Fractionation
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.
Read more here
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
Read more here
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Fractionation
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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.
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