Marcus Bantscheff

Mass spectrometric proteome analyses of synovial fluids and plasmas from patients suffering from rheumatoid arthritis and comparison to reactive arthritis or osteoarthritis

Differential proteome analysis is used to study body fluids from patients suffering from rheumatoid arthritis (RA), reactive arthritis (reaA) or osteoarthritis (OA). Mass spectrometric structure characterization of gel‐separated proteins provided a detailed view of the protein‐processing events that lead to distinct protein species present in the respective body fluids. (i) Fibrin(ogen) β‐chain degradation products, presumably plasmin‐derived, appeared solely in synovial fluids (SF) from both patient collectives, (ii) calgranulin B (MRP14) was exclusively identified in SF samples derived from 5 out of 6 patients suffering from RA. Calgranulin B was not observed in synovial fluids from OA patients, nor in plasmas from either patient group. In all cases where calgranulin B was detected, calgranulin C was identified as well. (iii) Serum amyloid A protein spots were determined in plasmas and synovial fluids from patients with RA, but not in patients with OA. In addition to disease‐relevant differences, interindividual differences in haptoglobin patterns of the patients under investigation were observed. Hence, in‐depth proteome analysis of body fluids has proven effective for identification of multiple molecular markers and determination of associated protein structure modifications, that are thought to play a role for specifically determining a defined pathological state of diseased joints.

Mass Spectrometry-Based Proteomics in Preclinical Drug Discovery

Preclinical stages in the drug discovery process require a multitude of biochemical and genetic assays in order to characterize the effects of drug candidates on cellular systems and model organisms. Early attempts to apply unbiased proteomic techniques to the identification of protein targets and off-targets as well as to elucidate the mode of action of candidate drug molecules suffered from a striking discrepancy between scientific expectations and what the technology was able to deliver at the time. Dramatic technological improvements in mass spectrometry-based proteomic and chemoproteomic strategies have radically changed this situation. This review, therefore, highlights proteomic approaches suitable for preclinical drug discovery illustrated by recent success stories.

Proteome analysis of diseased joints from mice suffering from collagen-induced arthritis.

Abstract Strains of mice that are susceptible to autoimmunity have provided experimental models to analyze the molecular basis for the complex multifactorial inheritance of human autoimmune disease. In this study proteins associated with collagen-induced arthritis (CIA) in mice were experimentally identified using a global proteomics approach. Two-dimensional gels of proteins from inflamed and non-inflamed joints showed a distinguished protein profile visualizing about 530 Coomassie-stained protein spots in the pH 4–7 range. A total of 76 spots were identified by peptide mass fingerprinting with good confidence. They included proteins of cytoskeletal origin, chaperones, enzymes and also some signal transduction molecules. Comparison to gels from non-inflamed paws pointed to proteins that were differentially expressed between the control and diseased state. Ferritin light chain and antioxidant protein 2 were slightly more abundant, lymphoid enhancer binding factor 1 slightly, but significantly, less abundant in inflamed paws. Fourteen of the identified proteins were the products of genes that had increased transcript levels in the diseased state. However, on the protein level no significant differences were found in comparison to the controls. This study provides us with the framework for more detailed approaches to understanding the complex disease arthritis that go beyond global proteomics.

