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A high-efficiency Azide-A-DSBSO cross-linking-MS protocol for identifying thousands of in vivo protein interactions in single day

ABSTRACT: Enrichable and mass spectrometry (MS)-cleavable crosslinkers have gained popularity in crosslinking-MS (XL-MS) in the past decade. The azide-tagged, acid-cleavable disuccinimidyl bis-sulfoxide (DSBSO) crosslinker offers both these advantages, enabling deep XL-MS interactomics studies in intact cells. However, current DSBSO XL-MS workflows take several days, which limits the experimental throughput. Here, we developed an in-cell DSBSO XL-MS protocol that allows sample ready for MS analysis within 8 hours. By systematically optimizing digestion, click chemistry enrichment, LC parameters, and MS fragmentation, we increased the number of crosslink spectrum matches (CSMs) from single-shot experiments more than twofold compared to previous single-shot measurements. This allowed us to efficiently generate deep protein-protein interaction datasets from intact human mitochondria, Bacillus subtilis, and HEK293T cells.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human) Bacillus Subtilis

TISSUE(S): Cell Culture, Early Embryonic Cell

SUBMITTER: Pin-Lian Jiang

LAB HEAD: Fan Liu

PROVIDER: PXD065905 | Pride | 2025-11-21

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	DS14_DSBSO_FAIMS.csv	Csv
	DS21-2.csv	Csv
	DS21_human_2025.02.01.filtered_cross-linked_spectra.csv	Csv
	DS23_interday_intraday_beads.csv	Csv
	DS34_DSBSO_washing_buffer.csv	Csv

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