Proteomics

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Cross-linking mass spectrometry analyses of three different kinetochore protein complexes (KMN, NDC80C, MIS12C) using an MS-cleavable cross-linker, BuUrBu (DSBU)

ABSTRACT: In order to develop a simplified cross-linking mass spectrometry protocol, we applied one-step size-exclusion chromatography (SEC) for peptide purification after proteolysis with Lys-C and trypsin. Three benchmark protein complexes (KMN, NDC80C, MIS12C) from human kinetochore were cross-linked with an MS-cleavable cross-linker (BuUrBu). After proteolysis, digested peptides were purified with SEC omitting the step of C18 column to avoid loss of cross-linked peptides. Our protocol showed improvement of existing protocols, and we could successfully identify over 700 cross-links per experiment of each of the three protein complexes. We also observed that about half of the non-redundant cross-links were not lysine-lysine cross-links but cross-links between lysine and side chains with alcohols (serine, threonine, tyrosine).

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Dongqing Pan  

LAB HEAD: Tanja Bange

PROVIDER: PXD010070 | Pride | 2019-04-01

REPOSITORIES: Pride

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Dataset's files

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20180131_Q2_U1_1-10_AB_SA_DOP_20180126_Mis12-F10-id.raw Raw
20180131_Q2_U1_1-10_AB_SA_DOP_20180126_Mis12-F4-id.raw Raw
20180131_Q2_U1_1-10_AB_SA_DOP_20180126_Mis12-F6-id.raw Raw
20180131_Q2_U1_1-10_AB_SA_DOP_20180126_Mis12-F8-id.raw Raw
20180131_Q2_U1_1-2_AB_SA_DOP_20180126_Mis12-F11-id.raw Raw
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Publications

Simplified Protocol for Cross-linking Mass Spectrometry Using the MS-Cleavable Cross-linker DSBU with Efficient Cross-link Identification.

Pan Dongqing D   Brockmeyer Andreas A   Mueller Franziska F   Musacchio Andrea A   Bange Tanja T  

Analytical chemistry 20180829 18

Chemical cross-linking combined with mass spectrometry (MS) is a powerful approach to identify and map protein-protein interactions. Its applications support computational modeling of three-dimensional structures and complement classical structural methodologies such as X-ray crystallography, NMR spectroscopy, and electron microscopy (EM). A plethora of cross-linkers, MS methods, and data analysis programs have been developed, but due to their methodological complexity application is currently r  ...[more]

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