- Daniel Wendscheck – Native mass spectrometry coupled to mathematical modelling reveals dynamics of protein subunit exchange and highlights importance of response factors
- Heather Chassaing – Comprehensive mapping of disulfide linkages in etanercept using an electron activated dissociation (EAD) based LC-MS/MS methodology
- Lennart Sänger – Characterization of interactions between Lassa virus nucleoprotein, matrix protein and RNA essential for RNP assembly and recruitment
- Frederik Lermyte – New insights into the intrinsic electron-based dissociation behaviour of cytochrome c oligomers
- Weijing Liu – Establishing a decision tree for native mass spectrometry analysis of membrane proteins in complex membrane mimetics
- Maria Mateos-Jimenez – Developing a carbene footprinting/top-down mass spectrometry strategy for analyzing protein-protein interactions
- Ilnaz Soleimani Mashhadi – Native Mass Spectrometry for Structural analysis of Viral Glycoproteins
- Steven Daly – THE PHOTO-SYANPT: HYPHENATIO OF ION MOBILITY AND ACTION SPECTROSCOPY TO STUDY PEPTIDE AGGREGATION
- Aisha Ben-Younis – Cyclic ion mobility–mass spectrometry and electron capture dissociation probe dimerisation of aggregation-prone IAPP
- Mikuláš Vlk – Utilization of Ion Mobility – MASS SPECTROMETRY for Detection and Characterization
of Aβ(1-42) Oligomers
- Alan Kádek – Fighting fire with fire: HDX-MS of human papillomavirus with heparin
- Martin Hubálek – The effect of domain order on modulation of fusion two-domain proteins structure and its dynamics studied by hydrogen-deuterium exchange
- Jakub Sýs – The single amino acid substitutions in Mason-Pfizer Monkey Virus matrix protein modulate its proteolytic cleavage rate
- Cornelia Wagner – HDX-MS drives Lead Selection and Characterization in Pharmaceutical Research
- Fojtík Lukáš – Monitoring of conformational changes in transmembrane proteins using HDX and FFAP radical labeling
- Dietmar Hammerschmid – Selective protein capture under HDX-MS quench conditions for probing complex biological systems
- Thomas BOTZANOWSKI – HDX-MS to study G-protein-coupled receptors in a Contract Research Organization
- Yuqi Shi – Optimization of HX-MS Workflow for Membrane Protein Melibiose Transporter MelB
- Dmitry Loginov – FPOP analysis of α-synuclein
Native mass spectrometry coupled to mathematical modelling reveals dynamics of protein subunit exchange and highlights importance of response factors
Daniel Wendscheck1,2, Mio Heinrich4,5, Franz-Georg Wieland4,5, Adrian Hauber4,5, Julian Bender2, Friedel Drepper1,3, Jens Timmer3,4,5, Bettina Warscheid1,2,3
1. Biochemistry and Functional Proteomics, Institute of Biology II, University of Freiburg, Schänzlestraße 1, 79104 Freiburg, Germany
2. Biochemistry II, Theodor Boveri-Institute, Biocenter, University of Würzburg, 97074 Würzburg, Germany
3. Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Germany
4. Institute of Physics, University of Freiburg, Hermann – Herder Str. 3, 79104 Freiburg, Germany
5. Freiburg Center for Data Analysis and Modelling (FDM), University of Freiburg, Germany
Introduction: Many proteins fulfil their biological functions not alone, but as part of macromolecular machines. As any other machine, such protein machines are assembled, subunits are dynamically exchanged and eventually they are disassembled and recycled. Studying these events on the molecular scale is key to understanding basic cellular processes. One such molecular machine is the peroxisomal translocon centred around the integral membrane protein Pex14p, which plays a pivotal role in the import of newly synthesized matrix proteins into peroxisomes. Its propensity for homo-oligomerisation is thought to be essential for adaptation of the peroxisomal translocon to folded cargos of different size. Here, we apply time-resolved native mass spectrometry (MS) and mathematical modelling to characterise subunit exchange of Pex14p oligomers.
Methods: To monitor subunit exchange between Pex14p homo-oligomers, we recombinantly expressed and purified two differentially truncated variants of Pex14p. Equimolar mixtures of the two variants were immediately analysed by continuous nanoflow electrospray ionisation and native MS to gain time-resolved mass spectra over a period of 10 minutes. Mass spectra were deconvoluted using the UniDec software suite and peak areas were integrated to calculate separate MS intensities for each species over time. We then applied a mathematical model to the experimental data, which considers all possible reactions during homo-oligomer assembly and disassembly. It also takes into account the individual response factors of all different oligomeric states. The model was tested with additional datasets acquired at lower and higher protein concentrations.
Preliminary data: Using differentially truncated protein variants to induce a detectable mass shift is a cheap, fast, and robust strategy on par with established approaches such as isotopic labelling or orthologous proteins. Pex14 predominantly exists in a trimeric form, whereas monomers and dimers were also detected but to a lower degree using standard native MS. After mixing of the two mass-different variants, hybrid species consisting of both truncated variants were observed and the equilibrium of subunit exchange reaction was reached after ~7 minutes. Our mathematical model uses flexible scaling factors to translate ion intensities to solution concentration for each molecular species. Applying this model, we found that dimers and monomers occur as transient states compared to trimers. However, Pex14 trimers rapidly exchange subunits. Thus, such dynamic Pex14 trimers render a rigid import complex rather unlikely. Moreover, Pex14 trimers might provide building blocks for structural flexibility for peroxisomal import of proteins of various sizes. Using flux-plots for each observable species, we found that the apparent equilibrium after ~7 minutes is maintained by fast fluxes of assembly and disassembly reactions resulting in a net zero flux. The generated model produces similar results for the scaling factors at Pex14p concentration differing by a factor of four. In agreement with published work, we derived higher scaling factors for higher oligomeric states, possibly a result of higher ionisation efficiencies compared to monomeric species. Overall, we suggest that the received scaling factors can be used as an estimate for response factors. By this, we propose a novel approach for estimating response factors, which are challenging to determine by existing protocols and thus often left unconsidered.
Novel aspect: We combined an improved native MS protocol with mathematical modelling to assess kinetics of a homo-oligomerisation process and estimate response factors.
Comprehensive mapping of disulfide linkages in etanercept using an electron activated dissociation (EAD) based LC-MS/MS methodology
Heather Chassaing1, Zhengwei Chen2, Lei Xiong2
1 Sciex, Villebon sur Yvette, France
2 SCIEX, Sciex, Redwood city, USA
The disulfide linkages of etanercept, a dimeric fusion protein, were characterized utilizing both collision induced dissociation (CID) and electron activated dissociation (EAD) in combination with a multi-enzyme approach (trypsin + Glu-C). The performance of CID and EAD in terms of peptide backbone coverage for disulfides at different complexity level were compared. For simple disulfides with relatively short peptides, both CID and EAD works equally well in generating fragments for good peptide backbone sequence coverage. However, when the length of peptides increases, EAD provides unique benefits in generating wealth of fragments for every peptide involved in the disulfides. Particularly, for disulfide peptides containing more than 2 disulfide bonds, CID generated very limited peptide backbone fragments; whereas EAD generated abundant fragments for the solid identification of every peptide. In addition, as a soft fragmentation technique, EAD also was able to successfully preserve the labile O-glycan on the disulfides, providing additional benefit of accurately locating the O-glycosylation site.
