Mass spectrometry technology can not be ignored in experiments such as protein identification, but the current application is not particularly extensive. So, how to solve the problem? This article collects the related newbies' common problems and solutions for this technology. Hope to help you better understand this technology.
Question 1. What is the difference between first-level mass spectrometry and second-level mass spectrometry?
Answer: The identification method of primary mass spectrometry mainly refers to fetal fingerprint (PMF), that is, the mass spectrometer is used to accurately measure the molecular weight of enzymatic fragments and search and compare to achieve protein identification. Some peptides were further broken and the fragments were further analyzed and compared to identify the sequence of the peptide and combine the results of PMF to achieve protein identification. Secondary mass spectrometry can obtain short sequences of some peptides, which has higher reliability. With the increasingly stricter data requirements of magazines, secondary mass spectrometry identification is the general trend of protein identification, and even if the results of primary mass spectrometry are currently performed, some PMF results need to be selected for secondary mass spectrometry verification.
Question 2. What if the species studied is not a model organism?
Answer: You can refer to the closest kinship model organisms for comparison to achieve the successful identification of the protein. If the mass spectrum is very good and no identification result indicates that this is a brand new protein, you can use de-novo and other technologies for in-depth analysis and identification.
Question 3. How to evaluate the effect of mass spectrometry?
Answer: PMF generally scores more than 60 points (P <0.05) even if it is successfully identified, while tandem mass spectrometry, it scores more than 60 points or although it does not exceed 60 points, but at least one peptide scored more than 30 points is considered successful identification.
Question 4. What are some special mass spectrometry methods for?
A: Other uses of mass spectrometry include phosphorylation site analysis, protein sequencing, mixed protein identification (shotgun technology), and accurate molecular weight determination, disulfide bond position analysis, etc.
Question 5. What dyeing method is better?
Answer: It is best to use Coomassie brilliant blue method for staining, silver staining is also possible, but the identification success rate is slightly lower, and the use of tandem mass spectrometry to identify silver-stained proteins can greatly improve the identification success rate.
Question 6. Can the gel be stored for a long time? How should it be kept? Does it affect the identification effect?
A: If it is stored for more than one month, it can be wrapped in plastic wrap and kept in the refrigerator. If it is less than one month, it can be kept at room temperature without affecting the identification effect of mass spectrometry.
Question 7. What should I do to take the glue points for mass spectrometry identification? What are the precautions?
A: When taking the point, cut the front end of the 0.2ml pipette tip a little, use the pipette tip to remove the gel and transfer it to a 0.5ml centrifuge tube, and repeatedly wash the rubber particles with water for two to three times, and finally dry it All water in the centrifuge tube is capped and stored. The centrifuge tube is best to use imported centrifuge tube to avoid plastic pollution; the water is best to use deionized water or double-distilled water; when taking a point, wear a mask and gloves to avoid keratin contamination.
Question 8. Can the gel be stored for a long time? How to store it? How to ship it?
A: The protein gel can be stored for a long time without affecting the identification effect of mass spectrometry. Generally speaking, if it is within a week or two, it is best to wrap it with plastic wrap and store it in a 4 degree refrigerator. If it is stored for more than one month, you can After removing the protein spots, put them in the refrigerator at -20 degrees or -80 degrees, it will not affect the subsequent mass spectrometry identification effect. When transporting the glue point, use normal temperature to transport, and there will not be much impact within three to five days.
Question 9. What should I do to take the glue points for mass spectrometry identification? What are the precautions?
A: When taking the point, cut the front end of the 0.2ml pipette tip a little, use the pipette tip to remove the gel and transfer it to a 0.5ml centrifuge tube, and repeatedly wash the rubber particles with water for two to three times, and finally dry it Keep and mark all moisture in the centrifuge tube. The centrifuge tube is best to use imported centrifuge tube to avoid plastic pollution; the water is best to use deionized water or double-distilled water; when taking a point, wear a mask and gloves to avoid keratin contamination.
Question 10. How to choose the first and second mass spectrometry?
Answer: The identification method of primary mass spectrometry is often called peptide-mass mapping (PMF), and the identification method of secondary mass spectrometry is often called tandem mass spectrometry. In early protein identification, because of the cost of identification, first-order mass spectrometry can be used for protein identification of sequencing organisms, and second-order mass spectrometry is often used for non-sequencing organisms. Now, the price of secondary mass spectrometry is also greatly reduced, and there is not much difference from the first mass spectrometry, and the reliability and identification success rate are greatly improved. The company generally recommends customers (whether it is a sequencing organism or a non-sequencing organism) to directly Do tandem mass spectrometry without considering peptide fingerprint identification.
Question 11. What is the general process of mass spectrometry identification, and what data does the company generally provide?
Mass spectrometry identification mainly includes three steps: proteolysis, mass spectrometry data acquisition, and library search. For the identification of each protein spot, three types of data are generally provided, including: mass spectrogram (PDF format, including 1 primary mass spectrum and 5-10 secondary mass spectra), mass spectrum peak list (text format, mainly It is the information of some mass spectrometry fragments), search results (generally provided in the PDF format or excel format of the self-built library for localized search, if it is the web page format generally provided by Mascot online search).
Question 12. What are the general factors for unsuccessful mass spectrometry identification?
Answer: The unsuccessful mass spectrometry identification can be divided into two categories, one is that the mass spectrometry is not good, and the other is that there is no protein database to refer to. The reason for the poor mass spectrometry effect can be subdivided into protein spots that are too light to the protein content Too low, too small to obtain protein spots is not conducive to operation, although two-dimensional electrophoresis separation, but it is a mixed protein spots of a few proteins, proteolysis and mass spectrometry operation errors, etc., to avoid these factors, try to take darker protein spots , And the area of ​​the protein spot needs to be appropriately larger (for some small but very clear protein spots, it is appropriate to select the tip with a larger aperture when taking the spot, and it does not matter if the blank space on the side is appropriately taken) In order to avoid taking protein spots that may be mixed, it has rich experience in the operation of enzymatic hydrolysis mass spectrometry. However, the poor identification results due to the lack of a reference database can be solved by replacing the larger database, using the EST database, and building a local mass spectrometry database based on the protein database of a single species.
Question 13. What is the difference between tandem mass spectrometry identification and mass spectrometry sequencing?
Mass spectrometry identification often uses software to automatically search and match existing databases to obtain results, while mass spectrometry sequencing needs to directly calculate the peptide sequence based on the molecular weight difference between adjacent or similar peaks in the mass spectrum peak map.
Question 14. Is the unknown protein sequenced by mass spectrometry or a protein sequencer?
The simpler method now is mass spectrometry, and it is not expensive to measure a sample. The specific price varies from laboratory to laboratory, 100-500 yuan. If mass spectrometry cannot be determined, it can also be combined with N-terminal sequencing, which can measure more than 10 amino acids. The combination of the two data can basically determine the target protein, and the current proteome database is quite powerful.
Finally, a brief summary of the above content, mass spectrometry is best to use Coomassie brilliant blue method for staining, when the protein gel is stored for more than one month, you can remove the protein spots and place them at minus twenty degrees or minus eight In a ten-degree environment, with the development of technology, the current price of secondary mass spectrometry is not much different from that of primary mass spectrometry. It is recommended to directly do secondary mass spectrometry.
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