Project description:High molecular weight prefractionated pea proteins (Time course), cross-linked by Transglutaminase (several concentrations). TMT labelled. See publication for details
Project description:AONE, a biological orthogonal probe of ONE, was used to identify the K-C cross-linking of raw246.7 cell lysate and living cell proteome.At the same time, we detected the quantitative changes of cross-linked peptides and the competitiveness of ONE to K and C sites.
Project description:Cross-linking of isotope-labelled RNA coupled with mass spectrometry (CLIR-MS) was used to study the UV cross-linking behavior of in vitro reconstituted FOX1 RRM in complex with its cognate RNA sequence, the Fox binding element (FBE), (U)GCAUGU. The FBE heptanucleotide was subsequently mutated, and the impact on UV cross-linking and affinity investigated. Cross-linking was performed using irradiation under 254 nm light, relying on the inherent reactivity of ribonucleotides.
Project description:Dynamic proteins and multi-protein complexes govern most biological processes. Cross-linking/mass spectrometry (CLMS) is increasingly successful in providing residue- resolution data on static proteinaceous structures. Here we investigate the technical feasibility of recording dynamic processes using isotope-labelling for quantitation. We cross-linked human serum albumin (HSA) with the readily available cross-linker BS3-d0/4 in different heavy/light ratios. Wefound two limitations. First, isotope labelling reduced the number of identified cross-links. This is in line with similar findings when identifying proteins. Second, standard quantitative proteomics software was not suitable for work with cross-linking. To ameliorate this we wrote a basic open source application, XiQ. Using XiQ we could establish that quantitative CLMS was technically feasible. Biological significance Cross-linking/mass spectrometry (CLMS) has become a powerful tool for providing residue- resolution data on static proteinaceous structures. Adding quantitation to CLMS will extend its ability of recording dynamic processes. Here we introduce a cross-linking specific quantitation strategy by using isotope labelled cross-linkers. Using a model system, we demonstrate the principle and feasibility of quantifying cross-linking data and discuss challenges one may encounter while doing so.We then provide a basic open source application, XiQ, to carry out automated quantitation of CLMS data. Ourwork lays the foundations of studying themolecular details of biological processes at greater ease than this could be done so far.
Project description:Ion mobility separates molecules in the gas-phase on their physico-chemical properties, providing information about their size as collisional cross-sections. The timsTOF Pro combines trapped ion mobility with a quadrupole, HCD cell and a time-of-flight mass spectrometer, to interrogate ions at high speeds with on-the-fly fragmentation. Ion mobility is perfectly suited for cross-linking mass spectrometry, which aims to uncover spatial restraints for proteins. The used cross-linking reagents covalently link amino acids in close proximity, resulting in peptide pairs after proteolytic digestion. This technique however suffers in terms of the low abundance of cross-linked peptides, which is partially resolved with enrichable cross-linking reagents. This class of reagents enables selective enrichment of cross-linking reagent modified peptides, resulting in selection of the cross-linked peptide pairs and peptides connected to a partially hydrolyzed reagent – termed mono-links. Even though mono-links carry limited structural information, for experiments aiming to uncover protein interactions in an unbiased manner they are unwanted byproducts. Gas-phase separation by IM has the potential to separate the two products and, indeed, we find separation between mono-links and cross-linked peptide pairs for PhoX cross-linked proteins. Moreover, an easy-to-interpret division at a CCS of 500 Å and a mono-isotopic mass of 2 kDa is present, which can be used for precursor selection. From our experiments on low complexity protein systems, sequencing of 50 - 70% of the mono-links is prevented, allowing the mass spectrometer the focus on sequencing the relevant cross-links. Application to a highly complex lysate focusing provides a 10-50 % increase in detected cross-links.
Project description:Chemical cross-linking coupled with mass spectrometry has emerged as a powerful strategy which enables global profiling of protein interactome with direct interaction interfaces in complex biological systems. The alkyne-tagged enrichable cross-linkers are preferred to improve the coverage of low-abundance cross-linked peptides, combined with click chemistry for biotin conjugation to allow the cross-linked peptides enrichment. Herein, together with in vivo chemical cross-linking by alkyne-tagged cross-linker, we explored the click chemistry-based enrichment approaches on protein and peptide level with three cleavable click chemistry ligands, respectively. By comparison, the approach of protein-based click chemistry conjugation with acid-cleavable tag was demonstrated to permit the most cross-linked peptides identification. The advancement of this strategy enhanced the proteome-wide cross-linking analysis, constructing a 5,518 protein-protein interactions network among 1,871 proteins with wide abundance distribution in cell. Therefore, all these results demonstrated a guideline value of our work for efficient cross-linked peptides enrichment, thus facilitated the in-depth profiling of protein interactome for functional analysis.
Project description:Quantitative cross-linking/mass spectrometry (QCLMS) provides increasing structural detail on altered protein states in solution. Accurate quantitation is a value in itself but may also be central to elucidating small differences between protein states. Hence, QCLMS could benefit from data independent acquisition (DIA) which generally provides higher reproducibility than data dependent acquisition (DDA) and higher throughput than targeted methods. Therefore we here open DIA to QCLMS by extending a widely used DIA software, Spectronaut to now also accommodate cross-link data. A mixture of seven proteins cross-linked with bis[sulfosuccinimidyl] suberate (BS3) was used to evaluate this workflow. Out of the 414 identified unique residue pairs, 292 (70%) were quantifiable across triplicates with a coefficient of variation (CV) of 9.8%, with manual correction of peak selection and boundaries for PSMs in the lower quartile of individual CV values. This compares favourably to DDA where we previously quantified only 63% of the identified cross-links across triplicates with a CV of 14%, for a single protein and complete manual data curation. DIA QCLMS is promising to detect differential abundance of cross-linked peptides in complex mixtures despite the encountered ratio compression when increasing sample complexity through the addition of E. coli cell lysate as matrix. In conclusion, DIA software Spectronaut can now be used in cross-linking and DIA is indeed able to improve QCLMS.
Project description:An in planta chemical cross-linking-based quantitative interactomics (IPQCX-MS) workflow has been developed to investigate in vivo protein-protein interactions and alteration in protein structures in a model organism, Arabidopsis thaliana. A chemical cross-linker, azide-tag-modified disuccinimidyl pimelate (AMDSP), was directly applied onto Arabidopsis tissues. Peptides produced from protein fractions of CsCl density gradient centrifugation were dimethyl-labelled, from which the AMDSP cross-linked peptides were fractionated on chromatography, enriched, and analysed by mass spectrometry. ECL2 and SQUA-D software was used to identify and quantitate these cross-linked peptides, respectively. These computer programs integrates peptide identification with quantitation and statistical evaluation. This workflow eventually identified 354 unique cross-linked peptides, including 61 and 293 inter- and intra-protein cross-linked peptides, respectively, demonstrating that it is able to in vivo identify hundreds of cross-linked peptides at an organismal level by overcoming the difficulties caused by multiple cellular structures and complex secondary metabolites of plants. Co-immunoprecipitation and super-resolution microscopy studies have confirmed the PHB3- PHB6 protein interaction found by IPQCX-MS. The quantitative interactomics also found hormone-induced structural changes of SBPase and other proteins. This mass spectrometry-based interactomics will be useful in the study of in vivo protein-protein interaction networks in agricultural crops and plant-microbe interactions.