Introduction to Protein Quantification Service

  • As we all know, dynamic changes of proteome abundance in cells have important effects on various life processes. For example, in the occurrence and development of many diseases, the expression of some proteins is often accompanied by abnormal. Protein Quantification Service is the precise quantification and identification of all proteins expressed in a genome or in a complex mixture system. Currently, quantitative proteomics techniques are mainly divided into label and label free quantitative proteomics.

     

     

    SILAC quantitative proteomics is a kind of commonly used label free quantitative proteomics at present:

    SILAC

    The basic theory of SILAC (Stable isotope labeling with amino acids in cell culture) is to use a medium containing essential amino acids of light, medium and heavy isotope type for cell culture (commonly used stable amino acids Lys and Arg). In the process of cell subculture, Isotopic amino acid will be used for protein synthesis, five or six generations later, all the proteins will be labeled by isotope, after the amount of mixed marker protein separation and mass spectrometry analysis, by comparing the level of mass spectrogram in the area of the three type of isotope peptides relative quantitative size, at the same time the secondary spectrum of peptides sequence determination in order to identify proteins.

     

    SILAC labeling technology is in vivo labeling technology, so it hardly affects the function of cells, that's why it has been widely used in proteomics related fields.

     

    Label free quantitative proteomics is to get mass spectrum analysis for protein enzymolysis peptides through the liquid mass spectrometry, which doesn’t need expensive stable isotope labels do internal standard, just simply analyze large-scale identification of proteins produced by the mass spectrometry data, compare different samples peptides corresponding signal strength, in order to quantify the protein corresponding to peptide segment.

    The following are two methods of Label free quantitative proteomics:

    ITRAQ Analysis

    The iTRAQ (isobaric tags for relative and absolute quantification) technology is a relative and absolute quantitative technique developed by AB SCIEX to mark the same kind of isotope in vitro. This technique which is a high throughput screening technique commonly used in quantitative proteomics in recent years, uses a variety of isotope reagents to mark protein polypeptide N terminal or lysine side chain groups. Through high precision mass spectrometer tandem analysis, protein expression of up to 8 samples can be compared at the same time. It can be used to compare proteins in different samples, for example, to study differences in protein expression levels in tissue samples under diverse pathological conditions or at different stages of development.

    ITRAQ reagent is a small molecule and same heavy element chemical substance, including three parts: one side is the reporter group, the other side is peptide reactive group, and the middle part is balance group.

     

     

    TMT quantitative proteomics

    TMT (Tandem Mass Tag) technology is an in vitro labeling technology of polypeptide developed by Thermo Scientific. The technology uses labels of 2, 6 or 10 isotopes to specifically label the amino groups of polypeptides, and can compare the relative contents of proteins in 2, 6 or 10 groups of different samples at the same time by mass spectrometry analysis.

    The TMT label is composed of three parts: molecular weight reporter, molecular weight standardized part and reactive group, forming two, six or 10 heterotopic labels with equal relative molecular weight.

     

    Compared with the experimental procedure of iTRAQ, the difference is that the reagent used to mark the peptide segments is different, but other steps are the same.

     

     

    Nowadays, the development of proteomics technology has become an important support for the rapid development of modern biotechnology and will lead to the breakthrough of biotechnology. It can be said that proteomics research is not only a milestone of life science research into the post-genome era, but also one of the core contents of life science research in the post-genome era. In the future development, proteomics research field will be more extensive.