The latest version of DIA-NN software identifies more than 5000 proteins – 200 times a day on the Evosep One

More than 5000 proteins identified – 200 times a day

Proteomics is a field in continuous development, where robust and optimized processes throughout the entire workflow – from sample preparation to acquisition strategies and downstream data processing are the goal. In recent years, data independent workflows have gained increased interest due to its high degree of reproducibility, better sensitivity and accuracy for quantification compared to classic data-dependent workflows.

Active developments in DIA acquisition strategies have moved the field further in the past years, latest with the development of diaPASEF, which utilizes the trapped ion mobility separation (TIMS) on the Bruker timsTOF Pro mass spectrometer. Most importantly, diaPASEF yields 2-5x improvement in sensitivity and thus increasing the effective duty cycle. To gain biological insight from the increased sensitivity, the introduction of neural-network based processing of raw data, have been a game-changer in the last couple of years.

High sensitivity diaPASEF proteomics with DIA-NN and FragPipe

A collaboration between the Ralser group at the Crick Institute, UK and the Nesvizhskii group at University of Michigan, USA introduce the latest addition to the DIA-NN software, which was originally conceived to maximize the performance of fast proteomics experiments. They have developed a neural network-based ion mobility module, which they benchmark the performance of in combination with FragPipe-generated libraries. They reprocessed the diaPASEF reference dataset (read more about diaPASEF with Evosep One here), where 200 ng of HeLa peptides were analyzed in triplicates with the 60, 100 and 200 samples per day methods.

They built a spectral library with FragPipe from 24 ddaPASEF runs containing 8201 proteins. Using this library, DIA-NN quantified on average 5056 proteins from 200ng HeLa digest analyzed with the ultra fast 200 SPD Evosep One method. Notably, they obtained very high data completeness of 94%. By extending the gradient time with the 100 and 60 SPD methods, the proteomic depth increased to 6104 and 6737 proteins respectively. The median CV were below 8% for all three methods.

The future of proteomics with fast chromatographic gradients

This paper nicely demonstrates the hidden potential of fast gradients, utilized by continued instrument developments focused on sensitive and faster scanning and improved sensitivity in data analysis. With a depth of more than 5000 proteins identified routinely 200 times a day, we are facing possibilities of applying these super-fast gradients for discovery proteomics with large cohorts of clinical samples.

Read full publication on biorxiv here: https://www.biorxiv.org/content/10.1101/2021.03.08.434385v1.full

More research using DIA

Here you can see publications available on DIA research featuring Evosep One. For a full overview of publications published using the Evosep One Technology visit our Literature room here

TitleSubjectMaterialYearSummaryInstituteEvosep methodMS instrumentationLearn More
A Compact Quadrupole-Orbitrap Mass Spectrometer With Faims Interface Improves Proteome Coverage In Short Lc Gradients, , , , , , , , , 2020Test of Orbitrap Exploris with DIA and TMT, proteome and phospho., ,

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Increasing Throughput: From Pre-Clinical Models To Protein Complexes, 2019Comparison study with >400 samples, same coverage in shorter time

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A paired liver biopsy and plasma proteomics study reveals circulating biomarkers for alcohol-related liver disease, , , 2020This publication from Matthias Mann groups in Copenhagen and Münich describes a paired liver biopsy and plasma proteomics study, which make use of Boxcar DIA scanning for a large clinical cohort of nearly 600 patients. They have developed a machine learning model based on their biomarker panel, which for the first time outperforms existing tests, …

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Multi-level proteomics reveals host-perturbation strategies of SARS-CoV-2 and SARS-CoV, , , 2020This publication describes the molecular functions of viral proteins and their interactions with the host proteome of SARS-CoV-2. The impact of viral infection on the proteome and phosphoproteome were analyzed in a time-resolved manner by DIA. The analysis revealed key pathways perturbed during the infection identifying potential vulnerable points of SARS-CoV-2.,

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timsTOF Pro with PASEF and Evosep One: Maximizing throughput, robustness and analytical depth for shotgun proteomics, 2018Read this application note to learn more about the timsTOF Pro with PASEF and the Evosep One for maximizing throughput, robustness and analytical depth for shotgun proteomics. Increased peak capacity via trapped ion mobility spectrometry allows the analysis of 200 samples/day, at formidable analytical depth, with unparalleled robustness., ,

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Oncogenic Mutations Rewire Signaling Pathways by Switching Protein Recruitment to Phosphotyrosine Sites, , , , 2019In this publication, the Olsen Group have combined DIA with short LC gradients to describe how cancer mutations close to tyrosine phosphorylation sites in the EGF receptor rewire signaling pathways by switching protein interactors. The performed 1200 pulldown experiments in just 20 days of LC-MS instrument time.

