Peptide fractionation is important in proteomics workflows to reduce sample complexity. This leads to increased dynamic range of the identified peptides compared to conventional single-shot analysis.

Explore how additional number of injections per fractionated sample is a good match with the short overhead time between injections on the Evosep One.

Fractionation Increases Dynamic Range and Understanding of Biology

Pre-fractionation is a widely used method to reduce the complexity of samples and increase the efficiency of the MS analysis. Offline peptide fractionation at high pH prior to low pH online separation has shown great promise in recent years despite the lack of full orthogonality between separations in the two LC dimensions. This is due to the high resolution provided and combined with short gradients on the Evosep One, where the overhead time between injections is reduced, this makes it affordable to analyze several fractions per sample. High pH reversed phase fractionation is often combined with TMT workflows for maximum depth of coverage.

In addition to peptide fractionation, subcellular fractionation has gained interest to separate cellular components and understand protein function.

Optimal analytical strategies for sensitive and quantitative phosphoproteomics

This publication from the Olsen group at University of Copenhagen, benchmark strategies for quantitative phosphoproteomics analysis with limited peptide input amounts.

They compare two TMT-based fractionation strategies using high-pH reversed-phase chromatography, against a label-free approach using data independent acquisition.

Based on this research, they present a decision tree to guide phosphoproteomics users to find the best workflow, to reach optimal depth of coverage, based on experimental design and input amount.

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Comprehensive maps of the subcellular proteome

Subcellular organization is essential to the function of all living cells as proteins localize to various compartments to fulfill their function. Therefore protein localization must be tightly regulated to ensure correct protein function.

The Borner group from the Max Planck Institute of Biochemistry, Martinsried has developed a dynamic organellar maps (DOMs) method for systems-level organellar mapping of the proteome, where cells are mechanically lysed and the released organelles are separated by differential centrifugation. Following subcellular fractionation and protein digestion, peptides are further fractionated using SDB-RPS and the resulting three fractions are analyzed with DIA

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Peptide fractionation is important in proteomics workflows to reduce sample complexity and increase the efficiency of the MS separation. This leads to increased dynamic range of the identified peptides compared to conventional single-shot analysis.

More research with Fractionation

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

TitleSubjectMaterialYearSummaryInstituteEvosep methodMS instrumentationLearn More
Evosep One solves the proteomics dilemma – covering high throughput analysis and proteome depth, 2023The Evosep One offers a diverse range of applications, utilizing its six standard methods. It caters to various use cases, spanning from comprehensive proteome analysis with fractionation strategies to ultra high-throughput single-shot analysis., , , ,

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Evaluation of Disposable Trap Column nanoLC–FAIMS–MS/MS for the Proteomic Analysis of FFPE Tissue, , , 2021This publication by the Kuster group presents a robust workflow to support the analysis of large cohorts of patient samples using formalin-fixed paraffin-embedded FFPE tissue. They make use of online fractionation by FAIMS requiring 50% less sample than conventional high pH fractionation., ,

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Inhibition of eEF2K synergizes with glutaminase inhibitors or 4EBP1 depletion to suppress growth of triple-negative breast cancer cells, , 2021In this publication by the Zacksenhaus group, they use LC-MS to investigate the global changes in the proteome following eEF2K and 4EBP1 knock-down in triple-negative breast cancer cell lines using TMT and high pH fractionation.

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Hemorrhage and saline resuscitation are associated with epigenetic and proteomic reprogramming in the rat lung, , , 2021In this study, the Sillesen group investigates the alteration of the proteome in the context of hemorrhage and saline resuscitation in trauma patients. They used rat lungs as a model and pooled 10 rats in one combined TMT experiment subjected to high pH offline fractionation.

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Molecular origin of blood-based infrared fingerprints, , , 2021This publication led by the Zigman group describes the molecular understanding of infrared molecular fingerprints (IMFs) by examining a prospective clinical study with both IR spectroscopy ad MS-based proteomics. They find the disease-related differences in IMFs of blood sera are dominated by contributions from the protein fractions, rather than metabolites.

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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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Evosep One Enables Robust Deep Proteome Coverage Using Tandem Mass Tags While Significantly Reducing Instrument Time, , 2019Comparison study with TMT, same coverage in shorter time,


In-Depth Characterization of Staurosporine Induced Proteome Thermal Stability Changes, , , 2020Deep CETSA MS profiling of Staurosporine

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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., ,


Covalent Protein Painting Reveals Structural Changes In The Proteome In Alzheimer Disease, , , , 2020Development of Covalent Protein Painting (CPP), a structural proteomics approach to quantify changes in protein fold or altered protein-protein interaction for any protein in a proteome. CPP directly determines the relative surface accessibility of amino acid side chains by measuring the molar fraction of a chemical functionality that is accessible for chemical modification on the …



application note
Evosep One solves the proteomics dilemma – covering high throughput analysis and proteome depth
application note
end-to-end, fully automated digestion protocol and evotip pure workflow on the opentrons ot-2
application note
fully automated, rapid, robust sample loading on evotip pure with the agilent assaymap bravo
EVOSEP ONE: A Standardized Separation
Tool for Clinical Omics