Characterization of interactions between Lassa virus nucleoprotein, matrix protein and RNA essential for RNP assembly and recruitment
Lennart Sänger1,2,3, Harry M. Williams1,2, Dingquan Yu2,4, Dominik Vogel1, Jan Kosinski2,4, Maria Rosenthal1, Charlotte Uetrecht2,3,5,6
1 Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Straße 74, 20359 Hamburg, Germany
2 Centre for Structural Systems Biology, Notkestraße 85, 22607 Hamburg, Germany
3 Leibniz Institute of Virology (LIV), Martinistraße 52 20251 Hamburg, Germany
4 European Molecular Biology Laboratory Hamburg, Notkestraße 85, 22607, Hamburg, Germany
5 University of Siegen, Am Eichenhang 50, 57076, Siegen, Germany
6 European XFEL GmbH, Holzkoppel 4, 22869, Schenefeld, Germany
Introduction: Lassa virus is a negative-strand RNA virus with only four structural proteins. It is endemic to West Africa and causes Lassa hemorrhagic fever. The nucleoprotein (NP) encapsidates the viral genome, forming together with the L protein the ribonucleoparticles (RNP), which have to be continuously restructured during viral genome replication and transcription. Z protein is important for RNP recruitment, mediates viral particle assembly and budding, and has been shown to interact with the L protein.
Objectives: The interaction of NP, viral RNA and Z is only poorly understood. Here, we characterize the interactions between Lassa virus NP, Z and RNA using an integrative structural approach of structural mass spectrometry, structure prediction and electron microscopy.
Materials & methods: NP and Z were subjected to native mass spectrometry (nMS) revealing protein interactions and stoichiometry of complexes. Conformational changes of NP upon RNA binding were observed in real time using nMS and binding interfaces were mapped by AlphaFold Multimer and mutational analysis.
Results: In presence of RNA, even short ones, the trimeric NP oligomer undergoes conformational changes leading to its disassembly into RNA bound NP monomers, which subsequently form higher order NP-RNA assemblies, reminiscent of RNPs. Additionally, we show that LASV NP can directly interact with the Z protein via the NP C-terminal domain both in presence and absence of RNA. We identified two arginines in the Z protein that were important for NP-Z interaction. We furthermore demonstrate that this interaction is strongly pH-dependent, suggesting a possible disassembly determinant during virus entry via the endosomal pathway.
Conclusion: Our findings support a model in which Z binds to the RNP at neutral pH via direct interaction to NP. Direct binding to NP could facilitates targeted transport of the RNP to the plasma membrane at high concentration of Z protein during late stages of the viral life cycle. Upon virus entry, the RNPs are released from the virion matrix triggered by the low pH in the endosome allowing the RNPs to enter the cytosol after membrane fusion for viral genome replication and transcription.
New insights into the intrinsic electron-based dissociation behaviour of cytochrome c oligomers
Sarah Brandner1, Tanja Habeck1, Frederik Lermyte1
1 Technical University of Darmstadt
Introduction: Native MS is a vital technology for structural biology, and combined with electron-based fragmentation can provide insights into protein higher-order structure. This nearly always involves interaction with either free electrons or radical anions. However, between 2003 and 2017, four reports were published demonstrating the intrinsic ability of the iron-containing proteins cytochrome c and ferritin to undergo radical-based gas-phase fragmentation without exogenous electrons, likely during dissociation of noncovalent oligomers. For cytochrome c, this effect has so far only been reported to occur in the ion source, preventing in-depth study of reactions of specific precursors. Here, we report the first observation of this phenomenon after isolation of specific cytochrome c oligomers, providing experimental support and new insights into the proposed reaction mechanism.
Methods: All reagents and solvents were obtained from Merck (Darmstadt, Germany) and used without further purification. Solutions were sprayed via direct infusion with a nano-electrospray ionisation setup and in-house pulled glass needles coupled to a Waters Synapt XS ion mobility – mass spectrometer. The needles were pulled with a Sutter P-97 micropipette puller. Collision-induced unfolding (CIU) data were processed with CIUSuite 2. Fragment spectra were analysed with MassLynx 4.2 and signals were assigned manually. For relative quantitation, the average ion currents of the extracted ion chromatograms of the fragments were calculated and divided by the average total ion current.
Preliminary data: Following literature procedures, we performed nano-electrospray ionisation of a highly concentrated (37.5 µM) cytochrome c solution. After optimisation of sample preparation, protein oligomers (up to trimers) were visible in the spectrum, as well as c– and y-type fragments. In particular, the presence of c-type fragments indicates a radical pathway. As in earlier reports, fragmentation occurred spontaneously, without precursor isolation or interaction with exogenous electrons. We next decided to isolate the two most intense charge states of the dimer and trimer, in order to investigate whether this intrinsic electron-based fragmentation process can occur in vacuo. Previous work has indicated that the process is more efficient in the ion source (during or shortly after desolvation). However, although we routinely had to average many spectra acquired over the course of several minutes (up to more than an hour in some cases) we were indeed able to monitor the spontaneous radical fragmentation of quadrupole-selected 11+ and 13+ dimer, as well as 14+ and 16+ trimer in vacuo. This experiment finally demonstrated that this process can indeed occur in the gas phase and not exclusively in the ESI source, with asymmetric charge partitioning during dissociation of the oligomers likely providing the potential difference that induces electron transfer between monomers. To provide further evidence for this mechanism, we prepared a solution with conditions known to induce oligomerisation and diluted this to 10 µM before ESI. This resulted in markedly higher c-fragment signal intensity compared to our initial experiments, which supports the hypothesis that specific, welldefined solution-phase oligomers are the origin of the intrinsic capability of cytochrome c to undergo electron-based fragmentation in the absence of exogenous electrons. Finally, we were able to rationalise preferred fragmentation sites through noncovalent interactions in published cytochrome c dimer and trimer crystal structures.
Novel aspect: First in-depth investigation of the intrinsic electron-based fragmentation of specific oligomers of the electron transfer protein cytochrome c.
