What is Proteomics ?
Why should proteins be analyzed?
Analyses of proteins that play important roles in living organisms have made rapid advances through creation of a technical platform based on mass spectrometry and an information platform based on accumulation of genome data. Various sample preparation methods for extracting proteins from biological samples (blood, urine, tissue, etc.) are being developed for analyses. These methods differ according to the purpose of each analysis. In addition, bioinformatics techniques are being developed for efficient processing of large amounts of analytical results and data.
Proteins do not function alone, but bind to other proteins (protein complexes) or low-molecular compounds to play their roles (functions). Put in human terms, homeostasis in society (cells, organs, and the whole body) is maintained in cooperation (communication) with other members of society. To analyze functions (roles) of a specific protein A, it is important to identify other protein molecules that bind to protein A. In terms of human society, such analysis is like investigating relationships among friends. For example, discovery of a protein B that binds to protein A leads to further analyses of protein B-related proteins. This further analyses will reveal a wide range of relationships (a network), leading to discovery of proteins that cause diseases. Since such proteins serve as targets for the development of compounds (drugs) that suppress or activate protein functions, protein identification techniques are useful for detecting targets for drug discovery.
Protein identification method by mass spectrometry
In order to be identified by mass spectrometry, the proteins must be processed into low-molecular peptides by a digestive enzyme. The PMF (peptide mass fingerprint) method is used to identify proteins by measurement of the masses of the peptides obtained after digestion. The MS/MS ion search method is used to identify proteins by analyzing the amino acid sequences of peptides by MS/MS. The PMF method is useful for identifying proteins that have been highly purified by a method with high separation capability, such as two-dimensional electrophoresis. On the other hand, the MS/MS ion search method allows highly accurate identification even for mixtures of multiple proteins, because it determines the amino acid sequences of digested peptides by MS/MS. For mixed protein samples, peptides should be separated by liquid-chromatography before analysis. Furthermore, for global analysis of complex mixed protein samples, peptides should be separated by multidimensional LC. The relationships between sample states and analytical methods are shown in Table 1.
| Analytical method | Sample | Separation |
|---|---|---|
| PMF method | High-purity protein | Two-dimensional electrophoresis |
| MS/MS ion search method | Sample containing several types of proteins | Two-dimensional electrophoresis, SDS-PAGE |
| LC-MS/MS method | Sample containing a large variety of proteins | Two-dimensional electrophoresis, SDS-PAGE |
| 2DLC-MS/MS method | Sample containing a complex mixture of proteins | Affinity purification Crude |
Global quantitative comparison of protein expression
By comparing changes in proteins in biological samples (blood, urine, tissue, etc.) of healthy individuals and patients, it may be possible to use proteins characteristic of disease samples as markers for diagnosis of diseases. The objective of developing disease markers is to provide highly-accurate early diagnoses and improve therapeutic effects. Sample analysis before and after drugs are administered to patients provides data for evaluating drug efficacy. Therapeutic effects of medication vary among individuals. When the expression levels of a specific protein differ significantly between patient groups who respond and do not respond to medication, the protein serves as an indicator of therapeutic effects, can be used to select treatment methods for individuals, and provides useful data for medical treatment tailored to individual patients.
THERAVALUES provides global comparative quantitative analysis of protein expression by iTRAQ® method
Posttranslational modification analysis
Proteins that have been biosynthesized according to genetic information are in a state similar to that of newborn babies. When the babies grow up and come to play their social roles, they change their appearance (clothes) according to their jobs. Posttranslational modifications are like the clothes. Clothes make people's social roles easily recognizable and allow smooth communication. Various posttranslational modifications exist, such as phosphorylation (a phosphate group binds to serine, threonine, tyrosine, etc.), sugar chain modification (a sugar chain binds to asparagine, serine, threonine, etc.), and lipid modification (a fatty acid binds to serine, cysteine, etc.). Modifications activate proteins, serve as markers for binding to other proteins, and play a role in anchoring proteins. Thus, posttranslational modifications are essential for proteins to perform their original functions. Modification analyses are indispensable for detailed elucidation of protein functions.
THERAVALUES analyzes posttranslational modifications by LC-MS/MS combined with nano-LC, based on the MS/MS ion search method
- Ogino T, Fukuda H, Ohmi S, Kohara M, Nomoto A, Membrane Binding Properties and Terminal Residues of the Mature HCV Capsid Protein in Inseet Cells. J. Virol. 2004, 78, 21, 11766-11777