Identification of gel-separated proteins or purified proteins
If you want to know what proteins are in a specific gel band or if you want to characterize a purified protein, you cand send us either a cut gel piece or your protein of interest. Mass-spectrometry is a very sensitive method and can characterize proteins that are not visible in a Coomassie or silver-stained gel. Moreover, in vitro enzyme assays are possible to analyze with mass-spectrometry. For example, in vitro phosphorylation or ubiquitination, assays can be performed and mass-spectrometry used as a readout.
Global identification of peptides and proteins in each sample
Global proteomes are measured to determine the expression levels of proteins in each sample. You will always get qualitative and quantitative information from a single mass-spec run. Since mass-spectrometry is only semi-quantitative, relative comparisons across sample conditions are the standard procedure.
Label-free approaches: We apply data-dependent and data-independent data acquisition schemes tailored to the needs of every project.
Isobaric labelling approaches: For the most accurate quantification and for deep proteome analyses, we have established an isobaric labeling workflow with tandem mass tags (TMT).
In the kickoff meeting we are discussing with you which method is best suited for your project.
Global identification of posttranslational modifications (phospho, acetyl, and ubiquitination)
PTMs are very low abundant. For example, only around 1% of the peptide pool is phosphorylated in a human sample. Thus, enrichment protocols have been established. Currently, we offer phospho-enrichment, acetyl-enrichment, and ubiquitination-enrichment. Very often enrichments are combined with isobaric labeling.
Label-free relative quantification by parallel reaction monitoring
If you are specifically interested in a subset of proteins (up to 20 proteins and around 100 peptides), we can design a customized parallel reaction monitoring assay which has a higher sensitivity and lower missing values than the global peptide/protein identification approach.
Study of protein localization
Protein localization influences the function of protein. Under different conditions or in mutated states, proteins can de-localize. Differential centrifugation followed by mass spectrometry is a popular method to map proteins to organelles (DOI: 10.1002/pmic.201900330). A more recent approach is to perform global organelle profiling with immunocapture coupled to mass spectrometry (DOI: 10.1016/j.cell.2024.11.028). We have not yet an established protocol at the facility, but we are happy to collaborate with you on this matter.
Study of protein stability and protein turnover
Proteins are constantly synthesized and degraded. Stable isotope labeling in cell culture (SILAC) in combination with TMT sheds light into protein turnover (https://doi.org/10.1074/mcp.RA118.000583). With mass spectrometry, we can determine the half-life of many proteins in a single experiment.
Characterization of protein-protein interactions
Proximity labeling experiments like BioID (doi: 10.1083/jcb.20112098 and doi: 10.1038/s41586-021-03592-2) and TurboID (doi: 10.1038/nbt.4201) or affinity purification followed by mass spectrometry are commonly applied methods to elucidate protein-protein interactions. Affinity purification using antibodies is referred to immunopurificaiton or immunoprecipitation (IP). Proper controls are essential in these kinds of experiments to distinguish from unspecific binding partners. Feel free to contact us if you have questions regarding controls or the experimental setup.
The BioID, TurboID, or AP experiment is conducted prior to sending the samples to us. We then quantitatively analyze the peptide content in the sample and provide differential abundance estimates.
Characterization of protein-drug interactions
The MDP-Proteomics facility specializes in chemical proteomics applications. The central aim is to characterize small molecules. With competition pulldowns or activity-based (competition) protein profiling (ABPP), we are able to elucidate targets and off-targets of your molecule of interest. So far, we have established protocols for streptavidin pulldowns but are dependent on the probes from you. We are currently working on the establishment of several affinity matrixes.
Characterize the mode of action of a molecule
DecryptM (DOI: 10.1126/science.ade3925) aims to decrypt the mode of action of small molecules or antibodies by dose-resolved proteomics of post-translational modifications. For example, cells are treated with a kinase inhibitor at a minimum of ten full-log doses, and the phosphoproteome is monitored. CurveCurator (doi: 10.1038/s41467-023-43696-z) then assesses, classifies, and explores the dose-response curves’ significance. The beauty of this approach lies in the potency dimension. Dose-response curves with close coherence between drug-target affinity and drug-PTM-modulation illuminate the mode of action of a molecule by the guilt—by-association principle.
The DecryptE approach (doi: 10.1038/s41587-024-02218-y) is equivalent to the DecryptM approach but is a proteome-wide approach. Briefly, cells are treated with a minimum of 10 full-log doses of a molecule of interest, and the global proteome is measured with mass spectrometry.
Thermal protein profiling (TPP) is a third approach which informs the mode of action of molecules.
Individual requests
We do our best to support you with your individual project requests. Please contact Miriam Abele and discuss possibilities.
