Synapses are intercellular junctions between neurons in the nervous system. They are organised into a presynaptic terminal, the synaptic cleft and the postsynaptic density. Information that arrives in form of an action potential at the presynaptic terminal is transmitted to the postsynaptic neuron via chemical neurotransmitters, which are stored in synaptic vesicles (SVs) of the presynaptic cytoplasm.

SVs are small storage organelles located at the presynaptic cytoplasm of every neuron. They contain neurotransmitters such as glutamate or γ-aminobutyric acid and pass through a trafficking cycle in the nerve terminal. The SV cycle includes import of neurotransmitters into the vesicles, docking, priming and fusion with the presynaptic membrane to release neurotransmitters as well as endocytosis and recycling of the vesicles.
Whereas the essential proteins governing the SV cycle have been identified in the last decades, we still have only little knowledge about their exact operation and sequence in which they interact to carry out the individual steps of the SV cycle. Like other trafficking mechanisms, the vesicle cycle appears to be governed by non-covalent protein-protein and protein-lipid interactions that are assembled on demand and dissociate again when the task is completed. The resulting supra-molecular complexes involve interactions between soluble proteins and membrane components with as yet undefined stoichiometries, making it very difficult to identify their protein-protein interactions. In particular, membrane fusion involves not only interactions between membrane proteins, but also relies on formation of microdomains in which proteins and certain lipids are clustered and whose composition is largely unclear. Indeed, the inability to define these membrane protein assemblies and to study their properties constitutes a major problem preventing a complete molecular understanding of exocytosis and recycling.

So far, knowledge about protein complexes operating on the synaptic membranes is based on the study of isolated proteins (usually binary or ternary interactions) and pull-downs or immunoprecipitations in detergents that disrupt the native membrane environment. We develop new mass spectrometric techniques to address these protein assemblies. The analysis of SVs will serve as a starting point for a more comprehensive analysis of synaptic protein complexes at the presynaptic side. This will include for instance the still enigmatic architecture of docked synaptic vesicles; to date it is not known where exactly SVs bind the presynaptic membrane, which proteins interact or what protein stoichiometries the involved complexes have.

Medical relevance for neurodegenerative disease
SVs are unique in that they must be faithfully regenerated during each exo-/endocytotic cycle, requiring control mechanisms ensuring their complete and correct assembly which could be mediated by loose but specific “superclusters”. The investigation of these clusters and their interactions in the synapse will prove highly important to fully understand the mechanism of signal transduction in neurons. Only this knowledge can pave the way for new therapeutic interventions addressing neurodegenerative disorders and diseases. We hope to shed light on important protein-protein and protein-lipid interactions in neuronal synapses and thus extend our knowledge about the mechanisms of signal transduction in nerve cells allowing generation of functional models to provide therapeutic targets for clinical research.
The relevance of the identified protein-lipid interactions for neurodegenerative disease will therefore be investigated. For this, synaptic vesicles and synapses purified from healthy and diseased tissue will be compared yielding differences in their protein-protein and protein-lipid interactions. Another important aspect is the study of proteins that cause neurodegeneration such as α-synuclein. Misfolding and subsequent aggregation often play an important role in neurodegenerative disease. The interactions in misfolded and aggregated states will therefore be studied in detail to provide novel insights and contribute to the understanding of their interactions in the synapse which in most cases causes degeneration.

Structural mass spectrometry
We combine various cross-linking strategies with proteomics, lipidomics and mass spectrometry of intact protein complexes (“native” MS) to study protein-lipid assemblies in SVs and neuronal synapses. While proteomics and lipidomics are employed to identify the components of the protein-lipid-assemblies, structural mass spectrometric techniques deliver novel insights into protein-lipid-interactions. Native MS is the analysis of intact proteins and protein complexes in the gas phase of a mass spectrometer and reveals their composition, stoichiometry, topology, heterogeneity, subunit interactions as well as ligand binding. It covers a wide m/z range and allows the analysis of small protein subunits up to large protein assemblies in the same measurement. This is beneficial for heterogeneous protein assemblies such as large protein assemblies in SVs. Native MS will be complemented with various (chemical) cross-linking strategies to gain further insights into direct contact sites in the assemblies.

