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Data independent acquisition (DIA)

In the ever-lasting effort to boost the number of proteins identified in MS-based proteomics experiments, it has become clear that data independent acquisition (DIA) combined with short gradients, has become a winning combination.

Explore how our customers have used Evosep One to generate record high IDs per minute for large-scale sample cohorts.

POWERFUL COMBINATION OF DIA AND ROBUST SHORT GRADIENTS 

Data independent acquisition (DIA) strategies have appeared as a powerful alternative to classic data dependent acquisition (DDA) in shotgun proteomics. DIA offers systematic measurement of all peptide ions regardless of their intensity by co-fragmenting all co-eluting peptides in broader precursor isolation windows. This provides a wider dynamic range of the proteomes analyzed, improved reproducibility for identification and enables better sensitivity and accuracy for quantification.

The selectivity is further enhanced by the ion mobility dimension in the FAIMS Pro interface and the dia-PASEF scan modes, while requiring more elaborate processing algorithms for identification and quantification.

The steep development over the last years, have positioned DIA as an important acquisition strategy to generate record high identifications with short gradients on the Evosep One.

Highly reproducible data with standardized methods

Low analytical variability between samples is desired when analyzing large cohorts of clinical samples. The Evosep One carefully delivers calibrated flow control for standardized methods with matched columns. This is a crucial part of the entire workflow, which should be streamlined from sample preparation to optimized DIA acquisition methods for short gradients and advanced software solutions.

Such an example is illustrated here by quadruplicate injections of 500 ng HeLa analyzed with DIA on an Orbitrap Exploris 480 MS and analyzed with Spectronaut, version 14. Notably, the overall median CV is below 5%.

SPECIAL LIBRARY, LIBRARY-FREE OR HYBRID LIBRARY ANALYSIS – HOW TO GET STARTED 

Spectral libraries are typically required for DIA analysis. The best approach is to generate these based on extensive offline fractionation with the same LC gradient and column as used in the actual DIA experiment. With standardized methods and matched columns on the Evosep One, it is easy to transfer spectral libraries between laboratories.

To get started with DIA on your Evosep One, we recommend to make use of already published high quality spectral libraries. Below you can find an overview of relevant technology papers using spectral libraries, which should get you started well.

It is also possible to search your DIA data using a library-free approach. This is a simple workflow, which enables reproducible and precise quantification without the need for DDA based spectral libraries.

Ultimately, both strategies can be combined using a hybrid library, combining resource DDA datasets and the actual DIA runs from an experiment.

Improving proteome coverage in short lc gradients with dia and faims

In a collaboration with Thermo Fisher Scientific, the Olsen Group at University of Copenhagen have described the Orbitrap Exploris™ 480 mass spectrometer for high-throughput analyses with short gradients using the Evosep One.

The instrument combines a compact and robust quadrupole-orbitrap mass spectrometer equipped with a front-end High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Interface. 

Combining DIA with FAIMS using single CVs, the instrument surpasses 2500 peptides identified per minute. This enables quantification of more than 5000 proteins with short online gradients allowing acquisition of 60 samples per day.

The versatility of applications presented in the publication demonstrates the powerful combination of the Orbitrap Exploris™ 480 and the Evosep One.

Read more in publication here

Application of DIA-PASEF for high-throughput proteomics with the evosep one

A collaboration between researchers from ETH Zürich, University of Belfast, University of Toronto and Bruker Daltonik were led by the Mann group at the Max Planck Institute for Biochemistry, Münich to develop the diaPASEF workflow for the timsTOF Pro. This scan mode samples up to 100% of the peptide precursor ion current in m/z and mobility windows by making use of the correlation of molecular weight and ion mobility in a trapped ion mobility device.

They optimized the diaPASEF schemes to balance selectivity, sensitivity and precursor coverage for fast gradients with the Evosep One. This typically require shorter mass spectrometry cycle times to achieve enough data points per peak for accurate quantification, thus a cycle time of 0.9 s were employed.

In triplicate analysis with the 60 samples per day method, they quantified on average  4,813 proteins per run with a median coefficient of variation (CV) of 5.8%. When they increased the throughput to 100 and 200 samples per day, more than 4,000 and 3,000 proteins in triplicate were still quantified, respectively.

Read publication here

ACCELERATED DIA-PASEF WITH DIA-NN

The 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 reprocessed the dia-PASEF reference dataset (read more about dia-PASEF with Evosep One here), where 200 ng of HeLa peptides were analyzed in triplicates with the 60, 100 and 200 samples per day methods.