Establishing a decision tree for native mass spectrometry analysis of membrane proteins in complex membrane mimetics
Weijing Liu1, Christopher Mullen1, Donggyun Kim2, Vadim Cherezov2,Gregory J Dodge3, Barbara Imperiali3, Hiruni S. Jayasekera4, Michael Marty4, Rosa Viner1
1 Thermo Fisher Scientific, San Jose, CA
2 Thermo Fisher Scientific, Bridge Institute, University of Southern California, Los Angeles, CA
3 Department of Biology, Massachusetts Institute of Technology, Cambridge MA
4 University of Arizona, Tucson, AZ, USA
Introduction: Membrane proteins (MPs) represents 60% clinical drug targets owing to their active involvement in cellular processes. The complexity of membrane mimetics for MPs solubilization pose the challenges for native mass spectrometry (nMS) analyses. Common membrane mimetics including detergent micelles, nanodiscs and styrene maleic-acid lipid particles (SMALPs) are arguably critical for preserving native structure of MPs. Here, we aim to develop a decision tree for nMS characterization of MPs in different membrane mimetics. Firstly, online buffer exchange-nMS (OBE-nMS) enables quick assessment of MPs in either detergent or nanodiscs. Secondly, either charge detection mass spectrometry or proton transfer charge reduction is compelling to resolve MPs in SMALPs. Decision tree approach using variety of MPs demonstrates its complementarity to other structural biology tools.
Methods: Membrane proteins in nanodiscs were provided by Michael Marty, University of Arizona. GPCR and GPCR-Gs protein complexes were provided by Vadim Cherezov, University of Southern California. Wbap in SMALP was provided by Barbara Imperiali from MIT.. OBE-nMS was performed by using a Thermo Scientific™ Vanquish™ Flex UHPLC system, Q Exactive™ UHMR and NativePac OBE-1 column with ESI or NSI sources. Mobile phase consisted of 50-200 mM ammonium acetate with or without detergent present. Direct Mass Technology mode employs direct infusion with Nanoflex ion source coupled to UHMR MS equipped with an ExD cell (e-MSion, Inc.). Data were analyzed using Thermo Scientific™ BioPharma Finder™ 5.0 and STORIboard (Proteinaceous).
Preliminary Data: We systematically investigate the complexity of MPs in different membrane mimetics to establish a decision tree on native MS method selection. Firstly, detergent screening for OBE-nMS reveals LDAO is universal for characterizing both bacterial and mammalian MPs with partial denaturation. With this approach, we were able to detect 4 out of 5 different subunits plus PTMs a GPCR-Gs complex (>200 kDa) within 6 minutes. Furthermore, top-down OBE-nMS/MS could rapidly confirm the sequence and identify potential alkylation sites to aid protein alkylation optimization for X-ray crystallography. To establish OBE-nMS method for MPs in nanodiscs, initial testing employs injection of empty nanodisc with mobile phase containing detergent. Consequently, nanodiscs were dissociated into monomeric membrane scaffold protein and phospholipids. Removal of detergent from mobile phase and switching HESI to EASY-spray source intriguingly preserve the empty nanodisc integrity, and thus facilitate apply such method to successful characterization of MPs in nanodiscs. Rapid characterization of MPs in detergent and nanodiscs using OBE-nMS cannot deny its limitation on resolving charge state of complex samples. The aforementioned GPCR-Gs complex and Wbap in SMALP remain unresolvable in ensemble measurement due to their heterogeneity. Using CDMS reveals stoichiometry dependence of the GPCR-Gs complex on membrane mimetics. Additionally, the oligomerization is controversial of Wbap. CDMS results show its MW ~146 kDa in AmAc containing detergent and ~243kDa in AmAc only. The first MW unravels the dimeric status of this protein, and the second MW indicates the dimer stays in a bulky environment composed of numerous lipids and SMA. Results of GPCR-Gs and Wbap both reflect the structure sensitivity to the environment. Future work will evaluate proton transfer charge reduction performance on resolving MPs in complex mimetics. Eventually, we aim for a decision tree on native MS method selection for assessing MPs in different membrane mimetics for efficiently solving biological problems.
Novel aspect: Systematic evaluation and method development of non-standard membrane proteins in complex mimetics
Developing a carbene footprinting/top-down mass spectrometry strategy for analysing protein-protein interactions
Maria Mateos-Jimenez1, C. Logan Mackay1, Lynne Regan2, Anita C. Jones1, Clinton Veale3, David J. Clarke1
1 EaStCHEM, School of Chemistry, The University of Edinburgh, UK
2 Institute of Quantitative Biology, Biochemistry and Biotechnology, School of Biological Sciences, The University of Edinburgh, UK
3 Department of Chemistry, University of Cape Town, South Africa
Introduction: The chaperone HSP90 is essential for protein homeostasis and cell survival. In tumoral cells, HSP90 has been found significantly overexpressed associated with the cochaperone HSP70-HSP90 organising protein (HOP). The HOP-HSP90 interaction is characterised by a ’carboxylate clamp’ present on the TPR2A domain of HOP, which binds to the conserved C-terminal MEEVD motif of HSP90. Recent evidence suggests that inhibiting the HOP-HSP90 interaction constitutes an attractive approach for inhibiting HSP90 in cancer. A diazirine-based footprinting experiment followed by a top-down mass spectrometry strategy is being developed to study this interaction. The main advantages of a top-down approach are the selection of a single protein ion of interest for fragmentation and the potential of achieving a residue-level resolution.
Methods: The 16.9 kDa TPR2A domain of HOP was expressed in E. coli and purified by affinity chromatography. 4-(3-Trifluoromethyl)-3H-diazirin-3-yl)benzoic acid was purchased from Sigma-Aldrich. Carbene-modified TPR2A was achieved by photoactivation of 10 mM of the diazirine labelling agent. Top-down mass spectrometry is perfomed in a 12 T Bruker SolariX coupled to a TriVersa NanoMate nanoelectrospray. Top-down fragmentation was achieved using electron-capture dissociation (ECD); typical ECD conditions were bias 1.7 V, lens 8 V and a pulse length of 10 ms. Top-down fragmentation data were processed using DataAnalysis v4.4 (Bruker) and Prosight Lite.
Preliminary data: Here we present our latest results on the biophysical characterisation of the proteinprotein interaction. Firstly, the diazirine-based modification of the TPR2A protein has been optimised. The extent of protein modification was assessed by intact protein mass spectrometry and a range of modified protein ions ranging from 1 up to 4 covalent insertions were observed. Subsequent top-down fragmentation analysis is being developed. Individual protein ions were isolated using the mass selecting quadrupole in the FT-ICR and subjected to topdown fragmentation with ECD. The peak abundance of each fragment ion was considered equivalent to the gas-phase ion abundance. Typically, 60 % sequence coverage could be achieved using ECD and summing at least 500 scans. Ultimately, this analytical platform will be applied to study the HOP-HSP90 interaction and it may provide some insights to the design of potential disrupters of this proteinprotein interaction.
Novel aspect: New analytical platform is being developed for the assessment of diazirine-based protein footprinting with top-down mass spectrometry.