Q Exactive Plus hybrid quadrupole-orbitrap mass spectrometer

We use the Q Exactive Plus for protein and lipid identification as well as advanced studies including cross-linking or PTM analysis. The Q Exactive Plus is coupled with a Dionex Ultimate LC-system or can be operated in direct infusion mode.

Q-ToF Ultima mass spectrometer

modified for transmission of high mass complexes. We use the Q-ToF Ultima for analysis of intact protein-ligand complexes to identify their stoichiometries and protein interaction modules. We can further obtain information on ligand binding and protein complex stability.

Q-ToF Synapt G1

modified for transmission of high mass complexes. The built-in ion mobility cell offers additional possibilities, for instance to study conformation and stability of protein-ligand complexes.

LTQ Orbitrap XL hybrid ion trap mass spectrometer

The LTQ Orbitrap XL is primarily used for lipidomics in our lab. It enables MSⁿ-based identification of lipids and features two fragmentation techniques. Equipped with a Waters nanoAcquity UPLC system complex lipidomes are characterised.

* contributed equally, # co-corresponding

Frick M, Schwieger C, Schmidt C (2021)
Liposomes as carriers of membrane-associated proteins and peptides for mass spectrometric analysis.
Angew Chem Int Ed Engl.
doi: 10.1002/anie.202101242

Wittig S, Ganzella M, Barth M, Kostmann S, Riedel D, Pérez-Lara Á, Jahn R & Schmidt C (2021)
Cross-linking mass spectrometry uncovers protein interactions and functional assemblies in synaptic vesicle membranes. Nat Commun 12, 858
doi: 10.1038/s41467-021-21102-w

Melissa Frick, Carla Schmidt (2019)
Mass spectrometry-A versatile tool for characterising the lipid environment of membrane protein assemblies.
Chem Phys Lipids. 221:145-157.
doi: 10.1016/j.chemphyslip.2019.04.001

Bender J, Schmidt C (2019)
Mass spectrometry of membrane complexes. Biol Chem. 400(7):813-829. doi: 10.1515/hsz-2018-0443.

Wittig S, Haupt C, Hoffmann W, Kostmann S, Pagel K, Schmidt C (2019)
Oligomerisation of Synaptobrevin-2 Studied by Native Mass Spectrometry and Chemical Cross-Linking. J Am Soc Mass Spectrom. 30(1):149-160
doi: 10.1007/s13361-018-2000-4

Frick M, Hofmann T, Haupt C, Schmidt C (2018)
A novel sample preparation strategy for shotgun lipidomics of phospholipids employing multilamellar vesicles. Anal Bioanal Chem. 410(18), 4253-4258
doi: 10.1007/s00216-018-1113-8.

Haupt C*, Hofmann T*, Wittig S*, Kostmann S, Politis A, Schmidt C (2017)
Combining Chemical Cross-linking and Mass Spectrometry of Intact Protein Complexes to Study the Architecture of Multi-subunit Protein Assemblies. J Vis Exp. 129
doi: 10.3791/56747

Blees A, Januliene D, Hofmann T, Koller N, Schmidt C, Trowitzsch S, Moeller A, Tampé R (2017)
Structure of the human MHC-I peptide-loading complex. Nature. 551(7681):525-528
doi: 10.1038/nature24627

Hofmann T, Schmidt C (2019)
Instrument response of phosphatidylglycerol lipids with varying fatty acyl chain length in nano-ESI shotgun experiments. Chem Phys Lipids.
doi: 10.1016/j.chemphyslip.2019.05.007

Mazhab-Jafari MT, Rohou A, Schmidt C, Bueler SA, Benlekbir S, Robinson CV, Rubinstein JL (2016)
Atomic model for the membrane-embedded VO motor of a eukaryotic V-ATPase. Nature 539(7627):118-122
doi: 10.1038/nature19828

Liko I, Degiacomi MT, Mohammed S, Yoshikawa S, Schmidt C#, Robinson CV# (2016)
Dimer interface of bovine cytochrome c oxidase is influenced by local posttranslational modifications and lipid binding. Proc Natl Acad Sci U S A.  113(29):8230-5
doi: 10.1073/pnas.1600354113

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