By using a spectral 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.

Read publication here

The trend continues

DIA has gained in popularity over the last few years and in our 2019 survey we saw an increase in the use of DIA strategies compared to DDA over the answers from back in 2017.

This trend continues into 2022 when we asked this question once more. Here 80% replied that DIA/SWATH acquisition strategies are their preferred workflow.

We are excited to see this development as DIA is the ideal match for short standardized gradients on the Evosep One.

“The short gradients and low overhead of the Evosep One standard methods are an ideal match for the speed of
dia-PASEF® on Bruker’s timsTOF Pro mass spectrometers.

The combination offers an unprecedented combination of sample throughput and depth of coverage”,

Gary Kruppa, Vice President Proteomics, Bruker Daltonics

EVOSEP WEBINAR

MEET OUR USERS and their research using DIA

UNLEASH THE POWER OF DIA 

Available on demand

In this webinar we will dive into the exciting research possibilties that comes by using DIA combined with short gradients with Evosep One.

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More research with DIA

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

TitleSubjectMaterialYearSummaryInstituteEvosep methodMS instrumentationLearn More
Unleash the power of DIADIAWebinar2022In this webinar we dive into the exciting research possibilities that comes by using DIA combined with short gradients with Evosep One.

Watch webinar

Ultra-high sensitivity mass spectrometry quantifies single-cell proteome changes upon perturbationDIA, Single cell, TechnologyPublication2020In this publication, the Mann group in Münich describes their innovative workflow for single-cell proteomics. They apply a nearly loss-less miniaturized sample preparation workflow to our robust Whisper 40SPD method and combine it with diaPASEF on a modified timsTOF mass spectrometer for a total of 420 FACS sorted single cells.Whisper 40 SPDBruker timsTOF Pro, Bruker timsTOF SCP

Download publication or learn more on our website

DebaryOmics: an integrative –omics study to understand the halophilic behaviour of Debaryomyces hanseniiDIA, Phosphorylation, PTM, YeastPublication2021This publication by the Martinez group at the Technical University of Denmark, presents the first global phosphoproteomics analysis in Debaryomyces hansenii, a non-conventional yeast considered to be a well-suited option for a number of different industrial bioprocesses.30 SPDThermo Orbitrap Exploris 480

Read publication here

Targeted Workflows with Evosep OneDIA, Doping, Targeted workflowWebinar2021During this webinar, Michael MacCoss (University of Washington) and Vivian Delcourt (GIE-LCH) share how they use the Evosep One for targeted workflows.High Organic Method

Watch webinar

Plasma Proteomics with the Evosep OneDIA, Liver, PlasmaWebinar2021In this webinar you will learn how the Evosep One, with high reproducibility and robustness, is ideally suited for analysis of large sample cohorts of blood plasma.30 SPDBruker timsTOF Pro, Thermo Orbitrap Exploris 480

Watch webinar

AlphaTims: Indexing trapped ion mobility spectrometry – time of flight data for fast and easy accession and visualizationDDA, DIA, TechnologyPublication2021This publication by the Mann group, introduces the open-source AlphaTims package with a stand-alone graphical user interface or as part of their AlphaPept ecosystem. With this package, unprocessed data are indexed, accessed, and visualized by a combination of tools.200 SPD, 60 SPDBruker timsTOF Pro

Read publication here

Proteomics of resistance to Notch1 inhibition in acute lymphoblastic leukemia reveals targetable kinase signaturesAutomation, DDA, DIA, Phosphorylation, PTM, T-ALLPublication2021In 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.30 SPD, 60 SPDThermo Orbitrap Exploris 480

Read publication here

Unleashing the power of DIA combined with short gradients using Evosep OneDIA, TechnologyWebinar2021This 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.100 SPD, 200 SPD, 60 SPDBruker timsTOF Pro

Watch webinar

Unleashing the true power of DIA/SWATH data acquisition with short gradients – Rapid profiling of pre-clinical models and proteome dynamicsClinical research, DIA, TechnologyApplication note2019Achieving 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?

