How to prepare protein mass spectrometry samples?
The preparation of protein samples for mass spectrometry is a complex task that requires precise control of experimental conditions at each step. This includes protein extraction, purification, quantification, digestion, purification, and resuspension. Below are the general steps and tips.
1. Protein Extraction
Extract proteins from biological tissues or cells. Use appropriate buffers for cell lysis, such as RIPA buffer, PBS, etc. Common physical disruption methods include sonication, high-pressure homogenization, and freeze grinding.
2. Protein Purification
The extracted proteins contain various cellular components and require further purification. Common purification methods include salting out, electrophoresis, and chromatography.
3. Protein Quantification
Quantify protein concentration using BCA, Bradford, or Lowry methods.
4. Protein Digestion
Use enzymes (such as trypsin) to cleave proteins into peptides. Pay attention to the control of digestion temperature and time.
5. Peptide Purification
The digested peptides may be mixed with enzymes and other impurities from the digestion solution. Use adsorbents (such as C18 ZipTip) for peptide purification.
6. Peptide Resuspension
Resuspend peptides with an appropriate buffer (such as 0.1% formic acid) to prepare for mass spectrometry analysis. Avoid bubbles and excessive drying of the sample.
In actual practice, each step may need to be optimized based on specific experimental conditions and goals. For instance, the methods for protein extraction and purification may need selection based on the protein's source, properties, and purity requirements; the choice and conditions of digestion enzymes may depend on the protein's structure, stability, and subsequent analysis needs; the sample preparation method may need selection according to the mass spectrometry technology and equipment used.
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