[Protein Research Series] - 4 丨 quantitative proteomics method, you deserve to have
1 Background and meaning
The study of life phenomena and laws (especially disease prevention and pathology studies) from the perspective of the direct performer of life activities (especially disease prevention and pathology) has become the main means of studying life sciences. These studies are often inseparable from studies of the types and expression of proteins in cells, tissues or organs. Studies of changes in protein expression levels at different times and under different conditions, identification of functional modules and pathways, and monitoring of biomarkers for disease require identification and quantification of proteins. The emergence and maturity of bio-mass spectrometry technology provides a more reliable and dynamic range of research tools for protein differential expression analysis. Based on mass spectrometry, scientists have continued to develop new quantitative proteomics methods to understand the overall protein dynamics of cells, tissues or organisms.
2 Introduction to methodology
At present, there are five mainstream quantitative proteomics methods, namely Label-free, iTRAQ, SILAC, MRM (MRMHR), and SWATH. Briefly read as follows:
2.1 Label-free
Label-free quantification , that is, non-labeled quantitative proteomics, does not require specific labeling of the comparative sample. It is only necessary to compare the chromatographic mass spectrometry response signals of specific peptides/proteins between different samples to obtain the protein expression between samples. Changes, commonly used to analyze mass spectrometry data generated during large-scale protein identification and quantification.
Label-free is easy to operate and can be used to quantify the total protein difference of any sample. However, the stability and repeatability of the experimental operation are higher, and the accuracy is worse than the label. Therefore, Label-free technology is suitable for quantitative comparison of large sample sizes, as well as experimental designs that cannot be quantified by labeling.
2.2 iTRAQ
iTRAQ quantification is currently the most widely used technique for quantitative proteomics. The core principle of this technology is peptide labeling and quantification, which converts the content of peptides into 114, 115, 116 and 117 isotopes (or 113, 114, 115, 116). , 8 marks of 117, 118, 119, and 121), thereby simplifying the quantitative complexity, and finally returning to the quantitative value of the protein by the quantitative value of the polypeptide, thereby finally determining the difference in protein between different samples.
iTRAQ quantification does not depend on samples, can detect lower abundance proteins, cytoplasmic proteins, membrane proteins, nuclear proteins, extracellular proteins, etc., and quantitatively accurate, can simultaneously analyze 8 samples, and can simultaneously identify and Quantitative results are particularly useful for differential protein analysis using multiple treatments or samples from multiple treatment times. The Jinkairui Mass Spectrometry Platform uses iTRAQ quantification technology to identify up to 6,000 proteins (for example, human HepG2), and the correlation of protein expression between replicates can reach 0.95 or higher.
2.3 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 natural isotope (light) or stable isotope (neutral or heavy), respectively. After 5-6 doubling cycles, the stable isotope-labeled amino acids are completely Incorporating into the newly synthesized protein of the cell replaces the original amino acid. The lysed proteins of different labeled cells are mixed in the proportion of cell number or protein, separated and purified, and then identified by mass spectrometry. According to the area comparison of the two isotopic peptides in the first-order mass spectrum, the relative quantitation is carried out, which belongs to the metabolic labeling method in vivo.
SILAC belongs to the in vivo labeling technology, which is closer to the true state of the sample. The labeling efficiency is up to 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. The amount of protein. SILAC quantification is applicable to the analysis of living cultured cells, and the difference of whole cell proteins or subcellular proteins under different conditions of multiple samples or the same sample is compared.
2.4 MRM and MRMHR
MRM is a data acquisition method that sets mass spectrometry detection rules based on known information or hypothetical information, performs signal recording on the ions that conform to the rules, and removes a large number of interferences that do not conform to the regular ion signals, thereby obtaining mass spectrometry information, belonging to the target protein. group. The key is to first detect the specific parent ion, then only the selected specific parent ion for collision induction, and finally remove the interference of other daughter ions, and only collect the mass spectrometry signal for the selected specific ion. . Jin Kairui developed MRMHR technology on AB SCIEX's 5600-plus instrument based on his predecessor's work. Because MRMHR adopted high-precision data, the transition was more credible and the quantification was more accurate.
MRMHR technology eliminates a large number of interfering ions by two-stage ion selection, reduces the chemical background of the mass spectrometer, and significantly improves the signal-to-noise ratio of the target detection, thereby achieving high sensitivity of detection, reproducibility and high accuracy, especially Validation of differences in protein expression levels for known protein sequences allows detection of lower abundance proteins, but only about 20 target proteins can be detected in a single MRM assay.
2.5 SWATH
SWATH is a new mass spectrometry acquisition mode technology jointly developed by Dr. Ruedi Aebersold of the Federal Institute of Technology in Zurich, Switzerland, and AB-SCIEX in 2012. It is an extension of MS/MSALL technology. Compared with the traditional shot-gun technology, SWATH acquisition mode can scan all the peptide precursor ions in the scanning interval and perform secondary fragmentation to obtain complete peptide information, which is a true panoramic view. High-throughput mass spectrometry. Jin Kairui's existing Triple-TOF 5600-plus mass spectrometry system provides high accuracy and dynamic range for SWATH quantification, detecting protein abundance differences that span four orders of magnitude.
With the SWATH acquisition mode, complete quantitative and qualitative results can be obtained in one experiment without the need for method optimization. It collects information on all compounds in the sample and can trace, query and analyze all compounds. The quantitative method uses a high-resolution mode that eliminates interference, improves selectivity, and is comparable to triple quadrupole mass spectrometry. Sensitivity and dynamic range are comparable to SRM analysis. For samples of subcellular structure, bacteria, fungi, cell secretions, SWATH quantification is very effective.
3 Conclusion
Proteomics is a systematic analysis of proteins expressed in a cell or tissue. Analytical techniques include the separation of proteins and peptides, the science of identification and quantitative analysis of analytes, and the bioinformatics of data management and analysis. Key analytical tools. This technology has now been used as a universal discovery tool to dynamically detect the external or internal interference response of a cell or tissue, ie quantitative proteomics. Different quantitative analysis techniques can be selected for different samples and different experimental analysis purposes.
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