Download application note

Standardized workflow for precise mid- and high-throughput proteomics of blood biofluidsAutomation, Clinical research, DIA, PlasmaWebinar2021In 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.60 SPDThermo Orbitrap Exploris 480

Download publication

Watch webinar

Learn more

Integrative analysis of cell state changes in lung fibrosis with peripheral protein biomarkersClinical research, DIA, Fibrosis, PlasmaPublication2021In 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.100 SPD, 60 SPDThermo Q Exactive HF

Download publication

Covid-19 research with Evosep OneCovid-19, DIA, TechnologyWebinar2021This 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.60 SPDThermo Orbitrap Exploris 480

Watch webinar

SPIN – Species by Proteome InvestigationAncient proteomics, DIAPublication2021This 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.100 SPD, 200 SPD, 60 SPDThermo Orbitrap Exploris 480

Download publication

Spatial-proteomics reveal in-vivo phospho-signaling dynamics at subcellular resolutionDIA, FAIMS, Fractionation, Phosphorylation, Spatial proteomicsPublication2021This 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.60 SPDThermo Orbitrap Exploris 480

Download publication or read more on our website

High throughput proteome and phosphorpoteome sample processing coupled to fast gradient DIADDA, DIA, FAIMS, Phosphorylation, PTM, Technology, Tissue, TMTPublication2020High throughput workflows for proteomics100 SPD, 200 SPD, 60 SPDThermo Orbitrap Exploris 480

Download publication

New Orbitrap Exploris 480 Mass Spectrometer Coupled with Evosep OneDDA, DIA, FAIMS, TechnologyBrochure2020Brochure of Exploris 480 with Evosep data from piece no 1100 SPD, 200 SPD, 60 SPDThermo Orbitrap Exploris 480

Download brochure

A Novel LC System Embeds Analytes in Pre-formed Gradients for Rapid, Ultra-Robust ProteomicsDDA, DIA, Plasma, TechnologyPublication2018Description of Evosep60 SPDThermo Q Exactive HF-X

Download publication

High dynamic range proteome analysis with BoxCar DIA and super-resolution Orbitrap mass spectrometryDIA, Plasma, TechnologyPoster2020BoxCar DIA100 SPD, 30 SPDThermo Orbitrap Exploris 480

Download poster

Increasing proteome coverage using cysteine-specific DIA Mass spectrometry – Cys-DIADIA, PTMPublication2020DIA on cysteine-specific peptides60 SPDThermo Q Exactive HF-X

Download publication

Improving proteome coverage and peptide identification rates in short LC gradientsDDA, DIA, FAIMS, Phosphorylation, PTM, TechnologyVideo2020100 SPD, 200 SPD, 60 SPDThermo Orbitrap Exploris 480

Watch

High-throughput proteomics with Evosep OneDDA, DIA, FAIMS, Phosphoproteomics, PTM, TechnologyVideo2020100 SPD, 200 SPD, 60 SPDThermo Orbitrap Exploris 480

Watch

Rapid proteome analyses using the Evosep OneDDA, DIA, Phosphorylation, PTMVideo201860 SPDThermo Q Exactive HF

Watch

Using Artificial Intelligence on Ultrafast LC-MSMS-DIA runs for Bacterial Identification in UrineBacteria, Clinical research, DIAVideo2019View 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.  100 SPD, 200 SPD, 60 SPDThermo Orbitrap Exploris 480

Watch

High-throughput 4D-Proteomics – Application of dia-PASEF® and the Evosep One for short gradientsDIA, TechnologyApplication note2020diaPASEF app note  100 SPD, 200 SPD, 300 SPD, 60 SPDBruker timsTOF Pro

Download application note

Oncogenic Mutations Rewire Signaling Pathways by Switching Protein Recruitment to Phosphotyrosine SitesDIA, Liver, Lung, Protein interaction, TissuePublication2019In 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.60 SPDThermo Q Exactive HF-X

Learn more or download publication

Multi-level proteomics reveals host-perturbation strategies of SARS-CoV-2 and SARS-CoVClinical research, Covid-19, DIA, PTMPublication2020This 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-2.30 SPD, 60 SPDThermo Q Exactive HF-X

Download publication

A paired liver biopsy and plasma proteomics study reveals circulating biomarkers for alcohol-related liver diseaseClinical research, DIA, Liver, PlasmaPublication2020This 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, …30 SPDThermo Q Exactive HF-X

Download or read blog post

Increasing Throughput: From Pre-Clinical Models To Protein ComplexesDIA, Large-scaleVideo2019Comparison study with >400 samples, same coverage in shorter time60 SPDSCIEX

Watch

A Compact Quadrupole-Orbitrap Mass Spectrometer With Faims Interface Improves Proteome Coverage In Short Lc GradientsAutomation, DDA, DIA, FAIMS, Fractionation, Phosphoproteomics, PTM, Technology, Tissue, TMTPublication2020Test of Orbitrap Exploris with DIA and TMT, proteome and phospho.100 SPD, 200 SPD, 60 SPDThermo Orbitrap Exploris 480

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