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 converage 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 2021 when we asked this question once more. Here 75% replied that DIA/SWATH acquisition strategies are their preferred workflow.
“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
MEET OUR USERS and their research using DIA
find all publications, product brochures and presentations with Evosep One
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
|Title||Subject||Material||Year||Summary||Institute||Evosep method||MS instrumentation||Learn More|
|Unleashing the true power of DIA/SWATH data acquisition with short gradients – Rapid profiling of pre-clinical models and proteome dynamics||Clinical research, DIA, Technology||Application note||2019||Achieving 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?|
|Standardized workflow for precise mid- and high-throughput proteomics of blood biofluids||Clinical research, DIA, Plasma||Publication||2021||The Van Eyk group presents a standardized workflow for precise high-throughput proteomics of blood biofluids using the 60 SPD method. They established optimal conditions for sample preparation and DIA analysis in plasma, then automated and adapted this for depleted plasma and whole blood.||60 SPD||Thermo Orbitrap Exploris 480|
|Integrative analysis of cell state changes in lung fibrosis with peripheral protein biomarkers||Clinical research, DIA, Fibrosis, Plasma||Publication||2021||In 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 SPD||Thermo Q Exactive HF|
|SPIN – Species by Proteome Investigation||Ancient proteomics, DIA||Publication||2021||This 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 SPD||Thermo Orbitrap Exploris 480|
|Spatial-proteomics reveal in-vivo phospho-signaling dynamics at subcellular resolution||DIA, FAIMS, Phosphorylation, Spatial proteomics||Publication||2021||This 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 SPD||Thermo Orbitrap Exploris 480|
|Ultra-high sensitivity mass spectrometry quantifies single-cell proteome changes upon perturbation||DIA, Single cell, Technology||Publication||2020||In 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.||Whisper100 40 SPD||Bruker timsTOF Pro|
|High throughput proteome and phosphorpoteome sample processing coupled to fast gradient DIA||DDA, DIA, FAIMS, Phosphorylation, PTM, Technology, Tissue, TMT||Publication||2020||High throughput workflows for proteomics||100 SPD, 200 SPD, 60 SPD||Thermo Orbitrap Exploris 480|
|New Orbitrap Exploris 480 Mass Spectrometer Coupled with Evosep One||DDA, DIA, FAIMS, Technology||Brochure||2020||Brochure of Exploris 480 with Evosep data from piece no 1||100 SPD, 200 SPD, 60 SPD||Thermo Orbitrap Exploris 480|
|A Novel LC System Embeds Analytes in Pre-formed Gradients for Rapid, Ultra-Robust Proteomics||DDA, DIA, Plasma, Technology||Publication||2018||Description of Evosep||60 SPD||Thermo Q Exactive HF-X|
|High dynamic range proteome analysis with BoxCar DIA and super-resolution Orbitrap mass spectrometry||DIA, Plasma, Technology||Poster||2020||BoxCar DIA||100 SPD, 30 SPD||Thermo Orbitrap Exploris 480|
|Increasing proteome coverage using cysteine-specific DIA Mass spectrometry – Cys-DIA||DIA, PTM||Publication||2020||DIA on cysteine-specific peptides||60 SPD||Thermo Q Exactive HF-X|
|Improving proteome coverage and peptide identification rates in short LC gradients||DDA, DIA, FAIMS, Phosphorylation, PTM, Technology||Video||2020||100 SPD, 200 SPD, 60 SPD||Thermo Orbitrap Exploris 480|
|High-throughput proteomics with Evosep One||DDA, DIA, FAIMS, Phosphoproteomics, PTM, Technology||Video||2020||100 SPD, 200 SPD, 60 SPD||Thermo Orbitrap Exploris 480|
|Rapid proteome analyses using the Evosep One||DDA, DIA, Phosphorylation, PTM||Video||2018||60 SPD||Thermo Q Exactive HF|
|Using Artificial Intelligence on Ultrafast LC-MSMS-DIA runs for Bacterial Identification in Urine||Bacteria, Clinical research, DIA||Video||2019||View 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 SPD||Thermo Orbitrap Exploris 480|
|High-throughput 4D-Proteomics – Application of dia-PASEF® and the Evosep One for short gradients||DIA, Technology||Application note||2020||diaPASEF app note||100 SPD, 200 SPD, 300 SPD, 60 SPD||Bruker timsTOF Pro|
|Oncogenic Mutations Rewire Signaling Pathways by Switching Protein Recruitment to Phosphotyrosine Sites||DIA, Liver, Lung, Protein interaction, Tissue||Publication||2019||In 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 SPD||Thermo Q Exactive HF-X|
|Multi-level proteomics reveals host-perturbation strategies of SARS-CoV-2 and SARS-CoV||Clinical research, Covid-19, DIA, PTM||Publication||2020||This 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 SPD||Thermo Q Exactive HF-X|
|A paired liver biopsy and plasma proteomics study reveals circulating biomarkers for alcohol-related liver disease||Clinical research, DIA, Liver, Plasma||Publication||2020||This 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 SPD||Thermo Q Exactive HF-X|
|Increasing Throughput: From Pre-Clinical Models To Protein Complexes||DIA, Large-scale||Video||2019||Comparison study with >400 samples, same coverage in shorter time||60 SPD||Sciex|
|A Compact Quadrupole-Orbitrap Mass Spectrometer With Faims Interface Improves Proteome Coverage In Short Lc Gradients||Automation, DDA, DIA, FAIMS, Offline fractionation, Phosphoproteomics, PTM, Technology, Tissue, TMT||Publication||2020||Test of Orbitrap Exploris with DIA and TMT, proteome and phospho.||100 SPD, 200 SPD, 60 SPD||Thermo Orbitrap Exploris 480|
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