increased by reducing the impact of DDA precursor selection with replicate runs or fractionation. However, these strategies can affect the match between the library and SWATH measurement, and thus larger library sizes do not necessarily correspond to improved SWATH quantification. Here, three fractionation strategies to increase local library size were compared to standard library building using replicate DDA injection: protein SDS-PAGE fractionation, peptide high-pH RP-HPLC fractionation and MS-acquisition gas phase fractionation. The impact of these libraries on SWATH performance was evaluated in terms of the number of extracted peptides and proteins, the match quality of the peptides and the extraction reproducibility of the transitions. These analyses were conducted using the hydrophilic proteome of differentiating human embryonic stem cells.

2017-07-04 | PXD006190 | Pride

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