Optimized cross-linking mass spectrometry for in situ interaction proteomics

Abstract

Recent development of mass spectrometer cleavable protein cross-linkers and algorithms for their spectral identification now permits large-scale cross-linking mass spectrometry (XL-MS).Here, we optimized the use of cleavable disuccinimidyl sulfoxide (DSSO) cross-linker for labeling native protein complexes in live human cells. We applied a generalized linear mixture model to calibrate cross-link peptide-spectra matching (CSM) scores to control the sensitivity and specificity of large-scale XL-MS. Using specific CSM score thresholds to control the false discovery rate, we found that higher-energy collisional dissociation (HCD) and electron transfer dissociation (ETD) can both be effective for large-scale XL-MS protein interaction mapping. We found that the density and coverage of protein-protein interaction maps can be significantly improved through the use of multiple proteases. In addition, the use of sample-specific search databases can be used to improve the specificity of cross-linked peptide spectral matching.Application of this approach to human chromatin labeled in live cells recapitulated known and revealed new protein interactions of nucleosomes and other chromatin-associated complexes in situ. This optimized approach for mapping native protein interactions should be useful for a wide range of biological problems. 5 Experimental Section Reagents. Disuccinimidyl sulfoxide (DSSO), formic acid, dimethyl sulfoxide (DMSO), and LC-MS grade solvents were obtained from Thermo Scientific. Bovine serum albumin (BSA), (4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium chloride, magnesium chloride, potassium chloride, sucrose, ethylenediaminetetraacetic acid (EDTA), dithiothreitol, guanidinium hydrochloride, Staphylococcus aureus micrococcal nuclease, iodoacetamide, ammonium bicarbonate and formic acid were obtained from Millipore Sigma. Protease inhibitors 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF) and pepstatin were obtatined from Santa Cruz, bestatin from Alfa Aesar and leupeptin from EMD Millipore. Trypsin and GluC proteases were obtained from Promega. Chymotrypsin protease was obtained from Pierce Thermo. LysC was obtained from Wako Pure Chemical Industries.Cell culture and protein isolation. HEK293T cells were cultured in DMEM supplemented with 10% (v/v) fetal bovine serum, penicillin and streptomycin. HEK293T cells (50 million) were sedimented by centrifugation at 500 g and lysed in 500 µl 6M guanidine hydrochloride in 100 mM ammonium bicarbonate buffer, pH 8. The lysate was sonicated (E210 adaptive focused acoustic sonicator, Covaris) for 5 minutes at 4°C and homogenized by passing through a 27G needle. The lysate was then clarified by centrifugation at 18,000 g for 10 min at 4°C. Protein concentration of clarified lysates was determined using the bicinchoninic acid (BCA) assay, according to manufacturer's instructions (Thermo Scientific).Preparation of cross-linked bovine serum albumin and peptide purification. 50 µg of BSA was solubilized in 50 µl HEPES buffer (20 mM HE...

Integrative analysis reveals unique structural and functional features of the Smc5/6 complex

“…S1E). This is consistent with the accuracy of CL-MS observed in prior studies (26), and indicates high confidence in detected interactions.…”

A synthetic peptide library for benchmarking crosslinking-mass spectrometry search engines for proteins and protein complexes

“…For example, a lower value of 20 could be used for the stHCD data that gives rise to 183 correct crosslinks (11 more than with a score cut-off of 40) and 8 incorrect, resulting in a calculated FDR of 4.1%. A similar approach was taken by Ser et al 5 by spiking crosslinked BSA peptides into non-crosslinked proteome background peptides to calculated score filters that removed 99% and 90% of non-BSA crosslinks to determine FDR cut-offs for 1% and 10% FDR, respectively. The data included in the present publication could be even more useful for a systematic optimisation of search and validation strategies.…”

“…Initial applications of these techniques were limited to small proteins and protein complexes. More recently, due to major methodological and technological developments, XL-MS has been applied to living cells to investigate protein interactions and topological structures at the proteome-wide level [5][6][7][8] .…”

Optimized Workflow for Enrichment and Identification of Biotinylated Peptides using Tamavidin 2-REV for BioID and Cell Surface Proteomics

“…BioID-based proximity biotinylation of proteins occurs on lysine residues, which can inhibit efficient digestion with trypsin (39,40). Previous studies of cross-linking MS using lysine-reactive cross-linkers (e.g., DSSO) have shown that sequential digestion with Lys-C or other non-lysine-specific proteases (e.g., Glu-C) followed by trypsin increases the coverage of identified cross-linked peptides (41,42).…”