Methods & Services
The KIIDD Advanced Mass Spectrometry Facility in the School of Medicine offers various state-of-the-art MS-based proteomic approaches.
As routine procedure accounts identification of proteins from gel bands or solution, quantification (user provides labeled samples) and intact mass analysis, including appropriate LC-MS sample preparation and delivery of database search results.
For experiments where analytical requirement extends beyond the capabilities of the routine proteomics service KMS is happy to set up a collaboration. This may involve more extended analytical experiments including label free quantification, PTM/chemical modifications and native-state protein mass spectrometry as well as structural proteomics techniques (HDX, XL-MS, proximity labeling), analysis and data interpretation. Please contact Dr. Goran Stjepanovic in the first instance to discuss collaborations.
1. Intact protein mass analysis under denaturing conditions.
We provide a basic determination of intact proteins under denaturing conditions, without prior digestion or gas phase fragmentation of the molecule of interest. Native protein (complex) analysis are not offered as a routine service.
We can provide information on the stoichiometry of possible chemical or post-translational modifications as well as stability of protein sample, including any degradation/proteolysis that may have occurred. In addition, through MS/MS analysis (please see PTM Analysis), the location of the modification can be determined.
Please contact us for more information concerning sample submission and sample preparation.
2. Protein Identification
The facility provides reliable proteins identification from low-complexity (e.g. identification from gels, purified protein) as well as complex samples. Proteins are first proteolytically digested to peptides and then sequenced using a variety of MS/MS fragmentation techniques. Afterwards, peptides/proteins are identified by a Sequest (Proteome Discoverer) database search. To account for more complex samples or identifying hundreds of proteins present in cell line/tissue, different pre-fractionation methods and longer LC-gradients are used. Please contact us to discuss your MS-project in detail.
3. Quantitative Proteomics
Unbiased proteomic studies can be performed using our high resolution Orbitrap mass spectrometers to provide accurate quantification data. The Facility offers several quantification strategies:
- Label free quantification
Label-free quantification is a method in mass spectrometry that aims to determine the relative amount of proteins in two or more biological samples without any prior labeling. Comparing the ratio of ion abundances between identical peptides obtained in different experiment runs can be used to estimate differential expression. Label free quantification service is offered only in a collaborative setting.
- Labeling-based MS1 quantification (such as dimethyl and SILAC)
Metabolic labeling using SILAC
Stable isotope labeling using amino acids in cell culture (SILAC) is a highly accurate method to identify and quantify relative differential changes in complex protein samples under differential treatments. The SILAC method uses in vivo metabolic incorporation of isotopically labeled amino acids into proteins followed by mass spectrometry (MS). SILAC approaches are well suited for monitoring changes in post-translational modifications. The facility offers LC-MS/MS data acquisition and preliminary data analysis/quality control.
- TMT / iTRAQ protein quantification
Isobaric labeling provides a fast and easy way for relative and absolute protein quantification with high throughput. The facility offers data acquisition and analysis/quality control. In case you would like to perform such experiments, we would be happy to assist in the planning phase.
4. Post Translational Modifications (PTM) analysis
Identification of post-translational modifications on proteins e.g. phosphorylation, acetylation, methylation, lipidation, ubiquitination, glycosylation, cysteine redox state. We can perform site localisation and stoichiometry of post-translational modifications or chemical modifications on proteins from pure samples e.g. purified protein/protein complex and complex mixtures e.g. proteins extracted from cell line/tissue.
For complex mixtures e.g. proteins extracted from cell line/tissue or sub-stoichiometrically modified samples we can perform enrichment of the modification prior to analysis. We use MOAC columns packed with TiO2 for phosphopeptide enrichment.
5. Native mass spectrometry
During native MS, noncovalent interactions between proteins and their ligands, for example, other proteins, nucleotides, lipids, metals or other small molecules, are maintained. The obtained mass spectra therefore reveal the subunit composition and stoichiometry of the formed complexes.
Native MS further provides information on protein-ligand interactions including drug conjugates, subunit interactions and topology as well as heterogeneity of the assemblies. The High Mass Range MSn capability allows for identification of ligands bound to both cytosolic and membrane proteins.
Please contact us to discuss sample requirements and advanced applications such high-resolution native MS and characterization of native protein-ligand complexes. Native MS service is offered only in a collaborative setting.
For the identification of protein interaction partners, we can identify proteins following immunoprecipitation/co-affinity. Furthermore, we offer proximity-based labeling strategies (e.g. BioID, TurboID, APEX2) in combination with high resolution mass spectrometry to identify protein–protein interactions or in living cells. For determining the proteins abundances different quantification strategies are available, both label-free and label-based (see Quantitative Proteomics section). In case you would like to perform such experiments, we would be happy to assist in the planning phase.
The conformation of proteins can be analyzed using amide hydrogen exchange (HDX) mass spectrometry, which allows the analysis of protein dynamics, protein-protein interaction interfaces, ligand-induced allosteric conformational changes and protein stability studies. HDX data can be obtained from samples that are available in small amounts, and can complement high resolution methods as Cryo-EM and X-ray crystallography.
Our total HDX platform covers the complete process from sample labeling and optimization of digestion conditions to optimizing mass spec utilization and data curation. Automation allows for complete control of all key factors: reproducible accuracy in timing of liquid transfers, pH control and temperature control and in-line digestion.
Automated platform for HDX experiments
8. Cross-linking mass spectrometry (XL-MS)
XL-MS, is a complementary method to high resolution protein structure techniques such as Cryo-EM, NMR and crystallography. XL-MS provides distance constraints and is able to pinpoint residues in close proximity to the interaction interfaces between individual subunits of protein complexes of any size in solution. XL-MS delivers valuable complementary information to enable accurate 3D structural modelling of the proteins or protein complexes. Additionally, XL-MS is a potent tool to identify protein–protein interactions or to uncover protein assemblies in living cells, tissues, or organelles (please see Interactomics section).
The facility offers a complete toolset for performing XL-MS for defining protein-protein interactions (PPIs) and probing PPI interface.