Core facility Mass Spectrometry of Biopolymers (BioMS) provides service analyses of proteins and other biomolecules based on mass spectrometric detection. We perform identification, characterization, and quantification of purified proteins, bands from SDS-PAGE, immunoprecipitates, cell lysates, and similar samples. We also analyze post-translational modifications, including global phosphoproteomes. We are also developing methods for the analysis of nucleic acids and their complexes with proteins. In addition to routinely used proteomic procedures, we also offer structural mass spectrometry methods for characterizing protein-protein and protein-ligand interactions, such as analysis of the native mass of proteins and their complexes, chemical crosslinking, and hydrogen-deuterium exchange (HDX-MS).

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Services

Bottom-up proteomics

The analysis focuses primarily on the identification of proteins, their post-translational modifications, and/or the quantification of proteins, either using methods based on isotopic labelling or label-free methods. The procedure involves proteolytic digestion of proteins and the analysis of the resulting peptides. The analysis itself is based on liquid chromatography coupled with high-resolution mass spectrometric analysis of peptides. This allows for sensitive and reliable identification/quantification of proteins or their modifications in the analyzed sample. Protein samples in various forms can be analyzed. We can analyze dried protein samples, as well as proteins separated in SDS-PAGE gel or proteins dissolved in various buffers.

Note: If you are a new customer or are unsure whether your task is compatible with our capabilities, please contact the operators first for appropriate setup of the analysis or the entire proteomic experiment.

Operators:

• Jana Březinová
• Alena Meledina
• Michal Korecký
• Alena Křenková

Top-down proteomic analysis

Intact mass

Analysis of the intact mass of proteins or nucleic acids under denaturing conditions. It serves as a more precise equivalent to SDS-PAGE with a mass determination accuracy up to 1 Da.

Possible applications:

• Verification of correct protein expression based on total mass
• Detection and relative quantification of covalent modifications
• Detection of covalent protein-protein or protein-ligand complexes

It is possible to analyze both pure proteins and nucleic acids, as well as simpler mixtures (up to, for example, 10-20 pure proteins). More complex mixtures, or a very heterogeneous mixture of different proteoforms of one protein, can be problematic; however, it is possible to perform LC-MS separation of the sample before analysis. It is also possible to analyze larger proteins, such as antibodies. We also provide the calculation of Drug-to-Antibody Ratio (DAR) for antibody-drug conjugates as a service.

Requirements:

• Minimum 5 µg of protein in a 50 µl volume
• Precise composition of the buffer in the sample (common buffers are OK, but WITHOUT detergents!)
• Sample ideally reduced with 5 mM TCEP, if disulfide bonds do not need to be preserved
• Expected mass of the protein / nucleic acid / complex
• Expected mass of modifications / ligands

Operators:

• František Filandr
• Petra Junková

Native MS

Native mass spectrometry allows for the study of non-covalent protein-protein or protein-ligand complexes under biologically relevant "native" conditions. The analysis is performed using very gentle ionization, which allows the entire non-covalent complex to be transferred from solution to the mass spectrometer detector.

Possible applications:

• Confirmation of protein-protein or protein-ligand interaction
• Information about complex stoichiometry
• Presence and relative quantification of post-translational modifications
• Monitoring structural changes in proteins/complexes under different conditions

Requirements:

• Minimum 5-10 µM solution of protein/complex in a 20 µl volume
• Ultra-pure protein in 10-100 mM ammonium acetate, without any other substances except ligands; avoid DMSO (alternatives for dissolving ligands are MeOH or EtOH)
• Information about the precise composition of the buffer in the sample
• Expected mass of the protein / nucleic acid / complex
• Expected mass of modifications / ligands

Operators:

• Tereza Nešporová
• František Filandr

Structural Proteomics

These complementary methods allow for the study of protein structures and the characterization of not only protein-protein interactions but also the interactions of proteins with other biological molecules or small molecules.

HDX-MS

Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) is a structural proteomics method based on a chemical reaction that allows the exchange of covalently bound hydrogen atoms for deuterium from the solution. This exchange occurs under physiological conditions, and its rate depends on the local secondary structure of proteins. By performing a differential experiment under different conditions, it is possible to track the rate of exchange at the peptide level to localize structural changes on proteins due to the binding of ligand (identification of the binding site), another protein (identification of the interaction interface), or due to changes in physicochemical conditions (temperature, pH), including accompanying allosteric effects.

Requirements:

• At least 50 µg of protein for each of the compared conditions
• Any buffer without detergents (not applicable if necessary for membrane proteins
• Information about the precise composition of the buffer in the sample
• Hypothesis about the system's function, detailed information about the sample
• Sequences of all proteins

Operators:

• Tereza Nešporová

Crosslinking (XL-MS)

This method is based on crosslinking proteins using reagents of a specific length, which, after binding to the protein and subsequent mass spectrometric detection, provide information about the distances between two amino acid side chains. This technique can detect and structurally characterize protein-protein interactions or characterize distances within the structure of an individual protein. The output is a table listing reliable detected crosslinks and their graphical representation on the protein sequence and on a 3D model.

Requirements:

• Isolated proteins or their expected complexes in solution
• A suitable buffer for performing the crosslinking reaction and its precise composition. The suitable buffer depends on the crosslinking reagent used; reagents containing a reactive NHS group (DSBU, DSPU, DSSBU, DSS, BS3, DSSO) react with primary amines, therefore only buffers that do not contain them can be used! (suitable buffers include, for example, HEPES or PBS)
• Sequences of all proteins along with information about their modifications
• Sequences of all proteins

Operators:

• Petra Junková