Dina Schuster

All Publications

• A Prototype timsOmni Platform Enables Confident Annotation of the Key Hypervariable CDR3 Regions of IgG Immunoglobulins Using Low- and High-Energy Electron-Based Fragmentation ANALYTICAL CHEMISTRY Ollivier, S., Schuster, D., van Rijswijck, D. M. H., Stroganova, I., Smyrnakis, A., Fiala, J., Kosmopoulou, M., Suckau, D., Pengelley, S., Raether, O., Greisch, J., Papanastasiou, D., Heck, A. J. R. 2026

  Abstract

  The configuration of the first prototype timsOmni instrument, which integrates an Omnitrap linear ion trap into a timsTOF platform, is presented. A modified electrode design for the electron-based fragmentation (ExD) section of the Omnitrap is described, enhancing both the robustness and performance. Optimal characterization of antibodies requires characterizing light and heavy chains as pairs in addition to sequencing their variable domains and identifying any modifications. This is best addressed using protein-centric proteomics, as heterogeneity information such as the specific clonal origin of each identified fragment can be retained. Furthermore, by acting on intact proteins that retain part of their structure, such as disulfide bonds, it is possible to target key regions for fragmentation such as the hypervariable complementarity determining regions (CDR3) that are unique for each clone and necessary for target recognition. Therefore, as a proof of concept, we used the prototype timsOmni mass spectrometer for antibody analysis. Using solely electron-based fragmentation methods, we obtained full CDR3 sequences for paired heavy and light chains. Optimal results were achieved by performing Electron Induced Dissociation (EID) at ∼35 eV electron energy on native-like fragment antigen-binding (Fab) precursor ions. This approach yields both (a, x) and (c, z) fragment ion pairs with the potential to enhance both sequence coverage and annotation confidence. Overall, the timsOmni mass spectrometer presented here serves as an advanced and versatile platform for protein-centric proteomics.

  View details for DOI 10.1021/acs.analchem.5c07288

  View details for Web of Science ID 001734617400001

  View details for PubMedID 41945787

• Mass spectrometry-based strategies for membrane protein pharmacology. Trends in pharmacological sciences Reiter, A. H., Fehr, A., Florea, R., Floyd, B. M., Schuster, D. 2025

  Abstract

  Membrane proteins are essential for cellular physiology and the target of half of all FDA-approved drugs. However, their hydrophobicity and low abundance make large-scale expression and purification difficult, posing a challenge for drug discovery. Despite these problems, mass spectrometry (MS) has enabled workflows for higher-throughput ligand screening, simplified identification of membrane protein targets and ligandable sites, and direct analysis of drug binding in native environments. In this review, we highlight emerging MS-based strategies, adapted workflows, and novel technological advances in different MS-based fields, including affinity selection, probe-based and probe-free chemoproteomics, and native MS that collectively expand our ability to interrogate membrane proteins for drug discovery, target deconvolution, and mechanistic characterization.

  View details for DOI 10.1016/j.tips.2025.10.012

  View details for PubMedID 41271450

• Antibody-Drug Conjugate Stability Probed by Variable-Temperature Electrospray Ionization Mass Spectrometry JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY Fiala, J., Schuster, D., Heck, A. J. R. 2025

  Abstract

  Antibody-drug conjugates (ADCs) are effective anticancer biotherapeutics, often referred to as "magic bullets" due to their high specificity and cytotoxicity. This unique drug class consists of cytotoxic drugs coupled to monoclonal antibodies that target antigens on cancer cell surfaces. Different modes of drug conjugation are used to produce ADCs, whereby it has been shown that the employed linkage chemistries influence the drug load distribution as well as the stability of the product. While different methods to assess ADC stability are available, they mostly assess bulk properties and thus fail to assess stabilities at an individual stoichiometric drug-load level. Here, we demonstrate that variable-temperature electrospray ionization mass spectrometry can be used to study the heat stability of antibody-drug conjugates, resolving distinct stabilities for individual drug-loaded variants. As this stability is a key attribute of ADCs, we propose that variable-temperature electrospray ionization mass spectrometry may become an asset in the toolbox of analytical chemistry approaches to characterize ADCs in molecular fine detail.