Native Mass Spectrometry for Structural analysis of Viral Glycoproteins
Ilnaz Soleimani Mashhadi1, Juergen Muller-Guhl1,2, Pietro Scaturro3, Charlotte Uetrecht1,2,4
1 Centre for Structural Systems Biology (CSSB), Deutsches Elektronen-Synchrotron DESY & Leibniz Institute of Virology (LIV), Notkestraße 85, 22607 Hamburg, Germany.
2 Bernhard-Nocht Institute for Tropical Medicine, Hamburg, Germany.
3 Leibniz Institute of Virology (LIV) Martini Strasse 52, 20251 Hamburg, Germany.
4 University of Siegen, Siegen, Germany.
Introduction: Human viruses cause a broad range of diseases and are a main challenge to the healthcare system. Virus infectivity highly depends on its glycoproteins, which are necessary to invade the host cell. Glycoproteins existing on the surface of an enveloped virus facilitate the attachment of virion to the host cell through a cellular receptor and play an important role in both infection and immunity. Understanding structural and functional diversity of glycoproteins across different viral variants is crucial to develop the next generation of vaccines and antivirals. Due to the heterogeneity of glycoproteins, their analysis is a major challenge in structural biology.
Methods: High-resolution native mass spectrometry (MS) has already been used to analyze, compare and uncover the heterogeneity of biopharmaceuticals such as glycoproteins. In this study, we will take advantage of this technique to study intact glycoproteins directly from enveloped viruses. For this purpose, First, the virus particles were propagated and purified. Then, the purified particles were infused to high-resolution native mass spectrometry (MS), before and after deglycosylation, and the obtained spectra interpreted for glycoprotein identification.
Preliminary data: Using model systems, workflows to expel and fragment the glycoproteins from the envelope are developed. According to obtained data, by adjusting the correct MS parameters, glycoproteins can be repelled from the envelope and further fragmented, useful for characterization of their structure.
Novel aspect: Using whole virus particles for structural analysis of glycoproteins instead of pure recombinant proteins.
THE PHOTO-SYANPT: HYPHENATIO OF ION MOBILITY AND ACTION SPECTROSCOPY TO STUDY PEPTIDE AGGREGATION
Steven Daly1, Sjors Bakels1, Jan Commandeur1, Anouk Rijs2
1 MS Vision,Televisieweg 40, 1322 AM Almere, Netherlands
2 Division of BioAnalytical Chemistry, AIMMS Amsterdam Institute of Molecular and Life Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1108, 1081 HV, Amsterdam
A key physical process in the human body is the aggregation of peptides and proteins, the transition from soluble functioning proteins into insoluble amyloid aggregates. This unavoidable build-up of aggregates is directly linked to agerelated, neurodegenerative diseases, including Alzheimer’s and Parkinson’s disease. Gaining full understanding of the early, neurotoxic steps of the aggregation process is essential as this can lead to its control and prevention. However, this knowledge is obscured by a cascade of events occurring at various time and energy scales, producing complex and heterogenous mixtures of aggregates. Therefore, we are developing a novel, multidimensional spectroscopy- and mass spectrometry-based method, that allows us to probe the structure and kinetics of the initial steps of the aggregation process in a single measurement.
To this end we have developed the Photo-Synapt, a Waters Synapt G2 ion-mobility time-of-flight mass spectrometer was modified in to perform ion mobility slicing and to provide optical access and to allow for trapping of ions. This was done by adding two hexapoles coupled with pin traps in differentially pumped stages after the mobility stage. Mass- and ion mobility selected ions can therefore be stored. Optical access ports were added to allow irradiation by UV or IR photons. To precisely control the gas pressure in the irradiation cell additional gas inlets were attached.
We show that we can perform experimentations in all envisioned modes of operations: Normal measurements without trapping, UVPD on mass and mobility selected ions, and IRMPD spectroscopy on mass and mobility selected ions. The instrument itself is evolving, and some of the modifications to the original design are shown, which will increase performance, sensitivity and allow for greater overlap of ion cloud within the trap and lasers. We show that the Photo-Synapt is a powerful multipurpose tool for studying the structure of a wide range of biomolecular ions.
Cyclic ion mobility–mass spectrometry and electron capture dissociation probe dimerisation of aggregation-prone IAPP
Aisha Ben-Younis1, Alexander Zhyvoloup1, Hannah Britt1, Daniel Raleigh1,2, Konstantinos Thalassinos1,3
1 University College London, London, United Kingdom
2 Stony Brook University, Stony Brook, NY, USA
3 Birkbeck College, University of London, London
Introduction: Early oligomerisation of human islet amyloid polypeptide (hIAPP) is responsible for pancreatic β-cell death and is implicated in type II diabetes pathogenesis. S20G-hIAPP, the only known hIAPP, is associated with a higher propensity for disease. Rat IAPP (rIAPP) differs from the human sequence at just six residues but is non-amyloidogenic and non-toxic to β-cells. The question is therefore whether these sequence differences lead to structural differences in the dimeric forms of these peptides. Here, high resolution cyclic ion mobility–mass spectrometry (cIM–MS) with new electron capture dissociation (ECD) capability has been applied to investigate detailed gas-phase dynamic behaviour and observe conformational transitions of early oligomers of hIAPP, S20G-hIAPP, and rIAPP; and describe the self-assembly region for their dimers.
Methods: Lyophilised peptides were dissolved in 100% DMSO at a concentration of 1 mM and incubated for 24 h at 37°C. Samples were prepared in 100 mM ammonium acetate pH 7.4 at a concentration of 10 μM before direct infusion into a SELECT SERIES Cyclic IMS QToF (Waters Corp.) fitted with a post-mobility ECD cell (e-MSion Inc.). Data were analysed using UNIFI Scientific Information System (Waters Corp.) and our in-house software, AmphitriteX.
Preliminary data: Conformer-specific collision activation experiments for the +5 dimer species showed different gas phase behaviour and stabilities between amyloidogenic hIAPP and S20G-hIAPP, and non-amyloidogenic rIAPP species. Human IAPP dimers were seen to extend on activation before forming a super-extended state at high energies immediately before dissociation into monomer. S20G-hIAPP dimers behaved similarly to the wildtype, though forming more of the super-extended state. Rat IAPP dimers, however, tended to recompact with increasing activation and did not form the super-extended state. ECD fragmentation experiments localized the dimerisation interface of hIAPP to between residues Leu16 – Gly33, correlating well with previously published solution phase peptide mapping array studies. The dimerisation interface identified for S20G-hIAPP was less extensive, between N22-S29, and the peptide showed more fragmentation overall, indicative of a more extended structure. rIAPP dimerisation, however, was shown to be between N22-G33, indicating a different structural arrangement. cIM–MS has been used to find that the highly amyloidogenic hIAPP and S20G-hIAPP dimers, and non-amyloidogenic rIAPP dimers display different conformational behaviour, correlating to their solution-phase characteristics. Coupling ECD MS/MS to cIM–MS has allowed a detailed structural comparison of the amyloidogenic (hIAPP and S20G-hIAPP) and non-amyloidogenic (rIAPP) dimers. These results provide important insight into why hIAPP and S20G-hIAPP proceed to form β-sheet fibrils and rIAPP does not.
