How Many Ways of Labeling Do You Know in Biology?

Creative Proteomics
Sep 6, 2018 · 4 min read

There are many ways of labeling in biology study such as iTRAQ Labeling, TMT labeling, stable isotope labeling with amino acids and so on which have proved to be effective tools in solving many problems at the study of biological system. These labels are used as “ruler” to measure the distances between chosen groups and to measure the size, form and micro-relief of objects. By providing information about these factors, the label provides information that can help the scientist to under stand the structures of membranes, nucleic acids, enzymes and proteins and how they function. So let me introduce some ways of labeling to you to test how many ways of labeling do you know.

Do you know iTRAQ Labeling?

ITRAQ technology is a new and powerful method for both absolute and relative quantitative analysis of four samples. This method is based on iTRAQ reagent.ITRAQ is actually an isotope labeling reagent, including a report group with a charge, which has four relative molecular weights of 114, 115, 116 and 117, as well as a peptide segment reaction group and an equilibrium group. The equilibrium groups also have four relative molecular weights of 31, 30, 29 and 28 respectively. The equilibrium molecular weight of the equilibrium group ensures that the total relative molecular weight of the iTRAQ reagent after binding to each reporter gene is 145. The peptide segment reaction group can react with the N-terminal and lysine of the peptide segment to measure the E amino group, which can label all the peptides produced in the biological sample and enhance the coverage of the peptide segment of any given protein. At the same time, important structural information such as post-translational modification of some proteins is preserved.

Do you know TMT Labeling?

TMT Labeling is a polypeptide in vitro labeling technique developed by Thermo Scientific Company in the United States. The technique employs 10 isotope labels to label amino groups of peptides. The relative content of proteins in 10 groups of different samples can be compared by LC-MS/MS analysis. TMT Labeling is a common differential proteomics technique, which is widely used in the fields of disease marker screening, drug targeting, animal and plant disease resistance / stress resistance mechanism, animal and plant development differentiation mechanism and so on. TMT reagent consists of three parts which is quality reporting group, quality standardization group and amino reaction group. The mass reporting group has 10 different molecular weights and the mass standardized group 10 has different molecular weights, which can be matched with different reporting groups to ensure that the same peptides from different labeled sources have the same mass charge ratio in the first order mass spectrometry. The amino group can be covalently linked to the amino group of the N-terminal and the lysine side chain of the peptide segment, so that the peptide segment can be labeled. In the first order mass spectrometry, the same peptide segment in different samples labeled with any TMT reagent showed the same mass charge ratio. In secondary mass spectrometry, cleavable bond breaks release TMT reporter ions. 10 TMT reported ion peaks were produced in the low mass region of mass spectrometry, the intensity of which reflected the relative expression information of the peptide in different samples. In addition, the peak to mass ratio of the peptide fragment ion in the secondary mass spectrometry reflects the sequence information of the peptide segment. The qualitative and relative quantitative information of protein can be obtained by searching the original data of mass spectrometry.

Do you know stable isotope labeling with amino acids?

The basic principle of stable isotope labeling with amino acids is the corresponding amino acids in cell culture medium were replaced by natural isotopic or stable isotope labeled essential amino acids. After 5–6 doubling cycles, the stable isotope labeled amino acids were completely incorporated into the newly synthesized protein instead of the original amino acids. The cleavage proteins of different labeled cells were mixed according to the number of cells or the amount of protein. After being separated and purified, it was identified by mass spectrometry and compared with the area of two isotopic peptide segments in the first order mass spectrogram. It belongs to the method of metabolic marker in vivo. The stable isotope labeling with amino acids belongs to the technology of internal marking which is much closer to the real state of the sample and the labeling efficiency is as high as 100% and the labeling effect is stable. The stable isotope labeling with amino acids is not only suitable for whole cell protein analysis, but also suitable for the identification and quantification of membrane protein.

Proteomics is the systematic analysis of proteins expressed by a cell or tissue. Analytical techniques include the separation of proteins and peptides, the analytical science of identifying and quantifying substances and the bioinformatics of data management and analysis. Mass spectrometry is a key analytical tool. The same technique has now been used as a universal discovery tool to dynamically detect changes in the proteome of a cell or tissue that responds to external or internal interference, namely quantitative proteomics. Different quantitative analysis techniques can be selected for different samples and different purpose of experimental analysis. So are you clear about the above methods of labeling now?