  View details for DOI 10.1021/jasms.5c00109

  View details for Web of Science ID 001494638200001

  View details for PubMedID 40408263

• Mechanism of connexin channel inhibition by mefloquine and 2-aminoethoxydiphenyl borate PLOS ONE Lavriha, P., Han, Y., Ding, X., Schuster, D., Qi, C., Vaithia, A., Picotti, P., Korkhov, V. M. 2024; 19 (12): e0315510

  Abstract

  Gap junction intercellular communication (GJIC) between two adjacent cells involves direct exchange of cytosolic ions and small molecules via connexin gap junction channels (GJCs). Connexin GJCs have emerged as drug targets, with small molecule connexin inhibitors considered a viable therapeutic strategy in several diseases. The molecular mechanisms of GJC inhibition by known small molecule connexin inhibitors remain unknown, preventing the development of more potent and connexin-specific therapeutics. Here we show that two GJC inhibitors, mefloquine (MFQ) and 2-aminoethoxydiphenyl borate (2APB) bind to Cx32 and block dye permeation across Cx32 hemichannels (HCs) and GJCs. Cryo-EM analysis shows that 2APB binds to "site A", close to the N-terminal gating helix of Cx32 GJC, restricting the entrance to the channel pore. In contrast, MFQ binds to a distinct "site M", deeply buried within the pore. MFQ binding to this site modifies the electrostatic properties of Cx32 pore. Mutagenesis of V37, a key residue located in the site M, renders Cx32 HCs and GJCs insensitive to MFQ-mediated inhibition. Moreover, our cryo-EM analysis, mutagenesis and activity assays show that MFQ targets the M site in Cx43 GJC similarly to Cx32. Taken together, our results point to a conserved inhibitor binding site in connexin channels, opening a new route for development of specific drugs targeting connexins.

  View details for DOI 10.1371/journal.pone.0315510

  View details for Web of Science ID 001397799000129

  View details for PubMedID 39739741

  View details for PubMedCentralID PMC11687724

• Comprehensive Overview of Bottom-Up Proteomics Using Mass Spectrometry ACS MEASUREMENT SCIENCE AU Jiang, Y., Rex, D., Schuster, D., Neely, B. A., Rosano, G. L., Volkmar, N., Momenzadeh, A., Peters-Clarke, T. M., Egbert, S. B., Kreimer, S., Doud, E. H., Crook, O. M., Yadav, A., Vanuopadath, M., Hegeman, A. D., Mayta, M. L., Duboff, A. G., Riley, N. M., Moritz, R. L., Meyer, J. G. 2024

  View details for DOI 10.1021/acsmeasuresciau.3c00068

  View details for Web of Science ID 001239439700001

• Systematic identification of structure-specific protein-protein interactions MOLECULAR SYSTEMS BIOLOGY Holfeld, A., Schuster, D., Sesterhenn, F., Gillingham, A. K., Stalder, P., Haenseler, W., Barrio-Hernandez, I., Ghosh, D., Vowles, J., Cowley, S. A., Nagel, L., Khanppnavar, B., Serdiuk, T., Beltrao, P., Korkhov, V. M., Munro, S., Riek, R., de Souza, N., Picotti, P. 2024; 20 (6): 651-675

  Abstract

  The physical interactome of a protein can be altered upon perturbation, modulating cell physiology and contributing to disease. Identifying interactome differences of normal and disease states of proteins could help understand disease mechanisms, but current methods do not pinpoint structure-specific PPIs and interaction interfaces proteome-wide. We used limited proteolysis-mass spectrometry (LiP-MS) to screen for structure-specific PPIs by probing for protease susceptibility changes of proteins in cellular extracts upon treatment with specific structural states of a protein. We first demonstrated that LiP-MS detects well-characterized PPIs, including antibody-target protein interactions and interactions with membrane proteins, and that it pinpoints interfaces, including epitopes. We then applied the approach to study conformation-specific interactors of the Parkinson's disease hallmark protein alpha-synuclein (aSyn). We identified known interactors of aSyn monomer and amyloid fibrils and provide a resource of novel putative conformation-specific aSyn interactors for validation in further studies. We also used our approach on GDP- and GTP-bound forms of two Rab GTPases, showing detection of differential candidate interactors of conformationally similar proteins. This approach is applicable to screen for structure-specific interactomes of any protein, including posttranslationally modified and unmodified, or metabolite-bound and unbound protein states.