Quantitative mass spectrometry in proteomics

Investigating living systems at the protein level is continuing to provide important insights into many biological processes across all kingdoms of life. Mass spectrometry based proteomics has fundamentally changed the way in which biological systems are interrogated because of its ability to measure thousands of proteins and post-translational modifications in parallel. This enables investigations at all levels of biological complexity ranging from protein complexes to human patient populations. While five years ago, the majority of proteomic experiments mostly enumerated the protein constituents of a biological system, quantitative measurements are at the heart of practically every proteomic study today. This shift was once more significantly driven by developments in mass spectrometry which is now the de-facto standard for quantitative measurements in proteomics. Alongside, the miniaturization of associated sample preparation and separation techniques as well as the adoption and further development of data analysis strategies from other –omics research have helped transform proteomics from a qualitative to a quantitative science. At the same time, the areas of life science research to which proteomic methods are being applied are expanding rapidly. While a few years ago, most of the successful proteomic work was performed in rather simple models such as unicellular organisms or cell lines derived of animal and plant origin, it is becoming increasingly possible to work with primary samples including that of human patients. However, scientists engaged in such studies had to realize that the enormous molecular complexity and the dynamic nature of proteomes posed much larger challenges than those encountered for either genome or transcriptome studies. In particular, issues related to splice variants, post-translational modifications, dynamic range of protein expression spanning 10 orders of magnitude, protein stability, transient protein associations, and dependence on cell type or physiological state etc. are still limiting our technical ability to characterize proteomes comprehensively and reproducibly in a reasonable time. Unsurprisingly, numerous experimental strategies and schemes have been devised to address the many challenges and, as in any developing field, some have come and gone, while others have been adopted more broadly. It is probably safe to assume that we have not yet seen the end of the technical developments which, in turn, will allow more and more difficult biological systems to be approached by proteomics. Despite all the hurdles, mass spectrometry based quantitative proteomics increasingly impacts life science research in many areas including protein expression profiling, the analysis of signaling pathways or the development of protein biomarker assay systems to name a few. It is important to note that in each area, distinct scientific questions are being asked and, therefore, distinct proteomic approaches will have to be applied, many of which vary widely in their versatility, technical maturity, difficulty, and expense. Consequently, we must recognize that some scientific questions are, and will remain, much harder to answer by proteomics than others. Still, the strong trend towards the alternative scientific paradigm of system-level interrogation of biological phenomena is unstoppable and will without doubt continue to provide important complementary views to traditional and still highly successful hypothesis-driven research. In this special issue on quantitative mass spectrometry in proteomics, we have assembled a group of reputed scientists who all have significant statements to make regarding the topic of this issue. There are eight reviews that provide an in-depth appreciation of a state-of-the-art technology alongside six original research articles highlighting some of the current trends. We can of course not claim to cover the field comprehensively, but do believe that the content of this special issue has a lot to Published in the topical issue Quantitative Mass Spectrometry in Proteomics with guest editors Bernhard Kuster and Marcus Bantscheff.

Dimerization of signalling modules of the EvgAS and BvgAS phosphorelay systems

Biophysical and biochemical properties of signalling proteins or domains derived from the unorthodox EvgAS and BvgAS two-component phosphorelay systems of Escherichia coli and Bordetella pertussis were investigated. Oligomerization of the effector proteins EvgA and BvgA and of truncated EvgS and BvgS derived signalling proteins containing the receiver and histidine containing phosphotransfer (HPt) domains or comprising only the HPt domains were characterized by native gel electrophoresis, gel permeation experiments and analytical ultracentrifugation. The results obtained by the different methods are consistent with non-phosphorylated EvgA and BvgA proteins being dimers in solution with a dissociation constant significantly below 1 microM. In contrast, all sensor derived domains of EvgS and BvgS were observed to be monomers in vitro. No indications for a phosphorylation induced stimulation of oligomerization of the C-terminal histidine kinase domains could be detected. In agreement with these data, surface plasmon resonance studies revealed a 2:1 stoichiometry in the interaction of EvgA with the immobilized EvgS HPt domain and an affinity constant of 1. 24x10(6) M(-1).

Mass spectrometry approaches to monitor protein–drug interactions

Recent advances in mass spectrometry-based approaches have enabled the investigation of drug-protein interactions in various ways including the direct detection of drug-target complexes, the examination of drug-induced changes in the target protein structure, and the monitoring of enzymatic target activity. Mass spectrometry-based proteomics methods also permit the unbiased analysis of changes in protein abundance and post-translational modifications induced by drug action. Finally, chemoproteomic affinity enrichment studies enable the deconvolution of drug targets under close to physiological conditions. This review provides an overview of current methods for the characterization of drug-target interactions by mass spectrometry and describes a protocol for chemoproteomic target binding studies using immobilized bioactive molecules.

A Modular Probe Strategy for Drug Localization, Target Identification and Target Occupancy Measurement on Single Cell Level.

Late stage failures of candidate drug molecules are frequently caused by off-target effects or inefficient target engagement in vivo. In order to address these fundamental challenges in drug discovery, we developed a modular probe strategy based on bioorthogonal chemistry that enables the attachment of multiple reporters to the same probe in cell extracts and live cells. In a systematic evaluation, we identified the inverse electron demand Diels-Alder reaction between trans-cyclooctene labeled probe molecules and tetrazine-tagged reporters to be the most efficient bioorthogonal reaction for this strategy. Bioorthogonal biotinylation of the probe allows the identification of drug targets in a chemoproteomics competition binding assay using quantitative mass spectrometry. Attachment of a fluorescent reporter enables monitoring of spatial localization of probes as well as drug-target colocalization studies. Finally, direct target occupancy of unlabeled drugs can be determined at single cell resolution by competitive binding with fluorescently labeled probe molecules. The feasibility of the modular probe strategy is demonstrated with noncovalent PARP inhibitors.

