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A model dataset for quantitative cross-linking/mass spectrometry using isotope-labeled cross-linkers

ABSTRACT: 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. In order to investigate the technical feasibility of recording dynamic processes using isotope-labelling for quantitation, we generated a model dataset by cross-linking human serum albumin (HSA) with the readily available cross-linker BS3-d0/d4 in different heavy/light ratios.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Zhuo Chen

LAB HEAD: Juri Rappsilber

PROVIDER: PXD004107 | Pride | 2016-05-06

REPOSITORIES: Pride

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			Action	DRS
	Fischer_Rappsilber_QCLMS_xHSAd0-d4_1to1-I.raw	Raw
	Fischer_Rappsilber_QCLMS_xHSAd0-d4_1to1-II.raw	Raw
	Fischer_Rappsilber_QCLMS_xHSAd0-d4_1to1-III.raw	Raw
	Fischer_Rappsilber_QCLMS_xHSAd0-d4_1to2-I.raw	Raw
	Fischer_Rappsilber_QCLMS_xHSAd0-d4_1to2-II.raw	Raw

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Publications

Quantitative cross-linking/mass spectrometry using isotope-labelled cross-linkers.

Fischer Lutz L Chen Zhuo Angel ZA Rappsilber Juri J

Journal of proteomics 20130326

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. We found two limitations. First, isotop ...[more]

PMID: 23541715

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Quantitative cross-linking/mass spectrometry

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.

2015-06-08 | PXD002142 | Pride

Quantitative cross-linking/mass spectrometry analysis revealed unknown structure of complement iC3

Project description:We applied quantitative cross-linking/mass spectrometry (QCLMS) to interrogate the structure of iC3 (or C3(H2O)), the activated hydrolytic product of the abundant human complement protein C3. The slow but spontaneous and ubiquitous formation of iC3 from C3 initiates antibody-independent activation of the complement system that is a key first line of antimicrobial defense. QCLMS revealed structural differences and similarities between iC3 and C3, as well as between iC3 and C3b that is a pivotal proteolytic cleavage product of C3 and is functionally similar to iC3. Considered in combination with the crystal structures of C3 and C3b, our data support a model wherein the thioester-containing domain of C3 swings to the other end of the molecule creating, in iC3, a stable C3b-like platform for binding the zymogen, factor B, or the regulator, factor H. The integration of available crystallographic and QCLMS data allowed the determination of a 3D model for iC3. The unique arrangement of domains in iC3, which retains the anaphylatoxin (ANA) domain (while ANA is excised when C3 is enzymatically activated to C3b), is consistent with observed differences in activation and regulation between iC3 and C3b.

2016-06-17 | PXD003486 | Pride

Quantitative cross-linking/mass spectrometry reveals subtle protein conformational changes

Project description:We have developed quantitative cross-linking/mass spectrometry (QCLMS) to interrogate conformational rearrangements of proteins in solution. Our workflow was tested using a structurally well-described reference system, the human complement protein C3 and its activated cleavage product C3b. We found that small local conformational changes affect the yields of cross-linking residues that are near in space while larger conformational changes affect the detectability of cross-links. Distinguishing between minor and major changes required robust analysis based on replica analysis and a label-swapping procedure. By providing workflow, code of practice and a framework for semi-automated data processing, we lay the foundation for QCLMS as a tool to monitor the domain choreography that drives binary switching in many protein-protein interaction networks.