  View details for DOI 10.1038/s44320-024-00037-6

  View details for Web of Science ID 001220340600001

  View details for PubMedID 38702390

  View details for PubMedCentralID PMC11148107

• Protein-Centric Analysis of Personalized Antibody Repertoires Using LC-MS-Based Fab-Profiling on a timsTOF JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY Fiala, J., Schuster, D., Ollivier, S., Pengelley, S., Lubeck, M., Busch, F., Jankevics, A., Raether, O., Greisch, J., Heck, A. J. R. 2024; 35 (6): 1292-1300

  Abstract

  Endogenous antibodies, or immunoglobulins (Igs), abundantly present in body fluids, represent some of the most challenging samples to analyze, largely due to the immense variability in their sequences and concentrations. It has been estimated that our body can produce billions of different Ig proteins with different isotypes, making their individual analysis seemingly impossible. However, recent advances in protein-centric proteomics using LC-MS coupled to Orbitrap mass analyzers to profile intact Fab fragments formed by selective cleavage at the IgG-hinge revealed that IgG repertoires may be less diverse, albeit unique for each donor. Serum repertoires seem to be dominated by a few hundred clones that cumulatively make up 50-95% of the total IgG content. Enabling such analyses required careful optimization of the chromatography and mass analysis, as all Fab analytes are highly alike in mass (46-51 kDa) and sequence. To extend the opportunities of this mass-spectrometry-based profiling of antibody repertoires, we here report the optimization and evaluation of an alternative MS platform, namely, the timsTOF, for antibody repertoire profiling. The timsTOF mass analyzer has gained traction in recent years for peptide-centric proteomics and found wide applicability in plasma proteomics, affinity proteomics, and HLA peptidomics, to name a few. However, for protein-centric analysis, this platform has been less explored. Here, we demonstrate that the timsTOF platform can be adapted to perform protein-centric LC-MS-based profiling of antibody repertoires. In a side-by-side comparison of the timsTOF and the Orbitrap we demonstrate that the extracted serum antibody repertoires are alike qualitatively and quantitatively, whereby in particular the sensitivity of the timsTOF platform excels. Future incorporation of advanced top-down capabilities on the timsTOF may make this platform a very valuable alternative for protein-centric proteomics and top-down proteomics and thus also for personalized antibody repertoire profiling.

  View details for DOI 10.1021/jasms.4c00076

  View details for Web of Science ID 001242176100001

  View details for PubMedID 38662593

  View details for PubMedCentralID PMC11157643

• Structure of the connexin-43 gap junction channel in a putative closed state ELIFE Qi, C., Acosta Gutierrez, S., Lavriha, P., Othman, A., Lopez-Pigozzi, D., Bayraktar, E., Schuster, D., Picotti, P., Zamboni, N., Bortolozzi, M., Luigi Gervasio, F., Korkhov, V. M. 2023; 12

  Abstract

  Gap junction channels (GJCs) mediate intercellular communication by connecting two neighbouring cells and enabling direct exchange of ions and small molecules. Cell coupling via connexin-43 (Cx43) GJCs is important in a wide range of cellular processes in health and disease (Churko and Laird, 2013; Liang et al., 2020; Poelzing and Rosenbaum, 2004), yet the structural basis of Cx43 function and regulation has not been determined until now. Here, we describe the structure of a human Cx43 GJC solved by cryo-EM and single particle analysis at 2.26 Å resolution. The pore region of Cx43 GJC features several lipid-like densities per Cx43 monomer, located close to a putative lateral access site at the monomer boundary. We found a previously undescribed conformation on the cytosolic side of the pore, formed by the N-terminal domain and the transmembrane helix 2 of Cx43 and stabilized by a small molecule. Structures of the Cx43 GJC and hemichannels (HCs) in nanodiscs reveal a similar gate arrangement. The features of the Cx43 GJC and HC cryo-EM maps and the channel properties revealed by molecular dynamics simulations suggest that the captured states of Cx43 are consistent with a closed state.

  View details for DOI 10.7554/eLife.87616

  View details for Web of Science ID 001070995100001

  View details for PubMedID 37535063

  View details for PubMedCentralID PMC10400079

• Structures of wild-type and selected CMT1X mutant connexin 32 gap junction channels and hemichannels SCIENCE ADVANCES Qi, C., Lavriha, P., Bayraktar, E., Vaithia, A., Schuster, D., Pannella, M., Sala, V., Picotti, P., Bortolozzi, M., Korkhov, V. M. 2023; 9 (35): eadh4890

  Abstract

  In myelinating Schwann cells, connection between myelin layers is mediated by gap junction channels (GJCs) formed by docked connexin 32 (Cx32) hemichannels (HCs). Mutations in Cx32 cause the X-linked Charcot-Marie-Tooth disease (CMT1X), a degenerative neuropathy without a cure. A molecular link between Cx32 dysfunction and CMT1X pathogenesis is still missing. Here, we describe the high-resolution cryo-electron cryo-myography (cryo-EM) structures of the Cx32 GJC and HC, along with two CMT1X-linked mutants, W3S and R22G. While the structures of wild-type and mutant GJCs are virtually identical, the HCs show a major difference: In the W3S and R22G mutant HCs, the amino-terminal gating helix partially occludes the pore, consistent with a diminished HC activity. Our results suggest that HC dysfunction may be involved in the pathogenesis of CMT1X.

