PEG & ADC

Methodology of Quantitative Proteomics

The study of life phenomena and laws from the perspective of the direct executor of life activities—proteins, especially disease prevention and pathology research, has become the main means of studying life science. These studies are often inseparable from the study of the types and expressions of proteins in cells, tissues, or organs.

Studies on changes in protein expression levels at different periods and under different conditions, identification of functional modules and pathways, and monitoring of disease biomarkers, these studies require the identification and quantification of proteins.

The emergence and maturity of biological mass spectrometry technology provide more reliable and wider dynamic range research methods for protein differential expression analysis. Based on mass spectrometry technology, scientists continue to develop new quantitative proteomics methods to understand cells, tissues, or life The overall protein dynamics of the body.

Methodology Introduction

There are currently five mainstream quantitative proteomics methods, namely Label-free, iTRAQ, SILAC, MRM (MRMHR), and SWATH.

Label-free

Non-labeled quantitative proteomics does not require the specific labeling of comparison samples. It only needs to compare the chromatographic response signals of specific peptides/proteins between different samples to get the changes in sample protein expression. Usually used for large-scale analysis Mass spectrometry data generated during protein identification and quantification.

Label-free quantification does not require labeling treatment and is simple to operate. It can be used to quantify the total protein difference of any sample. However, it requires higher stability and repeatability of experimental operation, and its accuracy is also worse than that of labeled quantification. Therefore, the label-free technique is suitable for quantitative comparison of large sample sizes and for experimental design that cannot be achieved by label-free quantification.

iTRAQ

It is the most widely used technique in quantitative proteomics. The core principle of this technology is peptide labeling and quantification, which converts the content of peptides into the content of isotopes 114, 115, 116, and 117 (or 113, 114, 115, 116…), thus simplifying the complexity of quantification, and finally, the quantitative value of the peptide is regressed to the protein to obtain the quantitative value, so as to finally determine the difference of the protein between different samples.

iTRAQ does not rely on samples for quantification. It can detect low-abundance proteins, periplasmic proteins, membrane proteins, nucleoproteins, extracellular proteins, etc., with accurate quantification. It can analyze 8 samples at the same time and obtain identification and quantification at the same time. The results are particularly suitable for the analysis of differential proteins in samples that are processed in two ways or from multiple processing times.

SILAC

The basic principle of SILAC quantification is to replace the corresponding amino acids in the cell culture medium with essential amino acids labeled with a natural isotope (light) or stable isotope (neutral/heavy) respectively. After the cells undergo 5-6 doubling cycles, the amino acids labeled with stable isotope are completely The newly synthesized protein incorporated into the cell replaces the original amino acid.

The lysed proteins of differently labeled cells were mixed in equal proportion according to the number of cells or the number of proteins. After separation and purification, they were identified by mass spectrometry. The relative quantification was carried out according to the area comparison of two isotope-type peptides in the primary mass spectrometry, which belongs to the metabolic labeling method in vivo.

SILAC technical route

SILAC is an in vivo labeling technology, which is closer to the true state of the sample. The labeling efficiency is as high as 100%, and the labeling effect is stable. It is not only suitable for whole-cell protein analysis but also suitable for the identification and quantification of membrane proteins. Each sample only needs dozens of micrograms of protein.

SILAC quantification is suitable for the analysis of live cultured cells and compares the difference of whole-cell proteins or subcellular proteins of multiple samples or the same sample under different conditions.

MRM&MRMHR

MRM is a data acquisition method that sets mass spectrometry detection rules based on known information or assumed information, records the signals that conform to the rules, removes a large number of non-conformance ion signals, and obtains mass spectrometry information. It belongs to the target protein. group.

The key is to first detect the specific parent ion, then only the selected specific parent ion is collision-induced, and finally, the interference of other daughter ions is removed, and only the selected specific ion is collected by mass spectrum signal.

The MRMHR technology eliminates a large number of interfering ions through two-stage ion selection, reduces the chemical background of the mass spectrometer, and significantly improves the signal-to-noise ratio of the target detection object, thereby achieving high detection sensitivity, and has the characteristics of good reproducibility and high accuracy. It is especially suitable for the verification of protein expression differences of known protein sequences. It can detect lower abundance proteins, but only about 20 target proteins can be detected in one MRM experiment.

SWATH

It is a brand new mass spectrometry acquisition mode technology jointly launched by Dr. Ruedi Aebersold and his team from the Swiss Federal Institute of Technology Zurich in 2012 with AB-SCIEX, which is an extension of MS/MSALL technology.

Compared with the traditional shot-gun technology, the SWATH acquisition mode can scan all peptide precursor ions in the scanning interval through ultra-high-speed scanning and perform secondary fragmentation to obtain complete peptide information.

Conclusion

Proteomics is the systematic analysis of proteins expressed in a cell or tissue. The analysis technology includes separation science for separating proteins and peptides, analysis science for identifying and quantifying analytes, and bioinformatics for data management and analysis. Mass spectrometry is the Critical analysis tool.