Quantitative Proteomics Labeling

With the advancement of proteomics, qualitative analysis of protein types and modifications alone can no longer meet research needs. In this context, quantitative proteomics technology has emerged and has become one of the hot topics in life sciences in recent years. The quantification technology of proteomics is based on the known protein type, and quantifies its expression and abundance based on the signal intensity given by mass spectrometry. It can be divided into targeted and non-targeted. Among them, targeted quantitative technologies include multiple reaction monitoring (MRM) technology and parallel reaction monitoring (PRM) technology, and non-targeted quantitative technologies can be divided into non-labeled quantification and labeled quantification technologies based on whether or not they are marked. This article mainly introduces labeled quantitative technology.

 

The main strategy of labeling quantification is to introduce small molecules with stable isotope labels into different protein or peptide samples, and use the mass difference generated after isotope labeling to identify the source of the peptide. In the same mass spectrometry scan, the ionization efficiency and fragmentation mode of chemically identical labeled peptides are also the same, so comparing the signal strength of different isotopic labels can calculate the relative content of proteins in different samples. According to the different ways of introducing isotope labeling, it can be divided into in vivo labeling and in vitro labeling.

 

In Vivo Labeling Quantification

In vivo labeling can also be called metabolic labeling. The stable isotope amino acid cell culture technology (SILAC) is the most classic in vivo labeling quantitative technology. The basic principle of this technology is to separately add essential amino acids labeled with light and heavy isotopes (usually lysine and arginine) to cell culture medium. After 5-6 doubling cycles, newly synthesized protein amino acids in cells are almost completely labeled with stable isotopes. The precise quantification of proteins can be realized based on the peak intensity or area ratio of the two isotopic labeled peptide segments in the mixed sample.

 

1. Advantages

The labeling process is combined with the metabolic process of living cells, which can more truly reflect the protein expression in the body, and there is no need to separate and purify the protein, avoiding errors introduced by the separation step.

 

2. Disadvantages

The labeling efficiency may be affected by the metabolic state of the cell, and the labeling process is easily interfered by other metabolic pathways.

 

In Vitro Labeling Quantification

Metabolic reaction-based in vivo labeling quantitative technology has problems such as long time consumption and high price, so a series of in vitro labeling quantitative technologies have been developed. Among them, enzymatic labeling and chemical labeling after sample processing is the current research focus.

 

1. Enzymatic Labeling Technology

This technology replaces the 16O atom at the C-terminus of a set of peptide samples with 18O through the catalysis of trypsin, thereby causing a difference in molecular weight between the two sets of samples. By comparing the peak area of the labeled peptide segment and the unlabeled peptide segment, the protein sample can be quantified.

 

(1) Advantages: The reaction conditions are mild, the region specificity is high, and the operation is simple and cheap.

(2) Disadvantages: Poor labeling stability, poor repeatability, and low resolution.

 

2. Chemical Labeling Technology

Chemical labeling is currently the most widely used quantification method in the field of proteomics. It is not limited by the source of the sample and can design different chemical reactions based on different reaction groups on the peptide or protein molecule to achieve peptide and protein quantification. According to the different reaction groups, chemical labeling can be divided into aldehyde labeling, amine labeling, and shuttle labeling, etc. Among them, the most commonly used is isotope labeling relative and absolute quantification (iTRAQ) and tandem mass tag (TMT) technology based on amine labeling.

 

(1) Advantages: The labeling process is highly controllable, and the labeling matter and labeling matter quality can be flexibly selected, with high accuracy and accuracy.

(2) Disadvantages: Depend on chemical reactions, be affected by the environment, and the labeling process may introduce errors, which requires higher technical requirements for operators.

 

Protein labeling quantification has a wide range of applications in biomedical research, drug development, disease diagnosis, etc., especially in studying how proteins express and regulate under different conditions. The above several labeling methods each have their own advantages and disadvantages in quantitative proteomics. When choosing a labeling method, it is necessary to consider the specific research needs, experimental conditions, and sample characteristics.

 

MtoZ Biolabs uses Thermo Fisher's Q ExactiveHF mass spectrometry platform, Orbitrap Fusion mass spectrometry platform, Orbitrap Fusion Lumos mass spectrometry platform combined with Nano-LC, provides isotope-labeled protein labeling quantitative technology services (SILAC, iTRAQ and TMT), in addition to non-targeted unlabeled quantification (Label-free) and targeted quantification related services (MRM, PRM), can meet your different needs, welcome to consult.