  View details for DOI 10.1126/sciadv.adh4890

  View details for Web of Science ID 001168611600004

  View details for PubMedID 37647412

  View details for PubMedCentralID PMC10468125

• Multiomics-empowered Deep Phenotyping of Ulcerative Colitis Identifies Biomarker Signatures Reporting Functional Remission States JOURNAL OF CROHNS & COLITIS Janker, L., Schuster, D., Bortel, P., Hagn, G., Meier-Menches, S. M., Mohr, T., Mader, J. C., Slany, A., Bileck, A., Brunmair, J., Madl, C., Unger, L., Hennlich, B., Weitmayr, B., Del Favero, G., Pils, D., Pukrop, T., Pfisterer, N., Feichtenschlager, T., Gerner, C. 2023; 17 (9): 1514-1527

  Abstract

  Ulcerative colitis [UC] is a chronic disease with rising incidence and unclear aetiology. Deep molecular phenotyping by multiomics analyses may provide novel insights into disease processes and characteristic features of remission states.UC pathomechanisms were assessed by proteome profiling of human tissue specimens, obtained from five distinct colon locations for each of the 12 patients included in the study. Systemic disease-associated alterations were evaluated thanks to a cross-sectional setting of mass spectrometry-based multiomics analyses comprising proteins, metabolites, and eicosanoids of plasma obtained from UC patients during acute episodes and upon remission, in comparison with healthy controls.Tissue proteome profiling indicated colitis-associated activation of neutrophils, macrophages, B and T cells, fibroblasts, endothelial cells and platelets, and hypoxic stress, and suggested a general downregulation of mitochondrial proteins accompanying the establishment of apparent wound healing-promoting activities including scar formation. Whereas pro-inflammatory proteins were apparently upregulated by immune cells, the colitis-associated epithelial cells, fibroblasts, endothelial cells, and platelets seemed to predominantly contribute anti-inflammatory and wound healing-promoting proteins. Blood plasma proteomics indicated chronic inflammation and platelet activation, whereas plasma metabolomics identified disease-associated deregulations of gut and gut microbiome-derived metabolites. Upon remission several, but not all, molecular candidate biomarker levels recovered back to normal.The findings may indicate that microvascular damage and platelet deregulation hardly resolve upon remission, but apparently persist as disease-associated molecular signatures. This study presents local and systemic molecular alterations integrated in a model for UC pathomechanisms, potentially supporting the assessment of disease and remission states in UC patients.

  View details for DOI 10.1093/ecco-jcc/jjad052

  View details for Web of Science ID 001002042100001

  View details for PubMedID 36961872

  View details for PubMedCentralID PMC10588787

• Structural basis of calmodulin modulation of the rod cyclic nucleotide-gated channel PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Barret, D. C. A., Schuster, D., Rodrigues, M. J., Leitner, A., Picotti, P., Schertler, G. F. X., Kaupp, U., Korkhov, V. M., Marino, J. 2023; 120 (15): e2300309120

  Abstract

  Calmodulin (CaM) regulates many ion channels to control calcium entry into cells, and mutations that alter this interaction are linked to fatal diseases. The structural basis of CaM regulation remains largely unexplored. In retinal photoreceptors, CaM binds to the CNGB subunit of cyclic nucleotide-gated (CNG) channels and, thereby, adjusts the channel's Cyclic guanosine monophosphate (cGMP) sensitivity in response to changes in ambient light conditions. Here, we provide the structural characterization for CaM regulation of a CNG channel by using a combination of single-particle cryo-electron microscopy and structural proteomics. CaM connects the CNGA and CNGB subunits, resulting in structural changes both in the cytosolic and transmembrane regions of the channel. Cross-linking and limited proteolysis-coupled mass spectrometry mapped the conformational changes induced by CaM in vitro and in the native membrane. We propose that CaM is a constitutive subunit of the rod channel to ensure high sensitivity in dim light. Our mass spectrometry-based approach is generally relevant for studying the effect of CaM on ion channels in tissues of medical interest, where only minute quantities are available.

