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DSSO Crosslinking of Intact Tetrahymena Thermophila Cilia

ABSTRACT: The MS-cleavable crosslinker, DSSO, was added to cilia isolated from Tetrahymena thermophila. After digestion crosslinked peptides were enriched by size exclusion chromatography. Data were collected using an MS2/MS3 method. Analysis was done using the XlinkX node of PD2.3 and a fasta file generated from standard MS1/MS2 analysis of the crosslinked samples.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Tetrahymena Thermophila (ncbitaxon:5911)

SUBMITTER: Edward Marcotte

PROVIDER: MSV000089917 | MassIVE | Tue Jul 19 11:32:00 BST 2022

SECONDARY ACCESSION(S): PXD035387

REPOSITORIES: MassIVE

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Publications

Does AlphaFold2 model proteins' intracellular conformations? An experimental test using cross-linking mass spectrometry of endogenous ciliary proteins.

McCafferty Caitlyn L CL Pennington Erin L EL Papoulas Ophelia O Taylor David W DW Marcotte Edward M EM

Communications biology 20230415 1

A major goal in structural biology is to understand protein assemblies in their biologically relevant states. Here, we investigate whether AlphaFold2 structure predictions match native protein conformations. We chemically cross-linked proteins in situ within intact Tetrahymena thermophila cilia and native ciliary extracts, identifying 1,225 intramolecular cross-links within the 100 best-sampled proteins, providing a benchmark of distance restraints obeyed by proteins in their native assemblies. ...[more]

PMID: 37061613

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2022-08-01 | MSV000090056 | MassIVE

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2023-04-20 | MSV000091761 | MassIVE

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Project description: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.

2022-06-24 | PXD032821 | Pride

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2019-10-28 | PXD013735 | Pride

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2023-08-28 | PXD040267 | Pride

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