Novel aspect: cIM–MS with ECD has proved a powerful technique for the in-depth study of protein conformation within a single experiment.
Utilization of Ion Mobility – MASS SPECTROMETRY for Detection and Characterization
of Aβ(1-42) Oligomers
Mikuláš Vlk1, John A. Hey2, Walter Korfmacher2, Petr Kocis2, Alexander Muck3, Martin Hubálek1,
1 Institute of Organic Chemistry and Biochemistry of the CAS, Mass Spectrometry Group, Prague, Czech Republic
2 Alzheon, Inc., R&D, Framingham, MA, USA
3 Waters Corporation, Analytical Professional Services EMEA, Wilmslow, UK
Introduction: Quantification of Aβ(1-42) oligomers is a critical step in understanding the role of these species in Alzheimer’s disease (AD), developing potential treatments and early diagnosis. Quantification remains an analytical challenge due to rapid aggregation, structural heterogeneity, and low abundance of Aβ(1-42) oligomers in biofluids. We employed a state-of-the-art high-resolution cyclic ion mobility mass spectrometry (cIM-MS) system to develop a method for detection of soluble oligomeric species in vitro. Moreover, we applied the developed method to CSF samples spiked with Aβ(1-42) standard.
Methods: Samples of Aβ(1-42) were incubated under various conditions to enhance the generation of soluble oligomers. Aβ(1-42) was pre-treated by hexafluoroisopropanol to break down any pre-existing aggregates. Detection was performed using a SELECT SERIES Cyclic IMS instrument (Waters Corporation, UK) with a static nanoelectrospray ion source operated with in-house pulled borosilicate emitters. Buffer exchange was performed on Bio-Spin® P-6 Gel Columns (Bio-Rad Laboratories, USA). Desalting of CSF and protein removal was performed using Amicon Ultra-0.5 mL 3kDa and 30kDa Centrifugal Filters (Merck, Darmstadt).
Preliminary data: Thorough optimization of instrumental parameters was essential for transmitting labile non-covalent Aβ(1-42) oligomers. To prevent dissociation of such labile non-covalent complex species, ion optics tuning was a critical step in method optimization. Specifically, fine tuning of voltage gradients and RF values was vital for detection of up to hexamers. Furthermore, travelling-wave heights and velocities and gas flows enabled detection of up to dodecamer. A single-pass ion mobility method was developed to separate the isobaric oligomer ions.
We applied the optimized method combined with two-step desalting procedure by centrifugal filters with molecular weight cut-off 3 kDa and 30 kDa on a CSF sample spiked with Aβ(1-42) and control sample consisting of Aβ(1-42). Results show oligomers up to pentamer detected in control sample and up to dimer in spiked CSF sample. Results show that further sample preparation is needed in order to reduce matrix effects and to preconcentrate the oligomers.
Novel aspect: Our results show that Cyclic IMS can be employed to detect and characterize soluble Aβ(1-42) oligomeric species in vitro.
Fighting fire with fire: HDX-MS of human papillomavirus with heparin
Alan Kádek1,2,3, Sarah Nentwich2, Charlotte Uetrecht2,4,5
1 Institute of Microbiology, Czech Academy of Sciences – BIOCEV, 252 50 Vestec, Czech Republic
2 Leibniz Institute of Virology, 20251 Hamburg, Germany
3 European XFEL, 22869 Schenefeld, Germany
4 Centre for Structural Systems Biology, Deutsches Elektronen-Synchrotron DESY, 22607 Hamburg, Germany
5 Faculty V: School of Life Sciences, University of Siegen, 57076 Siegen, Germany
Introduction: Hydrogen/deuterium exchange mass spectrometry is a versatile tool in the modern structural biology portfolio. It is often considered as practically limitless in regards to the size of the studied systems, monitored conditions and the composition of the experimental buffers. While mostly true, DNA and other highly negatively charged compounds have long been known to be troublesome and are often avoided or approached with the utmost caution due to their precipitation at quench conditions. This poses a specific challenge for the study of viruses such as human papillomavirus, for which interactions with extremely negatively charged heparin sulfate are of high biological importance.
Methods: Human papillomavirus 16 samples were produced in the form of virus-like particles and pseudovirions by the collaborating laboratory of Prof. Mario Schelhaas essentially as described previously (Buck et al.: Methods in Molecular Medicine 2005). Heparin quantitation was successfully achieved using Heparin Red Ultra assay (Redprobes, Germany). HDX-MS was performed in an online setup using immobilized porcine pepsin microreactor columns mounted in an LC-MS system consisting of an Agilent Infinity 1260 and an Orbitrap Fusion mass spectrometer.
Preliminary data: An HDX-MS study will be reported of the conformational changes in human papillomavirus particles during the initial steps of its infection cycle, when the virus is being primed for entering the cell. Apart from several proteolytic cleavages and conformational changes, these first steps involve interaction with highly sulfated cellular surface proteoglycans and/or soluble heparin sulfate. Such extremely negatively charged compounds present in vast excess, however, produce highly incompatible conditions for standard localized HDX-MS workflows utilizing online digestion by immobilized proteases and LC-MS separation of the resulting peptides. Therefore, this contribution will primarily focus on the methodological pitfalls and challenges associated with these analyses and ways to overcome them. Specifically, previous efforts at DNA removal utilizing strong anion exchange by Sperry et al. (JASMS 2008) and protamine sulfate by Poliakov et al. (RCMS 2008) will be discussed, together with modifications necessary to enable their use in localized HDX-MS using online digestion, which ultimately enabled successful analysis of very limited amounts of viral particles in solution with vast excess of heparin necessary to trigger their conformational changes.
Novel aspect: A successful workflow for localized HDX-MS of virus samples under highly challenging conditions of vast excess of heparin.
The effect of domain order on modulation of fusion two-domain proteins structure and its dynamics studied by hydrogen-deuterium exchange
Jakub Sýs1,2, Kristýna Boušová3, Martin Hubálek2, Jiří Vondrášek3
1 Department of Biochemistry and Microbiology, University of Chemistry and Technology Prague, Technická 5, 166 28 Prague 6, Czech Republic
2 Department of Mass Spectrometry, Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science, Flemingovo náměstí 542, Prague 6, Czech Republic
3 Bioinformatics, Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science, Flemingovo náměstí 542, Prague 6, Czech Republic
Introduction: Is the function of the multidomain proteins only determined by the existence of domains that are embedded in the sequence or the position and order of the domains matters? In such protein, hypothetically, each domain structure or function could be influenced by close neighbours. To address this hypothesis, we used hydrogen‑deuterium exchange structural method (HDX-MS) to investigate possible perturbations in structure of PDZ3 domain of Zonula occludens‑1 protein (PDZ3). This domain has a natural peptide ligand with known binding site. We took PDZ3 domain and fused with TrpCage, in silico designed artificial domain, in both forward (PDZ3-TrpCage: PsLT) and reverse (TrpCage-PDZ3: TlLP) version of the sequence. Our aim was to determine peptide regions directly influenced by TrpCage position and find the differences between PsLT and TlLP proteins.
