Protein identification is based on experimental measurement of peptide molecular weights and matching the amino acid sequence of the protein under study with protein sequences deposited in libraries (databases). Two approaches are most commonly used in proteomic studies: top-down and bottom-up. The top-down method involves analyzing full protein sequences. Bottom-up analyses, on the other hand, use digestion of proteins with proteolytic enzymes. The peptides obtained by digestion are analyzed using a mass spectrometer. Each analysis leads to a comparison of experimental data with data obtained by theoretical digestion of proteins described in the database.
Bioanalytic: Identification of the proteins.
Tandem mass spectrometers are commonly used to determine protein sequences. They allow ion selection of pseudomolecular peptides and then fragmentation and measurement of the masses of the resulting fragments. Fragmentation using collision induced dissociation(CID ) is an invaluable tool in targeted and untargeted MS/MS analysis of small molecules. It is important to note that in proteomic analysis, data obtained using the aforementioned fragmentation method may leave gaps. For example, spectra obtained by CID fragmentation do not allow unambiguous differentiation of some amino acids: leucine from isoleucine (identical molar masses and CID fragmentation) and lysine from glutamine (very similar masses). For this reason, we perform proteomic analyses on a state-of-the-art high-resolution ZenoTOF 7600 (SCIEX), in which the Zeno ion trap and EAD (Electron-Activated Dissociation) technology together make a powerful combination. It enables the creation of unique fragments to differentiate problematic amino acids and increase the sensitivity and specificity of the analysis.
We perform analyses of:
- samples from solution;
- samples from a gel;
- samples from a complex matrix (by prior arrangement);
- samples from lyophilizer;
- samples previously prepared by the client.
Qualitative analysis of proteins includes:
- To confirm the presence of a protein, the sample is subjected to digestion and the result obtained from experimental data is compared with theoretical data;
- Comparison of experimental data on the masses of peptides included in the analyzed protein with the protein sequences described in the database;
- Analysis and identification of the protein mixture;
- Identification of post translational modifications (PTMs) (we use the tools: Skyline, Spectronaut).
- Reporting of the result including, but not limited to, confirmation of the coverage of the protein sequence obtained in the test sample with the theoretical protein and the list of modified peptides, location and % of modifications.
Comparative analyses include:
- Determining the composition of mixtures;
- Comparison and profiling of sample series - protein identification and trend analysis.
Our offer also includes:
- Support in the planning and execution of studies (the scope is determined individually);
- Assistance in the development of extraction methods from complex matrices - biological fluids, plant tissue, animal tissues (the scope is determined individually);
- Statistical analysis using tools: MarkerView (PCA), GraphPad Prism, Trends, Sciex OS;
- Quantitative analysis of proteins;
- Analysis and characterization of monoclonal antibodies.
Bioanalytic: Identification of the proteins.
