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

Wang, Ziyue; Mülleder, Michael; Batruch, Ihor; Chelur, Anjali; Textoris-Taube, Kathrin; Schwecke, Torsten; Hartl, Johannes; Causon, Jason; Castro-Perez, Jose; Demichev, Vadim; Tate, Stephen; Ralser, Markus

doi:10.7554/elife.83947

Cited by 45 publications

(39 citation statements)

References 37 publications

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“…For this application, the quantitative results we obtained with Speedy-PASEF were in high agreement with our previous study on the same cohort acquired with SWATH-MS 5 . Speedy-PASEF hence expands the compendium of fast proteomic methods such as Scanning SWATH or Zeno-SWATH 6,9 , a compendium that provides high-quality proteomes in combination with analytical flow rate chromatography and that has its strengths in large-scale and routine applications due to its fast throughput, high reproducibility, and robustness.…”

Section: Discussionmentioning

confidence: 99%

“…Analytical flow rate chromatography reduces sample carry-over, accelerates column equilibration, and allows fast chromatographic gradients. Moreover, it is robust when applied to large sample series 5,6,9 . It results in sharper peptide elution and hence higher peak capacity, leading to cleaner spectra, this being particularly beneficial for fast chromatographic gradients 5 .…”

Section: Introductionmentioning

confidence: 99%

“…Notably, some of the technological developments that led to higher throughput have also improved the consistency of protein identification, protein quantification and reduced batch effects, rendering proteomics increasingly routine-compatible [5][6][7][8][9][10][11][12][13][14][15][16][17] . Further, as both instrument time and hands-on time are key cost factors in mass spectrometry based proteomics, the fast sample throughput has rendered proteomic experiments more economical.…”

Section: Introductionmentioning

confidence: 99%

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Speedy-PASEF: Analytical flow rate chromatography and trapped ion mobility for deep high-throughput proteomics

Szyrwiel

Gille

Mülleder

et al. 2023

Preprint

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Increased throughput in proteomic experiments can improve accessibility of proteomic platforms, reduce costs and facilitate new approaches in systems biology and biomedical research. Here we propose Speedy-PASEF, a combination of analytical flow rate chromatography with ion mobility separation of peptide ions, data-independent acquisition and data analysis with the DIA-NN software suite, for conducting fast, high-quality proteomic experiments that require only moderate sample amounts. For instance, using a 500-μl/min flow rate and a 3-minute chromatographic gradient, Speedy-PASEF quantified 5,211 proteins from 2 μg of a mammalian cell-line standard at high quantitative accuracy and precision. We further used Speedy-PASEF to analyze blood plasma samples from a cohort of COVID-19 inpatients, using a 3-minute chromatographic gradient and alternating column regeneration on a dual pump system, for processing 398 samples per day. Speedy-PASEF delivered a comprehensive view of the COVID-19 plasma proteome, allowing classification of the patients according to disease severity and revealing plasma biomarker candidates. Speedy-PASEF thus facilitates acquisition of high-quality proteomes in large numbers.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Speedy-PASEF: Analytical flow rate chromatography and trapped ion mobility for deep high-throughput proteomics

Szyrwiel

Gille

Mülleder

et al. 2023

Preprint

Self Cite

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“…This greatly improves sensitivity (with increases in sensitivity of 5-10-fold, depending on fragment ion m/z) with no reported loss in acquisition speed or spectral resolution. Markus Ralser's group recently published an analysis of a K562 human cell lysate using this Zeno trap technology to acquire DIA-MS data (also termed Zeno SWATH DIA), in combination with fast chromatographic gradients, which highlighted the speed and sensitivity of the ZenoTOF 7600 system (21).…”

Section: Dia-ms Workflows Are Established On Multiple Different Mass ...mentioning

confidence: 99%

Substantial Downregulation of Mitochondrial and Peroxisomal Proteins during Acute Kidney Injury revealed by Data-Independent Acquisition Proteomics

Burton

Silva‐Barbosa

Bons

et al. 2023

Preprint

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Acute kidney injury (AKI) manifests as a major health concern, particularly for the elderly. Understanding AKI-related proteome changes is critical for prevention and development of novel therapeutics to recover kidney function and to mitigate the susceptibility for recurrent AKI or development of chronic kidney disease. In this study, mouse kidneys were subjected to ischemia-reperfusion injury, and the contralateral kidneys remained uninjured to enable comparison and assess injury-induced changes in the kidney proteome. A fast-acquisition rate ZenoTOF 7600 mass spectrometer was introduced for data-independent acquisition (DIA) for comprehensive protein identification and quantification. Short microflow gradients and the generation of a deep kidney-specific spectral library allowed for high-throughput, comprehensive protein quantification. Upon AKI, the kidney proteome was completely remodeled, and over half of the 3,945 quantified protein groups changed significantly. Downregulated proteins in the injured kidney were involved in energy production, including numerous peroxisomal matrix proteins that function in fatty acid oxidation, such as ACOX1, CAT, EHHADH, ACOT4, ACOT8, and Scp2. Injured mice exhibited severely declined health. The comprehensive and sensitive kidney-specific DIA assays highlighted here feature high-throughput analytical capabilities to achieve deep coverage of the kidney proteome and will serve as useful tools for developing novel therapeutics to remediate kidney function.

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“…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 11, 10, 12, 13 . However, one of the key aspects of setting up of DIA acquisition methods is understudied, namely determining the optimal number of data points per peak (DPPP) 14 .…”

Section: Introductionmentioning

confidence: 99%

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

Doellinger

Blumenscheit

Schneider

2022

Preprint

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The combination of short liquid chromatography (LC) gradients and data independent acquisition (DIA) by mass spectrometry (MS) has proven its huge potential for high-throughput proteomics. This methodology benefits from the speed of the latest generation of mass spectrometers, which enable short MS cycle times needed to provide sufficient sampling of sharp LC peaks. However, the optimization of isolation window schemes resulting in a certain number of data points per peak (DPPP) is understudied, although it is one of the most important parameters for the outcome of this methodology. In this study, we show that substantially reducing the number of DPPP for short gradient DIA massively increases protein identifications while maintaining quantitative precision as the number of data points per protein matters. Quantitative precision on protein level is maintained at low DPPP because the number of identified precursors per protein is largely enhanced. This strategy enabled us quantifying 6018 HeLa proteins (> 80,000 precursor identifications) with coefficients of variation below 20% in 30 min using a Q Exactive Orbitrap mass spectrometer, which corresponds to a throughput of 29 samples per day. This indicates that the potential of high-throughput DIA-MS has not been fully exploited and that high-throughput measurements are also possible with rather slow scanning mass spectrometers.

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

Cited by 45 publications

References 37 publications

Speedy-PASEF: Analytical flow rate chromatography and trapped ion mobility for deep high-throughput proteomics

Speedy-PASEF: Analytical flow rate chromatography and trapped ion mobility for deep high-throughput proteomics

Substantial Downregulation of Mitochondrial and Peroxisomal Proteins during Acute Kidney Injury revealed by Data-Independent Acquisition Proteomics

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

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