Quantitative analysis of proteins is important in many industries and applications. Proteins include a diverse group of compounds that participate in virtually every biological process. Proteins are building blocks, hormones, transport molecules, cell receptors, cell growth and differentiation factors. Because of this, the analysis of protein interactions is crucial in drug design and production.
Proteins are macromolecules - the basis of their structure are polypeptide chains containing from 100 to more than 10000 amino acids (10 - 100 kDa). Proteins are found in simple and complex forms. Complex proteins contain a non-protein component (sugar, lipids, metal ions or dyes) in their structure.
In our laboratory, proteomic analyses, including quantitative analysis of proteins, are performed by HPLC-MS or HPLC-MS/MS methods on a ZenoTOF 7600 high-resolution mass spectrometer (SCIEX). We process the obtained results using Sciex OS, Spectronaut, Skyline software.
Quantitative analysis of intact protein (whole protein in intact form) leads to the concentration (determined from a prepared curve from a standard (with or without matrix) and confirmation of the identification by superimposing (on a mirrored basis) the MS spectrum of the protein determined in the sample to the MS spectrum of the standard. For your needs, we can generate a list of protein-structured compounds (impurities) that were identified in the sample and determine the ratio of their signal area to the signal area of the analyzed protein.
The result of proteomic quantitative peptide analysis allows precise and much more sensitive determination of protein concentration. The analysis is based on MS/MS data obtained for peptides obtained by digestion with proteolytic enzymes. The reported result consists of the concentration of the protein under study and confirmation of its presence based on the determined amino acid sequence. For such analyses, it is recommended to add standards internal standards.
Our offer:
- For proteins and peptides with low masses - intact analysis with MS detection;
- Proteins with masses >20kDa require optimization of the digestion method and MS/MS detection. This results in specific signals from the analyzed protein in the mixture/matrix;
- From complex matrices (including serum, cell culture, fungal suspension), sample analysis involves several steps:
- Sample purification;
- Digestion to obtain unique peptides to distinguish the analyzed proteins from other proteins in the sample;
- Sample preparation requires appropriate method optimization and validation (let's set validation criteria individually);
- Reported quantitative result includes confirmation of the presence of the protein in native form or a peptide derived from it (usually confirmation includes at least two peptide sequences);
- Statistical analysis using tools: MarkerView (PCA), GraphPad Prism, Trends, Sciex OS.
To optimize and validate the method, it is necessary to provide a standard, in the case of proteins with high masses - it is recommended to add an internal standard.
See also:
Bioanalytic: Quantitative analysis of proteins.