Methods: We applied HDX analysis in a traditional HDX set-up and also in set-up with low temperature and short labelling times to characterize the deuterium accessibility of PDZ3 peptides and regions. Subsequently, we performed the experiments with peptide ligand (JAMAP6),
Results: The data shows that, in general, TrpCage destabilizes the PDZ3 structure. The peptide localiation also shows that some of the PDZ3 peptides with different behaviour in PsLT compared to TlLP were localized in ligand binding site suggesting that TrpCage affects also function of PDZ3. The results of the ligand binding experiments, surprisingly, counteract the effect of TrpCage more effectively in TlLP compared to PsLT. According to our results, we suggest the methodology to study the domain structural differences in the context of other domains using HDX-MS.
Novel aspect: The domain position in the sequence of multidomain protein affects the HDX, thus suggests the influence on the domain activity.
The single amino acid substitutions in Mason-Pfizer Monkey Virus matrix protein modulate its proteolytic cleavage rate
Jakub Sýs1,2*, Markéta Častorálová2, Jan Prchal2,3, Martin Hubálek1, Tomáš Ruml2
1. Mass Spectrometry, Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science, Czech Republic
2. Department of Biochemistry and Microbiology, University of Chemistry and Technology Prague, Czech Republic
3. Laboratory of NMR Spectroscopy, University of Chemistry and Technology Prague, Czech Republic
Introduction: N-terminal domain of polyprotein Gag of Mason-Pfizer Monkey Virus (M-PMV) Matrix protein (MA) is naturally myristoylated on its N-terminus. When MA is a part of polyprotein Gag, the myristate moiety is buried inside hydrophobic pocket and exposed probably upon interaction with host cell plasma membrane. This mechanism called myristoyl switch does not occur in M-PMV as readily as it does in human immunodeficiency virus type 1 HIV-1, suggesting that it may have an important role in M-PMV maturation by regulating the MA cleavage from Gag polyprotein. To address this hypothesis, we have tracked the rate of MA cleavage from several protein constructs on artificial liposomes mimicking plasma membrane. To support the observations, we also performed the proteins structural analysis in solution.
Methods: Proteolytic digestion of myristoylated (myrMAPPHis) and nonmyristoylated MAPPHis (nonmyrMAPPHis) M-PMV protein constructs, bearing the cleavage site for M-PMV viral protease (Pr13), was performed in solution and also on liposomes with treating of proteins by Pr13. The resulted products were then visualized by SDS-PAGE and the yields of MA were determined for each protein form. Hydrogen-deuterium exchange coupled with mass‑spectrometric detection of deuterium incorporation in proteins (HDX-MS) was applied to reveal the different accessibility of protein regions of different MAPPHis protein forms in solution (liposome free). The protein labelling was conducted at 4 °C with standard protein:D2O ratio 1:9 and samples were collected in narrow time course (2-120 seconds) to tract differences in protein structural dynamics.
Preliminary data: In contrast to rapidly degraded nonmyrMAPPHis even without liposomes, the myrMAPPHis, surprisingly, become cleaved more frequently only after addition of liposomes indicating the possible exposure of myristate upon interaction with plasma membrane. To support our findings, we have designed and examined also four mutants of MAPPHis M‑PMV with single amino acid substitutions with expectation to block or, conversely, facilitate the myristoyl switch by stabilization (A79V, A79L) or destabilization (I51A, I86A) of MA M‑PMV hydrophobic pocket. The mutants A79V and A79L were cleaved even less effectively than myrMAPPHis as well as the degradation of mutants I51A and I86A was faster compared to nonmyrMAPPHis. The different cleavage rates of proteins were confirmed also on structural level by using HDX-MS method. In contrast to myrMAPPHis wild type, the HDX-MS analysis of proteins I51A and I86A shows protease cleavage site more accessible also with the indication of formation of oligomers, thus at least their partial myr-switch event already in solution. Contrarily, the mutant A79V showed the clear evidence of stabilisation of hydrophobic pocket as an effect of introduced A79V mutation. Despite the equal cleavage rate of A79L compared to A79V, the HDX-MS of A79L mutant shows considerable destabilisation of protein core, but nevertheless indicates the preservation of secondary structure at protease cleavage site, which prevents its processivity. According to these findings, the data show that the protease cleavage site has different dynamics in proteins as a result of disruption/stabilisation of hydrophobic pocket.
Novel aspect: Using HDX-MS was essential to reveal the structural aspects which determine accessibility of protease cleavage site in examined protein forms.
HDX-MS drives Lead Selection and Characterization in Pharmaceutical Research
Cornelia Wagner1, Sarah Mundigl1, Urs Hanke1, Anton Speer1 and Maximiliane König1
1 Roche Pharma Research and Early Development, Large Molecule Research, Roche Innovation Center Munich, Germany
HDX-MS (hydrogen/deuterium exchange mass spectrometry) measures the backbone amide hydrogen exchange rate which depends on solvent accessibility and hydrogen bonding and thereby allows to study protein structure, dynamics and interactions in solution under native conditions.
In pharmaceutical research HDX-MS is a powerful technology to characterize the interaction of therapeutic proteins and their corresponding targets. During drug development epitope mapping greatly supports the selection of the lead molecule with the desired properties. Moreover HDX-MS reveals conformational changes of the therapeutic protein upon antigen interaction, after mutation or stress testing (e.g. temperature).
We have established a very robust and automated workflow that makes HDX-MS our method of choice for characterizing epitopes and associated structural changes quickly and comprehensively for multiple drug candidates. We show examples how HDX-MS analysis enabled us to understand the mode of action of our lead candidates.
Monitoring of conformational changes in transmembrane proteins using HDX and FFAP radical labeling
Fojtík Lukáš1,2*, Portašiková Jasmína1,2, Pompach Petr3, Kukačka Zdeněk1, Novák Petr1 and Man Petr1
Institute of Microbiology of the CAS, Prague, Czech Republic
Faculty of Science, Charles University, Prague, Czech Republic
Institute of Biotechnology of the CAS, Prague, Czech Republic
Introduction: The chloride channel family is divided into two groups: channels and antiporters. Channels are known for their large conformational changes of subunits during ion transport. In contrast, antiporters have only tiny movements near the transport pathway. Transporters are involved in many cellular processes, and their mutation can cause serious diseases. The CLC-ec1 is a Cl–/H+ antiporter of a single proton for two chloride ions from Escherichia coli and the only one with a high-resolution structure solved by X-ray crystallography. Ion transport is supposed to be accompanied by a structural change between inward and outward-facing conformation, but this transition has not been captured so far. During the transport cycle, the protonation of key Glu residues should induce an outward-facing state. This was mimicked through a mutation of three Glu residues for Gln. We used this mutation to reveal the role of protonation in the ion transport mechanism of CLC-ec1.