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Fast and reproducible phosphoproteomics using MagReSyn® Amine and Tim-IMAC HP magnetic beads and the Evosep One, , 2020Read this application note to learn about the automated workflows for sample preparation developed by the Olsen group in collaboration with Resyn Biosciences. Protein digestion and phosphoenrichment is fully automated for up to 96 samples in parallel with the Kingfisher Flex robot from Thermo Scientific.

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MaxQuant software for ion moiblity enhanced shotgun proteomics, , 2020MaxQuant for timsTOF analysis

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High-throughput 4D-Proteomics – Application of dia-PASEF® and the Evosep One for short gradients, 2020diaPASEF app note  , , ,

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Using Artificial Intelligence on Ultrafast LC-MSMS-DIA runs for Bacterial Identification in Urine, , 2019View the recording of our HUPO 2019 lunch seminar, where Florence Roux-Dalvai from Québec Research Center, Canada presents a new strategy for bacterial species identification in urinary tract infection using artificial intelligence on ultrafast LCMS DIA runs.  , ,

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Rapid proteome analyses using the Evosep One, , , 2018

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High-throughput proteomics with Evosep One, , , , , 2020, ,

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Improving proteome coverage and peptide identification rates in short LC gradients, , , , , 2020, ,

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Increasing proteome coverage using cysteine-specific DIA Mass spectrometry – Cys-DIA, 2020DIA on cysteine-specific peptides

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Development of a Novel LC Concept for Clinical Proteomics, , 2018Description of Evosep One

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High dynamic range proteome analysis with BoxCar DIA and super-resolution Orbitrap mass spectrometry, , 2020BoxCar DIA,

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Robust and Reprodusible Protein Quantification in Plasma Using the Evosep One and the Agilent 6495 Triple Quadrupole LC/MS, , 2020Agilent feasibility study

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Online parallel accumulation – serial fragmentation (PASEF) with a novel trapped ion mobility mass spectrometer, 2018Description of timsTOF

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A Novel LC System Embeds Analytes in Pre-formed Gradients for Rapid, Ultra-Robust Proteomics, , , 2018Description of Evosep

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New Orbitrap Exploris 480 Mass Spectrometer Coupled with Evosep One, , , 2020Brochure of Exploris 480 with Evosep data from piece no 1, ,

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Optimizing data processing parameters for samples acquired on instruments using Advanced Peak Determination, 2018Improving ID rates with APD on Q Exactive HF-X

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Introducing the Evosep One – Instrument Overview2017Evosep One instrument video

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Sample loading protocol for Evotips2020Evosep One Tip loading guide

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Gradient off-set focusing HPLC insturment for robust and high throughput clinical proteomics, , 2017Evosep One for clinical analysis

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Evosep One Field Test at the Max-Planck Institute of Biochemistry2017Evosep One case study with Philipp Geyer

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Tech note: Evosep One: A gradient off-set focusing HPLC instrument for robust and high throughput proteomics2017Evosep One descriptive technical note

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High throughput proteome and phosphorpoteome sample processing coupled to fast gradient DIA, , , , , , , 2020High throughput workflows for proteomics, ,

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diaPASEF: parallel accumulation–serial fragmentation combined with data-independent acquisition, 2020A collaboration led by the Mann group at the Max Planck Institute for Biochemistry, Münich describes the development of the latest addition to the PASEF technology. The diaPASEF workflow combines the advantages of DIA such as high degree of reproducibility and data completeness with the effective PASEF technology., ,

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ABPP-HT – high-throughput activity-based profiling of deubiquitylating enzyme inhibitors in a cellular context, 2020This publication from University of Oxford led by Adan Pinto-Fernandez, describes a new methodology named ABPP-HT (high-throughput-compatible activity-based protein profiling), implementing a semi-automated proteomic sample preparation workflow that increases the throughput capabilities of the classical ABPP workflow approximately ten times while preserving its enzyme profiling characteristics.

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Ultra-high sensitivity mass spectrometry quantifies single-cell proteome changes upon perturbation, , 2020In this publication, the Mann group in Münich describes their innovative workflow for true single-cell proteomics. They apply a nearly loss-less miniaturized sample preparation workflow to our robust Whisper100 40SPD method and combine it with diaPASEF on a modified timsTOF mass spectrometer for a total of 420 FACS sorted single cells.

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Spatial-proteomics reveal in-vivo phospho-signaling dynamics at subcellular resolution, , , 2021This publication from the Olsen group at the Novo Nordisk Foundation Center for Protein Research, describes a high-throughput workflow based on sequential cell fractionation to profile the global proteome and phospho-proteome dynamics across six distinct subcellular fractions.

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SPIN – Species by Proteome Investigation, 2021This publication from the Olsen Group, introduces “Species by Proteome INvestigation” (SPIN), a proteomics workflow capable of querying over 150 mammalian species 200 times a day with the Evosep One. Genetic species determination is an indispensable tool in forensics, archaeology, ecology, and food authentication., ,

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Powerful pairing of technologies and analytical approaches, 2019In this webinar, Thomas Angel from the Exploratory Biomarker Assay group at GSK presents his latest data and hands-on experience using the Evosep One.

