Proteomics

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Fast and highly efficient affinity enrichment of Azide-A-DSBSO cross-linked peptides.


ABSTRACT: Cross-linking mass spectrometry is an increasingly used, powerful technique to study protein-protein interactions or to provide structural information. Due to sub-stochiometric reaction efficiencies, cross-linked peptides are usually low abundant. This results in challenging data evaluation and the need for an effective enrichment. Here we describe an improved, easy to implement, one-step method to enrich azide-tagged, acid-cleavable disuccinimidyl bis-sulfoxide (DSBSO) cross-linked peptides using dibenzocyclooctyne (DBCO) coupled Sepharose������ beads. We probed this method using recombinant Cas9 and E. coli ribosome. For Cas9, the number of detectable cross-links was increased from ~100 before enrichment to 580 cross-links after enrichment. To mimic a cellular lysate, E. coli ribosome was spiked into a tryptic HEK background at a ratio of 1:2 ��������� 1:100. The number of detectable unique cross-links maintained high at ~100. The estimated enrichment efficiency was improved by factor 4 -5 (based on XL numbers) compared to enrichment via biotin and streptavidin. We were still able to detect cross-links from 0.25 ������g cross-linked E. coli ribosome in a background of 100 ������g tryptic HEK peptides, indicating a high enrichment sensitivity. In contrast to conventional enrichment techniques, like SEC, the time needed for preparation and MS measurement is significantly reduced. This robust, fast and selective enrichment method for azide-tagged linkers will contribute to map protein-protein interactions, investigate protein architectures in more depth and help to understand complex biological processes.

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

ORGANISM(S): Escherichia coli   Homo sapiens  

TISSUE(S): Tissue Not Applicable To Dataset

DISEASE(S): Not Available

SUBMITTER: Manuel Matzinger  

LAB HEAD: Karl Mechtler

PROVIDER: PXD016963 | Pride | 2020-05-11

REPOSITORIES: pride

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Publications

Fast and Highly Efficient Affinity Enrichment of Azide-A-DSBSO Cross-Linked Peptides.

Matzinger Manuel M   Kandioller Wolfgang W   Doppler Philipp P   Heiss Elke H EH   Mechtler Karl K  

Journal of proteome research 20200416 5


Cross-linking mass spectrometry is an increasingly used, powerful technique to study protein-protein interactions or to provide structural information. Due to substochiometric reaction efficiencies, cross-linked peptides are usually low abundance. This results in challenging data evaluation and the need for an effective enrichment. Here we describe an improved, easy to implement, one-step method to enrich azide-tagged, acid-cleavable disuccinimidyl bis-sulfoxide (DSBSO) cross-linked peptides usi  ...[more]

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