Methods: CLC-ec1 and CLC-QQQ mutant were expressed in the E. coli system and purified by IMAC chromatography in ƞ-decyl maltoside as a solubilization reagent. Solubilized protein was transferred to the saposin nanodisc by size exclusion chromatography. We then used hydrogen/deuterium exchange and radical labeling with Togni reagent (FFAP) coupled to high-resolution mass spectrometry to capture the structural changes alongside the backbone as well as on the side chains of aromatic residues
Results: The optimization of the digest workflow for both methods provided full sequence coverage with reasonable lengths of the peptides. As the best conditions for the HDX we determined online digest on the column of co-immobilized pepsin-nepentesin 2 whereas for FFAP we got better sequence coverage by offline digest via cyanogen bromide and trypsin. HDX and FFAP were subsequently used for the study of the structural differences between CLC-ec1 and the CLC-QQQ mutant at pH 7.4. To prove the role of protonation, a comparison experiment at four different pH levels, from 4.4 to pH 7.4, was performed for both proteins. Results showed a critical connection between protonation and structural changes in the ion transport path and conformational change in the transmembrane helixes.
Selective protein capture under HDX-MS quench conditions for probing complex biological systems
Dietmar Hammerschmid1, Anthony Keeble2, Polina Heatley1, Mark Howarth2, Eamonn Reading1
1 Department of Chemistry, King’s College London, 7 Trinity Street, SE1 1DB London, UK
2 Department of Pharmacology, University of Cambridge, Tennis Court Road, CB2 1PD Cambridge, UK
Introduction: Deuterium labeling in hydrogen/deuterium exchange mass spectrometry (HDX-MS) is typically performed on isolated proteins neglecting the cellular complexity, which has been shown to modulate protein structure and function. To capture protein dynamics in cellular-like contexts the labeling needs to be performed on increasingly complex biological systems, including live cells. Consequently, the labeled protein requires purification under extreme HDX quenching conditions, i.e. pH 2-3 and ≤0 °C, to avoid losing structural information due to D-to-H back-exchange. Currently, there is no robust strategy for protein enrichment in such an environment, as isolation of a target protein is extremely challenging under these conditions. Here, we established a system capable of rapid and selective protein capture under HDX quench conditions for downstream MS analysis.
Methods: Using an existing protein/peptide-tag system as a template we adapted its function to enhance the reaction efficiency under HDX quench conditions (pH 2.5 and 0 °C). We first selected rational mutations on both protein and peptide and then, secondly, developed a random mutational library of the protein using phage display screening to identify enhanced variants. The peptide was cloned into the maltose binding protein (MBP), and the binding efficiency between the protein and peptide-MBP was tested, and sample and quench buffer compositions were optimized to improve the reaction rate further. As a proof of principle, we tested the final construct’s ability to capture and elute the peptide-MBP protein under a range of quench and elution conditions using a bead-capture method.
Preliminary data: Post-labeling protein purification in HDX-MS is delicate, as subsequent steps are not only limited by solution conditions (pH 2.5 and 0 °C) but also by time. Through rational mutations and phage display screening of a new, evolved protein/peptide-tag system, we advanced the binding capability between both reaction partners under these challenging conditions. The new system is fast: Significant product formation already occurs after minutes, while the original constructs do not show any noticeable binding for hours. Product formation is high: We obtained binding between the protein and the peptide-tag of 20-30% and 50-75% between 5-10 minutes at protein concentrations of 1 and 10 mM, respectively. The peptide-tag is short and non-distributive: The peptide-tag is 13 amino acids long and can be cloned into a protein of interest (POI) alongside other common purification tags, providing flexibility in terms of the target. To establish an elution strategy, we equipped the protein partner with a unique cysteine opposite the reaction center which could be chemically crosslinked to beads via a disulfide bridge. Exposing these beads to a sample containing a POI coupled with the peptide partner (peptide-POI) would allow the entire complex, i.e. protein-peptide-POI, to be formed and, importantly, eluted under reducing conditions with TCEP. This elution strategy is compatible with downstream HDX-MS analysis as protease digestion is tolerant to relatively high concentrations of TCEP, and the protein partner is small and does not hamper the analysis of the target protein. This protein purification system offers new avenues in HDX-MS, as structural information of proteins can be retrieved from complexes within large protein networks and/or directly in cells (both currently under investigation). We envision that this workflow will progress our understanding of protein dynamics in complex protein machineries and beyond.
Novel aspect: Rapid protein capture and elution under HDX-MS quench conditions to enable protein purification post HDX-labeling.
HDX-MS to study G-protein-coupled receptors in a Contract Research Organization
Thomas BOTZANOWSKI1, Jérôme CASTEL1, Ieva BROOKS2, Gilbert BEY2, Renaud MORALES1, François DEBAENE1
1 Department of Biophysics, NovAliX, 67080 Strasbourg, France.
2 Department of Structural Biology, NovAliX, 67080 Strasbourg, France.
Introduction: NovAliX is a contract research organization (CRO) of 200+ employees providing expert driven innovative outsourcing and insourcing research services for drug discovery. NovAliX offers to the pharmaceutical industry a complete range of services spanning Biophysics, Biochemistry, Structural Biology, Chemistry & Drug Discovery. In order to extend our capabilities for the characterization of G-protein-coupled receptors and more particularly to capture protein motions/dynamics upon ligand binding and/or upon G-protein complex assembly, we have recently implemented an HDX-MS platform. In the present poster, we detail an extensive optimization of the feasibility stage for HDX-MS on a model GPCR and the mapping of small molecules binding sites. Present integrative workflow and development strategies of the Mass Spectrometry Unit are also highlighted.
Methods: All HDX-MS experiments were performed on an ultraperformance liquid chromatography system (ACQUITY UPLC M-Class System, Waters, UK) coupled to an ESI-Q-TOF mass spectrometer (Xevo G2-XS, Waters, UK). The present system is composed of a classical refrigerated HDX manager, an auxiliary solvent manager and a binary solvent manager. All injections were done manually using a 50 µL loop. Protein digestion was performed online and peptide trapping and separation steps were done respectively using a Vanguard column (BEH C18, 130 Å, 1.7 μm, 2.1 mm × 5 mm; Waters) and an Acquity UPLC column (BEH C18, 130 Å, 1.7 μm, 1.0 mm × 100 mm; Waters). All digestion tests and labelling experiments were done in duplicates and triplicates, respectively.