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A beginner’s guide to Evosep, 2021This webinar is part of our Evosep webinar series and hosted by Nicolai Bache, Evosep. Nicolai Bache introduces the basics of the Evosep technology, while Alexey Chernobrovkin and Raja Nirojogi share their experience with the Evosep One and present their own data.

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Covid-19 research with Evosep One, , 2021This webinar is part of our Evosep webinar series and hosted by Nicolai Bache, Evosep. Angela McArdle and Mathias Trost discuss their use of the Evosep One technology in their development of rapid Covid-19 assays.

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Integrative analysis of cell state changes in lung fibrosis with peripheral protein biomarkers, , , 2021In this study, the Theis and Schiller groups at the Helmholtz Zentrum München, investigated the correspondence of cell state changes in diseased organs to peripheral protein signatures in pulmonary fibrosis patient cohorts. From plasma proteome profiling of more than 122 patients, they propose CRTAC1 protein levels in plasma as a novel biomarker.,

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Standardized workflow for precise mid- and high-throughput proteomics of blood biofluids, , 2021In this seminar from the Canadian National Proteomics Network (CNPN) meeting 2021, Angela McArdle presents a standardized workflow for precise high-throughput proteomics of blood biofluids. They established optimal conditions for sample preparation and DIA analysis in plasma, then automated and adapted this for depleted plasma and whole blood.

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Evosep One: A Separation Tool Designed for Clinical Omics2019Evosep aims to improve quality of life and patient care by radically innovating protein-based clinical diagnostics. This brochure introduces our instrument Evosep One.

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The proteomics dilemma – High throughput analysis versus proteome depth2019With the five standard methods, the Evosep One covers a range of use cases from comprehensive proteome analysis with fractionation strategies to ultra high-throughput single-shot analysis., , , ,

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Improve and enhance your proteome coverage with the Performance column2019We have developed an additional Performance column for the 60 and 100 samples per day methods (SPD) to address applications with high demands.,

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Unleashing the true power of DIA/SWATH data acquisition with short gradients – Rapid profiling of pre-clinical models and proteome dynamics, , 2019Achieving sufficient statistical power for the analysis of a comprehensive set of target proteins requires processing of a large number of samples. With finite resources, researchers face a difficult choice: should they aim at deeper proteomic coverage or higher sample throughput?

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Towards a Standardized Omics Platform with the 300 samples per day method2020This application note describes the 300 samples per day standard method. Get an overview of the method, the chromatographic performance and the results expected from Hela standard injections.

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Towards a Standardized Omics Platform with the 200 samples per day method2019This application note describes the 200 samples per day standard method. Get an overview of the method, the chromatographic performance and the results expected from Hela standard injections.

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Towards a Standardized Omics Platform with the 100 samples per day method2019This application note describes the 100 samples per day standard method. Get an overview of the method, the chromatographic performance and the results expected from Hela standard injections.

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Towards a Standardized Omics Platform with the 60 samples per day method2020This application note describes the 60 samples per day standard method. Get an overview of the method, the chromatographic performance and the results expected from Hela standard injections.

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Towards a Standardized Omics Platform with the 30 samples per day method2020This application note describes the 30 samples per day standard method. Get an overview of the method, the chromatographic performance and the results expected from Hela standard injections.

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Unleashing the power of DIA combined with short gradients using Evosep One, 2021This webinar is part of our Evosep webinar series and hosted by Nicolai Bache, Evosep. Florian Meier and Vadim Demichev discuss their use of the Evosep One, using novel DIA methodologies., ,

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High Sensitivity Evotips2021This brochure describes the High Sensitivity Evotips for more efficient peptide recovery of small sample loads (< ~100 ng). They have superior performance at very low loads and almost identical retention times for other applications.

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A fleet of Evosep One instruments supports a proteomics factory at Rapid Novor2021This Case Study provides a glimpse behind the scenes at Rapid Novor, a core facility equipped with eight Evosep One instruments running 24/7. Read how instrument-to-instrument standardization played a vital role in the creation of de novo sequencing workflows in the laboratory.

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Proteomics of resistance to Notch1 inhibition in acute lymphoblastic leukemia reveals targetable kinase signatures, , , , 2021In this publication, the Olsen group highlights the potential of proteomics to dissect alterations in cellular signaling and identify druggable pathways in cancer. They identify protein kinase C delta (PKCδ) inhibition as a strategy to overcome resistance to Notch1 inhibition in T-cell acute lymphoblastic leukemia.,

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AlphaPept, a modern and open framework for MS-based proteomics, 2021This publication by the Mann group, introduces AlphaPept, a Python-based open-source framework for efficient processing of large high resolution MS data sets. They used the 200 samples per day method on the Evosep One to generate test data for computational functionality.

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