Proteomics

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Optimized parameter settings enhance proteome-wide formation of crosslinks on low-abundant proteins


ABSTRACT: Studies using crosslinking coupled to mass spectrometry on the proteome-wide level have spurred great interest as they facilitate structural probing of protein interactions in living cells or even organisms. Here we show, by using both an in-vitro mimic of a crowded cellular environment and eukaryotic cell lysates, that current proteome-wide crosslinking protocols have a bias for high abundant proteins. We demonstrate that this bias can be explained by kinetics that govern the formation of a crosslink between two polypeptides. We further show that optimized parameter settings, in particularly an excess of crosslinker, leadto a significant overall increase in the detection of lower abundant proteins within cellular lysates on a proteome-wide scale. Our study therefore explains the cause of a major limitation in current proteome-wide crosslinking studies and demonstrates a way forward how to address a larger part of the proteome by crosslinking

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Florian Stengel  

LAB HEAD: Florian Stengel

PROVIDER: PXD014759 | Pride | 2020-02-17

REPOSITORIES: Pride

Dataset's files

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Action DRS
16x_1.xls Xls
16x_2.xls Xls
1zu10_1.xls Xls
1zu10_2.xls Xls
1zu1_1.xls Xls
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Publications

Proteome-Wide Structural Probing of Low-Abundant Protein Interactions by Cross-Linking Mass Spectrometry.

Fürsch Julius J   Kammer Kai-Michael KM   Kreft Stefan G SG   Beck Martin M   Stengel Florian F  

Analytical chemistry 20200218 5


Proteome-wide cross-linking studies have spurred great interest as they facilitate structural probing of protein interactions in living cells and organisms. However, current studies have a bias for high-abundant proteins. In this study we demonstrate both experimentally and by a kinetic model that this bias is also caused by the propensity of cross-links to preferentially form on high abundant proteins and not by the inability to detect cross-links due to limitations in current technology. We fu  ...[more]

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