Dataset Information

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

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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]

PMID: 32011863

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Project description:Crosslinking mass spectrometry (XL-MS) is emerging as a unique method at the crossroads of structural and cellular biology, uniquely capable of identifying protein-protein interactions with residue-level resolution and on the proteome-wide scale. With the development of crosslinkers that can form linkages inside cells and easily cleave during fragmentation on the mass spectrometer (MS-cleavable crosslinks), it has become increasingly facile to identify contacts between any two proteins in complex samples, including in live cells or tissues. Photo-crosslinkers possess the advantages of high temporal resolution and high reactivity, thereby engaging all residue-types (rather than just lysine); nevertheless, photo-crosslinkers have not enjoyed widespread use, and have yet to be employed for proteome-wide studies, because their products are challenging to identify, and an MS-cleavable photo-crosslinker has not yet been reported. Here, we demonstrate the synthesis and application of two heterobifunctional photo-crosslinkers that feature diazirines and N-hydroxy-succinimidyl carbamate groups, the latter of which unveil MS-cleavable linkage upon acyl transfer to protein targets. Moreover, these crosslinkers demonstrate high water-solubility and cell-permeability. Using these compounds, we demonstrate the feasibility of proteome-wide photo-crosslinking mass spectrometry (photo-XL-MS), both in extracts and in cellulo. These studies provide a partial interaction map of the E. coli cytosol with residue-level resolution. We find that photo-XL-MS has a propensity to capture protein-protein interactions, particularly involving low-abundance uncharacterized proteins, suggesting it could be a powerful tool to shed light on the “darker” corners of the proteome. Overall, we describe methods that enable the detection of protein quinary interaction networks in their native environment at residue-level resolution proteome-wide, and we expect they will prove useful toward the effort to explore the molecular sociology of the cell.

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