Dataset Information

Reliable identification of protein-protein interactions by crosslinking mass spectrometry_CLMS_BS3&DSSO_datasets

ABSTRACT: Crosslink mass spectrometry dataset on soluble, SEC-fractionated high-molecular weight proteome of E. coli, crosslinked with BS3 or DSSO. This data was used to identify protein-protein interactions and to obtain information on protein (complex) topologies. In the end, the data was used for finding and evaluating a reliable approach for error control in crosslink mass spectrometry, particularly for PPI-studies. Cleared E. coli lysate was fractionated in the range of 3 MDa to 150 kDa (44 fractions). After taking aliquots for quantitative proteomics (JPST000843 ), the aliquots were split and the proteins in each fraction crosslinked (with BS3 or DSSO) and pooled again for each crosslinker set. Then, the pool was precipitated, derivatized and proteolysed in-solution. Digests were fractionated by SCX and hSAX (9x10 offline-fractionation matrix). Afterward, each peptide fraction was acquired via LC-MS on Q Exactive HF. Raw data was pre-processed (denoising, peak-list generation, error correction) and this data searched with xiSEARCH 1.6.746 using differing databases. FDR was calculated using xiFDR (version 2.0dev) under various settings to probe several FDR approaches.

ORGANISM(S): Escherichia Coli

SUBMITTER: Prof. Dr. Juri Rappsilber

PROVIDER: PXD019120 | JPOST Repository | Wed Apr 14 00:00:00 BST 2021

REPOSITORIES: jPOST

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Dataset's files

Source:

			Action	DRS
	B181228_08_HF_LS_IN_130_ECLP_BS3_01_SCX17_hSAX09_rep1.mgf	Mgf
	B181228_09_HF_LS_IN_130_ECLP_BS3_01_SCX17_hSAX09_rep2.mgf	Mgf
	B181228_10_HF_LS_IN_130_ECLP_BS3_01_SCX17_hSAX08_rep1.mgf	Mgf
	B181228_11_HF_LS_IN_130_ECLP_BS3_01_SCX17_hSAX08_rep2.mgf	Mgf
	B181228_12_HF_LS_IN_130_ECLP_BS3_01_SCX17_hSAX07_rep1.mgf	Mgf

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Publications

Reliable identification of protein-protein interactions by crosslinking mass spectrometry.

Lenz Swantje S Sinn Ludwig R LR O'Reilly Francis J FJ Fischer Lutz L Wegner Fritz F Rappsilber Juri J

Nature communications 20210611 1

Protein-protein interactions govern most cellular pathways and processes, and multiple technologies have emerged to systematically map them. Assessing the error of interaction networks has been a challenge. Crosslinking mass spectrometry is currently widening its scope from structural analyses of purified multi-protein complexes towards systems-wide analyses of protein-protein interactions (PPIs). Using a carefully controlled large-scale analysis of Escherichia coli cell lysate, we demonstrate t ...[more]

PMID: 34117231

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Project description:Sample preparation: P-AMPK+AMP, P-AMPK+ATPγS, and AMPK+ATPγS protein samples were crosslinked in HBST buffer (25 mM HEPES pH 7.5 at room temperature, 200 mM NaCL, and 1 mM TCEP). Non-crosslinked negative controls were generated using the same procedure without the addition of DSSO. Reactions were initiated by spiking 1 uL of 75 mM DSSO (Thermo-Fisher) in DMSO into a 50 uL solution containing 10 uM protein and mixing. The final molar ratio of crosslinker:protein was 150:1. The reactions were incubated at 25°C for 45 minutes prior to being quenched by the addition of Tris pH 8.0 to a final concentration of 50 mM. Each crosslink reaction was done in triplicate and the reaction replicates were pooled after quenching. 10 μL samples of the pooled samples were loaded for SDS-PAGE analysis with Coomassie staining to confirm the presence of crosslinked protein. The remaining 140 μL of crosslinked and non-crosslinked samples were then acetone precipitated at -20°C overnight and the protein was pelleted by centrifugation at 16,000 rcf for 5 minutes at 4°C. After decanting, the pellets were dried for 15 minutes at room temperature before being resuspended in 12.5 uL of resuspension buffer (50 mM ammonium bicarbonate, 8M urea, pH 8.0). ProteaseMAX (Promega) was added to 0.02% and the solutions were mixed on an orbital shaker operating at 400 RPM for 5 minutes. After resuspension, 87.5 uL of digestion buffer (50 mM ammonium bicarbonate, pH 8.0) was added. Cysteines in the protein samples were reduced and akylated as follows. DTT was added to 5 mM final concentration and the protein solutions were incubated at 56°C in an orbital shaker operating at 400 RPM and for 20 minutes. After reduction, 2.7 uL of 550 mM iodoacetamide was added and the solutions were incubated in the dark at room temperature for 15 minutes. Reduced and alkylated protein solutions were digested using trypsin at a ratio of 1:200 (w/w trypsin:protein) and incubating at 37°C. After overnight incubation at 37°C, the samples were acidified by the addition of trifluoroacetic acid (TFA, Thermo-Fischer) to 1%. Samples were then frozen and stored at -20°C until analysis.