1. Background and Significance
From the perspective of protein, the direct executor of life activities, studying life phenomena and laws (especially disease prevention and pathological research) has become the main means of studying life sciences. These studies are often inseparable from the study of protein types and expression levels in cells, tissues or organs. The study of changes in protein expression levels in different periods and under different conditions, identification of functional modules and pathways, and monitoring of disease biomarkers, all require the identification and quantification of proteins. The emergence and continuous maturity of biological mass spectrometry technology provides a more reliable and wider dynamic range for the analysis of protein differential expression. Based on mass spectrometry, scientists continue to develop new quantitative proteomic methods to understand the overall protein dynamics of cells, tissues or organisms.
2. Introduction to methodology
There are currently five mainstream quantitative proteomics methods, namely Label-free, iTRAQ, SILAC, MRM (MRMHR), and SWATH. It is briefly described as follows:
Label-free quantification, that is, non-labeled quantitative proteomics, does not require specific labeling of the comparative samples, but only needs to compare the chromatographic mass spectrometry response signals of specific peptides/proteins between different samples to obtain the protein expression between samples. Variation is commonly used to analyze mass spectrometry data generated during large-scale protein identification and quantification.
Label-free quantification does not require labeling, the operation is simple, and the total protein difference quantification of any sample can be performed, but the stability and repeatability of the experimental operation are required to be higher, and the accuracy is also lower than that of label quantification. Therefore, Label-free technology is suitable for quantitative comparisons of large sample sizes, as well as for experimental designs that cannot be achieved quantitatively with labels.
iTRAQ quantification is the most widely used technology in quantitative proteomics. , 117, 118, 119 and 121), which simplifies the complexity of quantification, and finally returns to the quantitative value of protein through the quantitative value of polypeptide, so as to finally determine the difference of protein between different samples.
iTRAQ quantification does not depend on samples, and can detect low-abundance proteins, cytoplasmic proteins, membrane proteins, nuclear proteins, extracellular proteins, etc., and the quantification is accurate. It can analyze 8 samples at the same time, and can simultaneously obtain identification and Quantitative results are particularly useful for differential protein analysis of samples with multiple treatments or from multiple treatment times. Gencare mass spectrometry platform uses iTRAQ quantitative technology to identify up to 6,000 proteins (take human HepG2 as an example), and the protein expression correlation between replicate samples can reach more than 0.95.
The basic principle of SILAC quantification is to replace the corresponding amino acids in the cell culture medium with essential amino acids labeled with natural isotopes (light) or stable isotopes (neutral or heavy), respectively. After 5-6 cell doubling cycles, the stable isotope-labeled amino acids are completely It is incorporated into the newly synthesized protein of the cell to replace the original amino acid. The lysed proteins of different labeled cells are mixed in equal proportions according to the number of cells or the amount of protein. After separation and purification, mass spectrometry identification is carried out, and relative quantification is carried out according to the area comparison of the two isotopic peptides in the first-level mass spectrum, which belongs to the in vivo metabolic labeling method.
SILAC belongs to the in vivo labeling technology, which is closer to the real 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 for the identification and quantification of membrane proteins. Each sample only needs tens of micrograms amount of protein. SILAC quantification is suitable for the analysis of living cultured cells, and the differential comparison of whole-cell protein or subcellular protein of multiple samples or the same sample under different conditions.
2.4 MRM and MRMHR
MRM is a data acquisition method that sets mass spectrometry detection rules based on known information or assumed information, records the signals of ions that conform to the rules, removes a large number of ions that do not conform to the rules, and obtains mass spectrometry information, which belongs to the target protein. Group. The key is to first be able to detect the specific precursor ions, then only the selected specific precursor ions are subjected to collision induction, and finally the interference of other product ions is removed, and only the selected specific product ions are collected for mass spectrometry signals. . Based on the previous work, Jin Kai Rui developed the MRMHR technology on the 5600-plus instrument of AB SCIEX. Because MRMHR adopts high-precision data, it is more reliable to choose Transition and the quantification is more accurate.
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 substance, so as to achieve high detection sensitivity, and has the characteristics of good reproducibility and high accuracy. It is suitable for the differential verification of protein expression with known protein sequences, and can detect low-abundance proteins, but only about 20 target proteins can be detected in one MRM experiment.
SWATH is a new mass spectrometry acquisition mode technology jointly launched by Dr. Ruedi Aebersold of ETH Zurich and his team and AB-SCIEX in 2012, which is an extension of MS/MSALL technology. Compared with the traditional shot-gun technology, the SWATH acquisition mode can scan all the peptide precursor ions in the scanning range at ultra-high speed and perform secondary fragmentation to obtain complete peptide information, which is a truly panoramic view. , high-throughput mass spectrometry. Gencare’s existing Triple-TOF 5600-plus mass spectrometry system enables SWATH quantification with high accuracy and dynamic range, and can detect extremely different protein abundances, spanning 4 orders of magnitude.
Using the SWATH acquisition mode, complete quantitative and qualitative results can be obtained in a single experiment without method optimization. It can collect information on all compounds in the sample, and can trace, query and analyze all compounds. The quantitative method adopts high-resolution mode, which can eliminate interference and improve selectivity, and the quantitative ability is comparable to triple quadrupole mass spectrometry, and the sensitivity and dynamic range are comparable to the level of SRM analysis. For samples such as subcellular structures, bacteria, fungi, and cell secretions, SWATH quantifies very well.
Proteomics is the systematic analysis of proteins expressed in a cell or tissue. Analytical techniques include separation science for separating proteins and peptides, analytical science for identifying and quantifying analytes, and bioinformatics for data management and analysis. Critical Analysis Tool. The same technique is now used as a universal discovery tool to dynamically detect changes in the proteome of a cell or tissue in response to foreign or internal perturbations, known as quantitative proteomics. For different samples and different experimental analysis purposes, different quantitative analysis techniques can be selected.