Difference between in vivo Labeling and in vitro Labeling

Creative Proteomics
Sep 6, 2018 · 4 min read

Proteomics is an emerging discipline that uses a variety of technical means to study proteomics. The aim is to analyze the dynamic changes of protein composition, expression level and modification state, to understand the interaction and relationship, to explain the function of guarantee and the regularity of cell activity. Protein quantification is the accurate identification and quantification of all proteins expressed in a genome or in a complex mixed system. Protein quantification can be used to screen and search for differentially expressed proteins between samples caused by any factor. At the same time, some key proteins can also be qualitatively and quantitatively analyzed. Among them, according to the quantitative method, it can be divided into relative quantitative and absolute quantitative. According to labeling method, it can be divided into in vivo labeling method (SILAC) and in vitro labeling method (ICAT,AQUA).The following is an introduction to in vivo and in vitro labeling methods representing SILAC and AQUA.

Do you know SILAC?

SILAC refers to Stable isotope labeling with amino acids in cell culture that uses the essential amino acids mainly Lys and Arg labeled with light, medium or heavy isotopes to culture medium cell to label newly synthesized proteins in cells. Generally, after five to six generations, the proteins in the cells will be labeled with isotopes. The protein samples of different treatments were mixed in the same quantity. The quantitative and qualitative results of the proteins were obtained by SDS-PAGE separation, gel cutting, enzyme digestion and LC-MS / MS analysis. SILAC is widely used in comparative proteomics, protein-protein interaction, protein-DNA interaction and protein-RNA interaction. SILAC has many advantages over in vitro labeling. High throughput SILAC could be used to label the proteins in the cell. At the same time, the multiple proteins could be simultaneously analyzed and identified by mass spectrometry. SILAC isotope labeling has high efficiency, stability and no influence by pyrolysis solution. The results are reproducible and reliable. The sensitivity of SILAC was high and the amount of protein needed in the experiment was significantly reduced, and the results were closer to the true physiological state.

Do you know AQUA?

The AQUA method is to add a known amount of synthetic peptide internal standard with stable isotope labeling or a peptide segment internal standard labeled by chemical method after enzymatic hydrolysis of the sample to be tested, and compare the signal intensity of the target peptide segment with the addition of the internal standard. The content of the target peptide was obtained. Because the internal standard peptide is easily obtained from commercial companies, it is widely used in absolute quantification. Based on the same principle, in vitro isotope labeling of the target peptide, such as 18O, dimethylation or mTRAQ labeled peptide, can also be added as an internal standard to the system to be tested to realize the quantification of the target protein.

However, the quantitative results are uncertain because the method is to add the internal standard after enzymatic hydrolysis of the sample to be tested and ignore the loss caused by the sample pretreatment process. In addition, the cost of this method is high, it is difficult to achieve a large number of target protein absolute quantitative analysis.

Do you know the difference between in vivo labeling and in vitro labeling?

Internal standard method refers to selecting the appropriate substance as internal standard material, adding quantitative to the measured sample meanwhile the ratio of the measured component and the peak area of the internal standard material, multiplying the correction factor, and determining the content of the internal standard substance added. The advantages of the internal standard method are that the chromatographic conditions have little influence on the results, the accuracy and accuracy are high, and the disadvantage is that it is difficult to select suitable internal standard materials.

External standard method is also called standard curve method which is used to measure the content of the sample according to the curve drawn by the standard sample.The advantages of external standard method are simple, suitable and large sample analysis. The disadvantage is that each chromatographic condition is very difficult to be the same, prone to error.

We generally use external standard method when we do in vitro drug analysis and in vivo drug analysis. Because the extraction procedure is cumbersome, all use the internal standard method to carry on the correction, but if the instrument is stable, the reproducibility of the method is good, which also may use the external standard method. There are also many external standard methods for drug analysis in foreign countries.

If the internal standard substance is used in new drug approval, the structural confirmation data of the internal standard substance should be submitted, and the internal standard reference substance should be submitted as well as the production. So even the experts of SDA do not recommend the internal standard method. The initial use of the internal standard is due to the inaccuracy of the injector and the internal standard can be used to correct the sampling error. The accuracy of HPLC with quantitative loop injection is very high, and the accuracy is reduced because of the error of the operation when the internal standard is added. In drug quality inspection, if the ingredients of raw drug or preparation are not many, use external standard method. And now more and more standards have abandoned the use of internal standards.

For drug analysis in vivo, the error of analytical method is not so important, and the error introduced in the process of sample treatment should be paid more attention to. Therefore, the internal standard is generally added in the process of processing to correct the error. At this time, the recovery rate is not absolute high requirements. Although an absolute recovery rate of 70% or more is required, sometimes it doesn’t matter if it’s lower. The key is the stability of the recovery and the reproducibility of the determination.

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