Munich Cluster for Systems Neurology
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Proteome Hub

Proteome Hub

Antibody facility

Proteomics techniques

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Antibody facility

1.0 Objective

The Antibody part of the Proteome Hub offers generation of monoclonal antibodies to study molecular mechanisms of neurodegenerative diseases. We provide advice on antigen choice and preparation, as well as on the antibody validation strategy.
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1.1 Organisation

Location and Coordinator:

Name Institution Location Mail phone
Regina Feederle Helmholtz Zentrum München HMGU 35.33/2052 regina.feederle@helmholtz-muenchen.de 089-3187 1360

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1.2 Technical portfolio

• Generation of antibody-producing monoclonal hybridoma cell lines
• High-throughput multiplexed flow cytometry screening (IntelliCyt iQue)
• ELISA binding, capture, competition assays (Tecan Spark ELISA reader)
• Antibody purification (GE ÄktaTM pure, GE ÄktaTM avant)
• Aggregate- and endotoxin-free antibody preparations for in vivo studies
• Antibody labelling with fluorochromes and enzymes

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Fig.1 Workflow for monoclonal antibody generation. Antibody validation is performed in partner lab.

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1.3 Project request procedure

Generation of new monoclonal antibodies is subject to a Monoclonal Antibody Core Facility Agreement for Academic Collaborations. For antibody projects and general inquiries, please get in touch via email or phone:
regina.feederle@helmholtz-muenchen.de
Phone: 089 3187 1360

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1.4 Fees

New antibody projects and supply of established antibodies are free of charge for SyNergy members within collaborative research projects. If resources run short, projects will be selected based on feasibility and scientific impact by the hub. Antibody and collaboration requests outside SyNergy are welcome.

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1.5 Publications

https://www.synergy-munich.de/members/associate-investigators/feederle/lmu_publikationen.html

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Proteomics techniques

2.0 Objective

The Proteome Hub provides several proteomics techniques to study biological mechanisms in neurological diseases.

The SyNergy Proteome Hub will provide equal access to all users. However, according to the large number of projects and samples and the limited measurement time on the available instruments, we have to apply a certain prioritization of submitted projects based on:
• Scientific relevance of the project with regard to SyNergy aims
• Required sample preparation and measurement time
• Required method optimization or test sample evaluation
• Date of sample submission
• Special urgency such as manuscript revisions

The Proteome Hub members will provide a detailed data analysis report with statistical evaluation in an excel sheet. Furthermore, we will provide support for additional data analysis such as pathway and protein cluster enrichment analyses, as well as figure and manuscript preparation.

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2.1 Organisation

Location and Coordinators:

Name Institution Location Mail phone
Stefan Lichtenthaler DZNE Munich and TUM CSD U1 room 133 stefan.lichtenthaler@dzne.de 089-4400 46425
Christian Behrends LMU Munich CSD 3rd floor, room 117 christian.behrends@mail03.med.uni-muenchen.de 089-4400 46509

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2.2 Technical portfolio

LC-MS/MS Instrumentation:
• 2 Easy nLC 1200 + Q Exactive HF
• 1 Easy nLC 1000 + Q Exactive

Methods:
• Label Free Quantification (LFQ) of proteins with Data Dependent Acquisition (DDA) and Data Independent Acquisition (DIA)
• Secretome Protein Enrichment with Click Sugars (SPECS) to study protein secretion in (primary) cell cultures
• Cerebrospinal Fluid (CSF) proteomics including murine CSF samples
• Proteomics of MACS or FACS sorted cells
• Affinity purification mass spectrometry to determine protein interactomes
• Proximity labeling methods (APEX2, TurboID) to study organelles and protein-protein interactions
• Ubiquitination profiling

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Fig.2: General proteomics workflow.

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Fig.3: Proteomic analysis of MACS sorted microglia in two Alzheimer’s disease mouse models.

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2.3 Project request procedure

Before starting a new project, Synergy members have to provide a detailed project proposal, which will be discussed together with the members of the SyNergy Proteome Hub. Based on this proposal, the feasibility of the study will be evaluated regarding sample amount, methods, sample preparation, analysis time, but also potential limitations and discussed with the applying group. This helps to properly plan and schedule the projects. Furthermore, we want to provide advice for sample collection and submission to prevent certain pitfalls such as albumin, FCS or chemical (detergent, PEG) contaminations of samples, which can render samples useless for proteomic analyses. Additionally, some projects require initial evaluation or optimizations with test samples to achieve optimal results.

Please contact a member of the SyNergy Proteome Hub:

Research group Neuroproteomics (DZNE and TUM):

Prof. Dr. Stefan Lichtenthaler
Email: stefan.lichtenthaler@dzne.de
Phone: 089-4400 46425

Dr. Stephan Müller
Email: stephan.mueller@dzne.de
Phone: 089-4400 46437

Research Group Behrends (LMU):

Prof. Dr. Christian Behrends
Email: Christian.Behrends@mail03.med.uni-muenchen.de
Phone: 089-4400 46509

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2.4 Fees

The service of the Synergy Proteome Hub is free of charge for SyNergy members within collaborative research projects.

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2.5 Publications

https://www.synergy-munich.de/members/investigators/lichtenthaler/lmu_publikationen.html

https://www.synergy-munich.de/members/investigators/behrends/lmu_publikationen.html

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