General proteomics is a kind of science which takes proteome as the research object, and studies the protein composition and change law of cells, tissues or organisms. Proteomics essentially refers to the large-scale study of protein characteristics, including protein expression levels, post-translational modifications, protein-protein interactions, etc., so as to obtain an overall and comprehensive understanding of disease occurrence, cell metabolism and other processes at the protein level.
The protein non-label quantitative technology (Label free) is to analyze the protein enzymolysis peptide by mass spectrometry through liquid chromatography-mass spectrometry. It does not need to use expensive stable isotope labels as internal standards. It only needs to analyze the mass spectrometry data generated when large-scale identification of proteins, and compare the signal intensity of the corresponding peptide segments in different samples, so as to relatively quantify the proteins corresponding to the peptide segments.
- No isotope labeling, simple operation, low cost;
- Low identification coverage and sensitivity;
- Not limited by the number of samples;
- The "with/without" comparative analysis of protein can be carried out;
- The requirements for mass spectrometry equipment are high, and chromatography and mass spectrometry need to have good stability and repeatability at the same time.
DIA technology mainly adopts data independent acquisition mode (Data-independentAcquisition,DIA) and combines with traditional data dependent acquisition mode (Data-dependent Acquisition,DDA) to construct reference spectrum library. The whole mass spectrum scanning range is divided into several windows by liquid chromatography-mass spectrometry (LC-MS/MS), and all ions in each window are fragmented in turn to collect all sub-ion information. The obtained data were analyzed by mainstream analysis software for proteomics identification, quantification and differential protein analysis. The technology does not need to specify the target peptide, can obtain all the fragment information of all ions in the sample without omission, and the data utilization is greatly improved.
- High identification sensitivity: more low-abundance proteins can be identified to improve the reliability of quantitative analysis;
- Repeatability improvement: Compared with DDA mode, DIA has fewer missing values and higher data repeatability;
- Suitable for large sample size: not limited by sample size, can be used for large-scale sample number detection;
- High quantitative accuracy: lower CV value, improved quantitative accuracy;
- Data can be traced back: digital protein information, can be traced back.
Direct DIA(Direct Data-independent Acquisition) is a DIA technology that uses a non-database-building method. Compared with the traditional DIA analysis strategy, Direct DIA no longer uses DDA hierarchical database building, but uses machine deep learning to generate a database directly by searching DIA original file spectrogram. Deep learning scoring looks for spectral peak fragmentation patterns, predicts retention times, and removes false positive results. Compared with DDA, it has the advantages of less missing value, more number of identified proteins and accurate quantification. Compared with traditional DIA technology, this greatly reduces the cost, and the gap between the number of identified proteins and traditional DIA is getting smaller and smaller.
- DIA holographic scanning, good data integrity, high protein coverage;
- Do not build DDA database, improve efficiency, reduce the cost of the experiment;
- Less missing data, good reproducibility, quantitative accuracy.
TMT technology is a quantitative proteomics method, which realizes the simultaneous quantitative analysis of proteins in multiple samples based on the principle of chemical labeling. The technology uses specific chemical reagents (TMT tags) to label proteins in different samples with different mass-to-charge ratios, and then quantitatively analyze them by tandem mass spectrometry (tandem mass spectrometry).
- High throughput: TMT technology can analyze multiple samples at the same time, improve the efficiency of analysis;
- High sensitivity: TMT technology has high sensitivity and can detect low-abundance proteins;
- High quantitative accuracy: TMT technology provides accurate quantitative results and can be used for quantitative comparison and trend analysis.
Data acquisition mode
DIA (detection) DDA (database building)
Whether labeling reagent is required
Number of identified proteins
Lower, more missing values
High, few missing values
High, few missing values
High, few missing values
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