Label-based Protein Quantification Technology: iTRAQ, TMT, SILAC

Changes in the number of proteomes in a cell have a big effect on many aspects of life. For example, the occurrence and development of many diseases are often accompanied by abnormal expression of certain proteins. Quantitative proteomics is the accurate quantification and identification of all proteins expressed in a genome or all proteins in a complex mixed system. At present, quantitative proteomics technology is mainly divided into labeling strategies and non-labeling strategies, in which the labeling strategies are divided into in vivo labeling (such as SILAC), and in vitro labeling (such as iTRAQ, TMT).

Isobaric Tags for Relative and Absolute Quantitation (iTRAQ)

In 2004, the company AB SCIEX made iTRAQ, which is a quantitative proteomics research tool. This technique enables the comparison of proteins in many different samples. For example, it can study differences in protein expression levels in tissue samples under different pathological conditions or different developmental stages. At the same time, iTRAQ can accurately quantify and identify all proteins expressed in one genome or all proteins in a complex mixed system. iTRAQ can use eight different isotope reagents to simultaneously label and compare eight different protein samples. These reagents are composed of a report group, a balance group, and an amine-specific reactive group.

The workflow of iTRAQ

Briefly, samples are usually cleaved by trypsin, alkylated, and enzymatically digested into peptides. The generated peptides are differentially labeled with multiple tags of iTRAQ reagent before mixing labeled samples together to be analyzed by LC-MS/MS.

The advantages of TMT

1. High sensitivity: low abundances of protein can be detected.
2. Strong separation ability: can separate acid / alkaline proteins, proteins less than 10 KD or greater than 200 KD, insoluble proteins, etc.
3. It can be used to find any kind of protein, including membrane proteins, nuclear proteins, and proteins outside of cells.
4. High throughput: 10 samples can be analyzed at the same time, making it especially suitable for differential protein analysis of samples with multiple processing methods or multiple processing times.

Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)

SILAC is a powerful method for high-throughput quantitative proteomics. It is based on the idea that the growth of mammalian cells depends on amino acids that are called "essential." SILAC is applied to quantitative proteome research, which can analyze protein interactions and protein expression differences in cells, providing an effective solution for comprehensive and systematic qualitative and quantitative analysis of the complex mammalian cell proteome.

The workflow of SILAC

The general process of SILAC is shown in figure 3. By adding light, medium, or heavy amounts of stable isotope-labeled essential amino acids (mainly lysine and arginine) to the cell culture medium, and through the normal metabolism of the cell, the newly synthesized proteins are all tagged with stable isotopes. By mixing all types of proteins in equal amounts and separating them by SDS-PAGE, gum cutting, and enzyme digestion, LC-MS/MS analysis can obtain quantitative and qualitative results for related proteins.

The advantages of SILAC

1. SILAC is a living cell-level labeling technology. The labeling effect is stable and efficient, and its labeling efficiency is not affected by lysate.
2. SILAC requires fewer samples; usually only a few dozen micrograms of protein per sample is sufficient.
3. SILAC uses mass spectrometry to identify and quantify multiple proteins simultaneously.
4. SILAC uses in vivo labeling technology, which is close to the true state of the sample.