Dataset Information

Improved peptide backbone fragmentation is the primary advantage of MS-cleavable crosslinkers

ABSTRACT: Proteome-wide crosslinking mass spectrometry studies have coincided with the advent of MS-cleavable crosslinkers that can reveal the individual masses of the two crosslinked peptides. However, recently such studies have also been published with non-cleavable crosslinkers suggesting that MS-cleavability is not essential. We therefore examined in detail the advantages and disadvantages of using the commonly used MS-cleavable crosslinker, DSSO. Indeed, DSSO gave rise to signature peptide fragments with a distinct mass difference (doublet) for nearly all identified crosslinked peptides. Surprisingly, we could show that it was not these peptide masses that proved the main advantage of MS-cleavability of the crosslinker, but improved peptide backbone fragmentation which reduces ambiguity of peptide identifications. This also holds true for another commonly used MS-cleavable crosslinker, DSBU. We show, furthermore, that the more intricate MS3-based data acquisition approaches lack sensitivity and specificity, causing them to be outperformed by the simpler and faster stepped HCD method. This understanding will guide future developments and applications of proteome-wide crosslinking mass spectrometry.

INSTRUMENT(S): Orbitrap Fusion Lumos, Orbitrap Fusion, Q Exactive HF, Q Exactive

ORGANISM(S): Escherichia Coli Drosophila Melanogaster (fruit Fly) Mus Musculus (mouse)

SUBMITTER: Lars Kolbowski

LAB HEAD: Juri Rappsilber

PROVIDER: PXD032821 | Pride | 2022-06-24

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	Drosophila_DSBU_ID_sinz.fasta	Fasta
	Drosophila_DSBU_MS2_15ppm.conf	Other
	Drosophila_DSBU_xisearch_all_cov3_delt0.15_CSM_xiFDR2.1.2_mod.csv	Csv
	Ecoli_BS3_unique_no_extra_xifdr_filter_non-consecutive_proteome_extra_mods_CSM_xiFDR2.1.2.csv	Csv
	Ecoli_BS3_xiSEARCHconf.conf	Other

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Publications

Improved Peptide Backbone Fragmentation Is the Primary Advantage of MS-Cleavable Crosslinkers.

Kolbowski Lars L Lenz Swantje S Fischer Lutz L Sinn Ludwig R LR O'Reilly Francis J FJ Rappsilber Juri J

Analytical chemistry 20220525 22

Proteome-wide crosslinking mass spectrometry studies have coincided with the advent of mass spectrometry (MS)-cleavable crosslinkers that can reveal the individual masses of the two crosslinked peptides. However, recently, such studies have also been published with noncleavable crosslinkers, suggesting that MS-cleavability is not essential. We therefore examined in detail the advantages and disadvantages of using the commonly used MS-cleavable crosslinker, disuccinimidyl sulfoxide (DSSO). Indeed ...[more]

PMID: 35613060

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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.