2016-06-16 | PXD001675 | Pride

On the reproducibility of label-free quantitative cross-linking/mass spectrometry

Project description:Quantitative cross-linking/mass spectrometry (QCLMS) is an emerging approach to study conformational changes of proteins and multi-subunit complexes. Distinguishing protein conformations requires reproducibly identifying and quantifying cross-linked peptides. Here we analyzed the variation between multiple cross-linking reactions using bis[sulfosuccinimidyl] suberate (BS3)-cross-linked human serum albumin (HSA) and evaluated how reproducible cross-linked peptides can be identified and quantified by LC-MS analysis. To make QCLMS accessible to a broader research community we developed a workflow that integrates the established software tools MaxQuant for spectra pre-processing, Xi for cross-linked peptide identification and finally Skyline for quantification (MS1 filtering). Out of the 221 unique residue pairs identified in our sample, 146 (66% of those identified) could be imported into Skyline and 124 (84% of those imported) were subsequently quantified with coefficient of variation (CV) values of 14% (injection replica) and 32% (reaction replica). Thus our results demonstrate that the reproducibility of QCLMS is in line with the reproducibility of general quantitative proteomics and we establish a robust workflow for MS1-based quantitation of cross-linked peptides.

2018-01-02 | PXD007250 | Pride

Protein Structure Determination in Biological Matrices by Mass Spectrometry and Computational Biology

Project description:We have implemented the use of a heterobifunctional, UV photoactivatable cross-linker, which greatly increases the number of identified cross-links compared with homobifunctional, NHS-ester based cross-linkers. We have cross-linked human serum albumin in the context of human blood serum. We present a novel methodology that combines the use of this high-resolution cross-linking with conformational space search to investigate the structure of proteins in their native environment.

2015-09-22 | PXD001692 | Pride

Non-covalently associated peptides are observed during liquid chromatography-mass spectrometry and affect cross-link analyses

Project description:Cross-Linking/mass spectrometry draws structural information out of covalently linked peptide pairs. When these links do not match to previous structural models they may indicate changes of protein conformation. Unfortunately, such links can also be the result of experimental errors or artefacts. Here we describe the observation of non-covalently associated peptides during liquid chromatography-mass spectrometry analysis which can easily be misidentified as cross-linked. Strikingly, they often mismatch to the protein structure or may falsely suggest protein homo-dimerization. Non-covalently associated peptides presumably form during ionization and can be distinguished from cross-linked peptides by observing co-elution of the corresponding linear peptides in MS1, as well as the presence of the individual peptide fragments in mixed MS2 spectra. Interestingly, non-covalently associated peptides seem sensitive to ionization source design as we see them more prevalently on a Q Exactive than on an Orbitrap Velos mass spectrometer. Finally we show how more disruptive ionization settings such as moderate in-source fragmentation suppresses their presence.

2019-01-25 | PXD010895 | Pride

Clamping down on transcription: repression of archaeal RNAP by the novel viral regulator RIP

Project description:Little is known about how archaeal viruses perturbate the transcription machinery of their hosts. We provide the first example of a non-DNA binding archaeo-viral transcription factor that directly targets the host RNA polymerase and efficiently represses its activity. We have used cross-linking and mass spectrometry to identify how ORF145 binds in the DNA-binding channel of RNA polymerase.

2016-12-02 | PXD003282 | Pride

Data independent acquisition cross-linking/mass spectrometry

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.

2019-01-25 | PXD011036 | Pride

Aurora B kinase disrupts the extended conformation of lattice-bound Kinesin-13 MCAK

Project description:We reveal that MCAK has a compact conformation in solution using cross-linking and electron microscopy. When MCAK is bound to the microtubule ends, it adopts an extended conformation with the N terminus and neck region of MCAK interacting with the microtubule. Also Aurora Bphosphorylation does not alter MCAK conformation in solution. Aurora B interferes with the extended conformation of MCAK on microtubules to decrease the affinity of MCAK for microtubules and reduces its depolymerase activity in a graded fashion.

2019-01-07 | PXD008215 | Pride

Cross-linking/mass spectrometry analysis of the AOC complex

Project description:As a part of a study on how kinetochors are assembled at the centromeres of the chromosomes, cross-linking/mass spectrometry has been applied to investigate interactions between Okp1/ Ame1 heterodimer, which is part of the COMA complex, and CENP-A from Saccharomyces cerevisiae.

2018-10-30 | PXD009648 | Pride

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