Probing the tertiary structure of multidomain proteins by limited proteolysis and mass spectrometry

Limited proteolysis of the multidomain nitrogen regulatory protein C (NtrC) with thermolysin revealed well separated fragments using high-performance liquid chromatography (HPLC). Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and electrospray ionization mass spectrometry (ESI-MS) molecular weight determinations from the fragment mixtures showed that the cleavage products resembled the N-terminal receiver domain (R; amino acids (aa) 1–130), the still covalently linked output-and C-terminal domains (OC; aa 133–469), the C-terminal domain (C; aa 397–469), a core-fragment of the O-domain (O*), and intact NtrC. Borders of the separated domains were identified by mass spectrometric peptide mapping after on-target proteolysis of the HPLC-separated fragments. The flexible and, hence, accessible linker region between the R- and the O-domains of NtrC was shown to comprise the amino acids Val-131 and Gln-132. Thermolysin split the OC-fragment into the O- and the C-domains at accessible amino acid residues ranging from Thr-389 to Gln-396 identifying this partial sequence as a second hitherto unknown linker in NtrC. Individually expressed NtrCR, the R-domain of NtrC, afforded structure-dependent proteolytic fragments on tryptic digestion in solution. Mass spectrometric peptide mapping analyses determined the locations of cleavages in NtrCR in the A4-helix and the B4-β-sheet/loop region, providing information on surface-exposed partial structures of the R-domain. The combination of limited proteolysis with micro-preparative techniques and mass spectrometry provides an efficient tool for the rapid identification of protein tertiary structural features, affording lead information necessary for protein design and engineering and for structure/function studies.

Interrogating the Druggability of the 2-Oxoglutarate-Dependent Dioxygenase Target Class by Chemical Proteomics

The 2-oxoglutarate-dependent dioxygenase target class comprises around 60 enzymes including several subfamilies with relevance to human disease, such as the prolyl hydroxylases and the Jumonji-type lysine demethylases. Current drug discovery approaches are largely based on small molecule inhibitors targeting the iron/2-oxoglutarate cofactor binding site. We have devised a chemoproteomics approach based on a combination of unselective active-site ligands tethered to beads, enabling affinity capturing of around 40 different dioxygenase enzymes from human cells. Mass-spectrometry-based quantification of bead-bound enzymes using a free-ligand competition-binding format enabled the comprehensive determination of affinities for the cosubstrate 2-oxoglutarate and for oncometabolites such as 2-hydroxyglutarate. We also profiled a set of representative drug-like inhibitor compounds. The results indicate that intracellular competition by endogenous cofactors and high active site similarity present substantial challenges for drug discovery for this target class.

Identification of Plasmodium PI4 kinase as target of MMV390048 by chemoproteomics

Most antimalarial drugs face decreased efficacy due to the emergence of resistant parasites. Therefore, the discovery of new antimalarial medicines is focused on new drugs that act by novel mechanisms and are active against different P. falciparum development stages. Screening of a focused compound library for antiparasitic activity, lead to identification of a novel class of compounds with activity against P. falciparum, 2-aminopyridines. The selected hits were validated and subjected to a lead optimization program resulting in the pre-clinical candidate MMV390048. Here we report an unbiased chemoproteomics strategy for the identification of targets of MMV390048. An analogue of MMV390048 containing a primary amine function for immobilization in a permissive position was synthesized and covalently immobilized on sepharose beads. Affinity capturing of potential target proteins from a P. falciparum blood stage extract was performed in the absence and presence of an excess of MMV390048 in the extract to delineate target proteins for which capturing is competitively inhibited. All proteins captured by the beads were quantified by isotope tagging of tryptic peptides followed by LC-MS/MS. Notably MMV390048 competitively inhibited the binding of only a single protein, P. falciparum PI4 kinase, to the beads. However, the immobilization of a drug compound via a linker may not be compatible with the binding to all of its targets. Therefore, we also performed a capturing experiment with kinobeads, which represent a combination of immobilized promiscuous ATP competitive kinase inhibitors (Bantscheff et al., 2007). As in the previous experiment, PfPI4K was the only P. falciparum protein which exhibited a reduction of bead binding upon the addition of MMV390048 to the extract. Knowledge of the target will accelerate the drug discovery process.