Dataset Information

Comparison of MS Annika 3.0 against other crosslink search engines

ABSTRACT: The field of crosslinking mass spectrometry has seen substantial advancements over the past decades, enabling the structural analysis of proteins and protein-complexes and serving as a powerful tool in protein-protein interaction studies. However, data analysis of large non-cleavable crosslink studies is still a mostly unsolved problem due to its n-squared complexity. We here introduce a novel algorithm for the identification of non-cleavable crosslinks implemented in our crosslinking search engine MS Annika that is based on sparse matrix multiplication and allows for proteome-wide searches on commodity hardware. Application of this new algorithm enabled us to employ a proteome-wide search of C. elegans nuclei samples and we were able to uncover previously unknown protein interactions and conclude a comprehensive structural analysis that provides a detailed view of the Box C/D complex, enhancing our understanding of its assembly and functional dynamics. Our findings provide valuable insights into the intricate regulation of cellular homeostasis and immune responses, which are conserved across species, including humans. Moreover, our algorithm will enable researchers to conduct similar studies that were previously unfeasible.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Escherichia Coli Streptoccous Pyogenes

SUBMITTER: Micha Johannes Birklbauer

LAB HEAD: Viktoria Dorfer

PROVIDER: PXD055512 | Pride | 2024-12-19

REPOSITORIES: Pride

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			Action	DRS
	20210521_QExHFX2_RSLC3_ADH_acidic-lib_sep_rep1.mgf	Mgf
	20210521_QExHFX2_RSLC3_ADH_acidic-lib_sep_rep1.raw	Raw
	20210531_QExHFX2_RSLC3_ADH_acidic-lib_sep_rep2.mgf	Mgf
	20210531_QExHFX2_RSLC3_ADH_acidic-lib_sep_rep2.raw	Raw
	20210604_QExHFX2_RSLC3_ADH_acidic-lib_sep_rep3_pl3.mgf	Mgf

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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.

Dataset Information

Comparison of MS Annika 3.0 against other crosslink search engines

Dataset's files

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