Preliminary data: The first part of the work is dedicated to the optimization of digestion conditions of our model GPCR in order to maximize sequence coverage while minimizing the chromatographic carry-over. Many parameters were assessed such as injected quantity, reducing agent, denaturing agent and protease significantly improving the sequence coverage (from 60 to 93%) and the redundancy of the GPCR of interest. Prior labelling experiments, chromatographic carry-over was assessed using a 4 steps manual washing protocol. The second part of the work aims to investigate the dynamics of the model apoGPCR and to locate transmembrane domains based on HDX kinetics and AlphaFold model prediction. Finally, results of binding site mapping experiments for small molecules are shown and validated using dedicated softwares. G-protein / GPCR complex assembly is also an interesting topic and will be presented in case the ongoing HDX-MS experiments work.
Novel aspect: Extensive characterization of a GPCR by HDX-MS upon ligand binding and G-protein complex assembly.
Optimization of HX-MS Workflow for Membrane Protein Melibiose Transporter MelB
Yuqi Shi1; Hariharan Parameswaran2; Lan Guan2; Rosa Viner1
1 Thermo Fisher Scientific, San Jose, CA
2 Department of Cell Physiology and Molecular Biophysics, Texas Tech University Health Sciences Center, Lubbock, TX
Introduction: Membrane proteins are currently one of the most common targets for pharmaceuticals. However, due to a lack of automated methods for handling full-length membrane proteins in lipid bilayers, characterization of their structural dynamics by hydrogen/deuterium exchange mass spectrometry (HX-MS) is limited. Additionally, membrane lipids and detergents used to mimic the membrane environment and to solubilize membrane proteins can impair chromatography performance and cause ion suppression in the mass spectrometer resulting in poor sequence coverage. In this work, we optimized HX-MS workflow using a Trajan LEAP extended parallel system with filtration module for a melibiose transporter MelBst, which is a prototype of the Na+-coupled major facilitator superfamily (MFS) transporters.
Methods: MelBst and MelBst with/out ligand or Nanabody were incubated in buffered D2O and quenched by acid pH 2.3 at 0 °C at different time points, and then added to the base of a filter system containing ZrO2 for 60 s. A LEAP X-Press then compressed the filter assembly to separate proteins from phospholipids bond to ZrO2 particles and subjected to online digestion using a Nepenthesin-2 (Affipro) column. Separation and detection of peptides were achieved using an UltiMate™ 3000 RSLCnano and the Orbitrap EclipseTM MS. The HDX data were analyzed using BioPharma Finder 4.1 and HDExaminer. The differences observed between two protein states were subjected to statistical tests and significant differences were further mapped on a homology model for structural interpretation.
Preliminary Data: HX-MS workflow for membrane proteins continues suffering from low peptide recovery rate that results in poor sequence coverage. In this study, HX-MS workflow was extensively optimized, including MS parameters, quenching conditions, and protease columns. Digestion conditions were optimized from two aspects: type of protease column and digestion flow rate. Two protease columns, pepsin/XIII (1:1) dual protease column (NovaBiossay) and nepenthesin-2 protease column were assessed under same quenching and digestion conditions. Nepenthesin-2 protease column gave 33.9% sequence coverage with 121 peptides while pepsin/XIII dual protease column had slightly lower number of peptides (117) and sequence coverage (23.9%). Therefore, Nepenthesin-2 protease column was chosen for further condition optimization. Optimal digestion flow rate was selected by increasing the flow rate from 100 µl/min to 200 µl/min. The highest number of overlapping peptides and sequence coverage was obtained with 180 ul/min flow rate over 3 min. Quenching condition was optimized using two denaturing reagents, urea and guanidine HCl, in different concentrations. 4M guanidine HCl gave 46.2% sequence coverage with 140 peptides, 2M urea improved sequence coverage to 84.2% with 218 peptides, and 6M urea boost the coverage further to 91.1% with 246 overlapping peptides. In terms of MS parameter setting, to increase the number of identifications, dynamic exclusion was set to 40 seconds with single ion selection. Using optimized conditions, we achieved 96.9% sequence coverage with 346 overlapping peptides for the membrane protein MelBst. The peptide-level HX-MS experiment was performed under the optimal conditions for MelBst and MelBst-Nanobody complex. Binding site of Nb on the MelBst as well as the allosteric effects induced by Nbs binding with/without melibiose were identified. HDX results provided support to cryo-EM results and are useful for understanding the conformational changes induced by MelBst substate.
Novel Aspect: Optimization of HX-MS workflow for membrane protein using automated filtration module.
FPOP analysis of α-synuclein
D. Loginov1, P. Novak1
1. Institute of Microbiology of the Czech Academy of Sciences, BioCeV, Vestec, Czech Republic
Introduction: One of the main aspects of protein structural studies is gaining insights into process dynamics. Fast photochemical oxidation of proteins (FPOP) provides the required information through comparative analysis of the extent of modification of accessible residues in proteins during various events such as interactions, folding, and aggregation. FPOP utilizes hydroxyl radicals to label amino acid side chains exposed to solvent. Here, we applied FPOP to study α-synuclein (αS), the dominant component of Lewy bodies and a key pathological feature in synucleinopathies, including Parkinson’s disease, in both monomer and fibril forms. Accumulation of the latter plays a crucial role in the development of related diseases, making important to gain a better understanding of αS fibril formation.
Methods: The FPOP experiment was carried out in a continuous capillary flow system at 10 °C. Protein (0.5 mg/ml) was mixed with 7.5 mM H2O2, irradiated using 248 nm KrF laser (15 Hz, 20 ns pulse duration, 2.24 mJ/mm2) and quenched with 75 mM L- methionine. Excess of H2O2 was removed with catalase. Collected samples were precipitated with acetone. Pellets were dissolved in 6M guanidine chloride with acetonitrile 15% (v/v). Prior Lys-C/AspN digestion samples were diluted 3 times. Data were collected using timsTof Pro (Bruker Daltonics). Peaks X+, Data Analysis v.5.2 and custom-made Python scripts were used for data analysis. Commonly observed FPOP modifications were considered as variable modifications. Peptide FDR was set to 1%.
Preliminary data: Overall, 28 modifications on various residues were quantified, with 21 of them showing significant differences between α-synuclein and formed fibrils. As expected, a general trend of protection (less oxidation extent) of residues upon fibril formation was observed. A region corresponding to residues 66-82 has been shown to be crucial for αS misfolding and aggregation into fibrils. We detected statistically significant protection of several valine residues in this region (positions 66, 70, 74, and 77) in fibrils, although the differences were relatively small (up to 1%), which may be explained by the moderate reactivity of •OH to valine. Additionally, a 3.7% less modification extent in fibrils was observed for the peptide 59-80, where 6 different modified residues were separated only by ion mobility. According to FPOP data, the most protected residues were K34 (5.7%), K43/K45 (5.6%), and F94 (6.4%). The only residue found to be more oxidized in fibrils was E46 (1.4%), located on the exposed surface of fibrils and participating in salt bridge formation required for stabilization of fibril conformation.
Novel aspect: α-synuclein residues involved in formation of fibrils were identified using FPOP analysis.
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