  View details for DOI 10.1073/pnas.2300309120

  View details for Web of Science ID 001038886600004

  View details for PubMedID 37011209

  View details for PubMedCentralID PMC10104587

• Structure of<i> Mycobacterium tuberculosis</i> Cya, an evolutionary ancestor of the mammalian membrane adenylyl cyclases ELIFE Mehta, V., Khanppnavar, B., Schuster, D., Kantarci, I., Vercellino, I., Kosturanova, A., Iype, T., Stefanic, S., Picotti, P., Korkhov, V. M. 2022; 11

  Abstract

  Mycobacterium tuberculosis adenylyl cyclase (AC) Rv1625c/Cya is an evolutionary ancestor of the mammalian membrane ACs and a model system for studies of their structure and function. Although the vital role of ACs in cellular signalling is well established, the function of their transmembrane (TM) regions remains unknown. Here, we describe the cryo-EM structure of Cya bound to a stabilizing nanobody at 3.6 Å resolution. The TM helices 1-5 form a structurally conserved domain that facilitates the assembly of the helical and catalytic domains. The TM region contains discrete pockets accessible from the extracellular and cytosolic side of the membrane. Neutralization of the negatively charged extracellular pocket Ex1 destabilizes the cytosolic helical domain and reduces the catalytic activity of the enzyme. The TM domain acts as a functional component of Cya, guiding the assembly of the catalytic domain and providing the means for direct regulation of catalytic activity in response to extracellular ligands.

  View details for DOI 10.7554/eLife.77032

  View details for Web of Science ID 000849306700001

  View details for PubMedID 35980026

  View details for PubMedCentralID PMC9433096

• protti: an R package for comprehensive data analysis of peptide- and protein-centric bottom-up proteomics data BIOINFORMATICS ADVANCES Quast, J., Schuster, D., Picotti, P. 2022; 2 (1): vbab041

  Abstract

  We present a flexible, user-friendly R package called protti for comprehensive quality control, analysis and interpretation of quantitative bottom-up proteomics data. protti supports the analysis of protein-centric data such as those associated with protein expression analyses, as well as peptide-centric data such as those resulting from limited proteolysis-coupled mass spectrometry analysis. Due to its flexible design, it supports analysis of label-free, data-dependent, data-independent and targeted proteomics datasets. protti can be run on the output of any search engine and software package commonly used for bottom-up proteomics experiments such as Spectronaut, Skyline, MaxQuant or Proteome Discoverer, adequately exported to table format.protti is implemented as an open-source R package. Release versions are available via CRAN (https://CRAN.R-project.org/package=protti) and work on all major operating systems. The development version is maintained on GitHub (https://github.com/jpquast/protti). Full documentation including examples is provided in the form of vignettes on our package website (jpquast.github.io/protti/).

  View details for DOI 10.1093/bioadv/vbab041

  View details for Web of Science ID 001153137500080

  View details for PubMedID 36699412

  View details for PubMedCentralID PMC9710675

• Child murder in the Early Bronze Age: proteomic sex identification of a cold case from Schleinbach, Austria ARCHAEOLOGICAL AND ANTHROPOLOGICAL SCIENCES Rebay-Salisbury, K., Janker, L., Pany-Kucera, D., Schuster, D., Spannagl-Steiner, M., Waltenberger, L., Salisbury, R. B., Kanz, F. 2020; 12 (11): 265

  Abstract

  The identification of sex-specific peptides in human tooth enamel by nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) represents a quantum leap for the study of childhood and social relations more generally. Determining sex-related differences in prehistoric child rearing and mortality has been hampered by the insufficient accuracy in determining the biological sex of juveniles. We conducted mass spectrometric analysis to identify sex-specific peptides in the dental enamel of a child from a settlement pit of the Early Bronze Age settlement of Schleinbach, Austria (c. 1950-1850 bc). Four perimortal impression fractures on the skull of a 5-6-year-old child indicate an intentional killing, with a co-buried loom weight as possible murder weapon. Proteomic analysis, conducted for the first time on prehistoric teeth in Austria, determined the child's sex as male. While we cannot conclusively determine whether the child was the victim of conflicts between village groups or was slain by members of his own community, we suggest that contextual evidence points to the latter. A possible trigger of violence was the follow-on effects of an uncontrolled middle ear infection revealed by an osteological analysis. The boy from Schleinbach highlights the potential for further investigation of gender-biased violence, infanticide and child murder based on the recently developed method of proteomic sex identification.

  View details for DOI 10.1007/s12520-020-01199-8

  View details for Web of Science ID 000587315100001

  View details for PubMedID 33123298

  View details for PubMedCentralID PMC7584537