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.
“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