How to Identify Proteins and Suggestions in Protein Sample Preparation
With the development of high-resolution mass spectrometer analysis techniques, protein mass spectrometry methods have become quite sophisticated. It is no longer difficult to identify low-abundance protein samples and multiple protein samples at one time. However, due to the high sensitivity of mass spectrometry, foreign-introduced proteins may cause false positives in mass spectrometry. Therefore, we must be careful as to avoid the introduction of foreign protein contaminants during the preparation of protein samples. In addition, the preparation of protein samples also requires the use of solutions and reagents that are compatible with mass spectrometry detection, which can greatly improve the accuracy of mass spectrometer identification.
Based on my experience, I’m going to introduce you every detailed process on protein identification via mass spectrometry, and what you should pay attention to in protein sample preparation.
General Processes in Protein Mass Spectrometry
Firstly, you need to beak the cell or tissue sample, and there are many ways to break it. Most cells can be lysed by using common cell lysis reagents such as RIPA buffer, adding protease inhibitors and reducing agents. The tissue can be broken by cryogenic homogenization, liquid nitrogen freezing, etc.
Secondly, centrifuge the sample at 12,000 rpm for 10 minutes, and remove the insoluble cellular components.
Liquid protein samples can be enriched, purified, and processed with IP using antibodies, and the protein to be detected can then be concentrated. Proteins can also be separated using 1D/2D-PAGE gel.
After the concentrated liquid protein was digested, LC-MS/MS was used for analysis. Protein gel samples are required to excise protein bands with the same molecular weight as the target protein, and the proteins are subjected to in-gel digestion and mass spectrometry detection.
ID Analyze the data identified by mass spectrometry to obtain highly reliable protein peptide sequences, and then compare them with protein database to obtain protein ID.
Sample Types
Cell samples: microbial cells such as animal cells, plant cells, fungi and bacteriaTissue samples: animal tissue, plant tissueBody fluid samples: serum, plasma, urine, etc.
Sample Quantity
Proteins: 100ug Cell Samples: 100ug Animal Tissues: 1 gPlant Tissues: 200 mg Blood (EDTA Anti-coagulation): 1 mLSerum: 0.2-0.5 mLUrine: 2 mL Yeast, Microbial Samples: 200 mg (dry weight)
Suggestions in Protein Sample Preparation
Concentrated Sample
The protein sample can be concentrated by various methods such as precipitation, membrane separation, separation column, freeze-drying, etc. The user should select the appropriate method according to the characteristics of the sample. The SDS sample solution can elute the protein from the chromatographic sample, thereby maintaining the small size of the sample.
Electrophoretic Gel
For SDS-PAGE, the traditional Laemmli-type, triglycine gels and various other modified gels, such as di-, tri-, or trihydrochloride gels, are compatible with protein identification. There is no limit to the concentration of various gels. The gel type and concentration chose by the user should be determined based on the proper separation of the sample.
Protein Gel Staining
- Coomassie Blue, SYPRO Ruby, and Silver stain samples can all be used in protein identification.• Silver staining samples may not be compatible with subsequent mass spectrometry analysis. I recommend that you use the following products or experimental procedures for silver staining experiments:ProteoSilver Plus, Sigma (Product # PROTSIL1 or PROTSIL2)Dodeca Silver Stain, BioRad (Product # 161-0481 or 161-0480)• In addition, please do not discolor the silver-stained samples.
Liquid Sample
Minimize the use of surfactants such as SDS during sample preparation and pay attention to reducing the salt concentration. If you submit an IP elution sample, I recommend using the HPH EB (0.5 M NH4OH, 0.5 mM EDTA) buffer system for elution of the last step protein to ensure that the elution buffer system is compatible with subsequent mass spectrometry experiments.
Cleavage Protein Band
Avoid direct contact with the gel with no matter your hand, the light box, or any other sources of possible keratin contamination during the cutting process. When cleaving the protein tape, try to avoid cutting the blank gel (it may reduce digestion efficiency and polypeptide recovery). Place each gel band in a clean, 1.5 ml microcentrifuge tube and label each sample carefully.