Protein Sequencing Requirements for Samples

The protein sequencing requires strict quality and treatment of the samples to ensure the accuracy and repeatability of the experimental results. The following are the main requirements for samples when performing protein sequencing:

 

Sample Purity

High purity: The proteins in the samples need to be as pure as possible, avoiding interference from nucleic acids, lipids, and other non-protein components.

Decontamination: Avoid contamination from exogenous proteins (such as enzymes, antibodies, etc.), which may come from experimental materials or the operation process.

 

Sample Concentration

Appropriate concentration: The protein concentration of the sample needs to meet the minimum requirements for mass spectrometry analysis, usually accurately measured using specific methods (such as BCA, Bradford, or Lowry). Too low or too high protein concentrations will affect the experimental results. Too low concentration may make it difficult to detect the target protein in mass spectrometry analysis, while too high concentration may overload the instrument or make sample treatment difficult.

 

Sample Quantity

Sufficient sample quantity: In addition to concentration, the total sample quantity (i.e., the total protein quantity) is also an important consideration. Depending on the mass spectrometer and analysis method used, the required sample quantity can range from micrograms to milligrams.

 

Sample Storage and Treatment

Proper storage: The sample should be stored under appropriate conditions, usually frozen at -80°C, and avoid repeated freeze-thaw cycles to prevent protein degradation or denaturation.

Gentle treatment: The sample treatment process should be as gentle as possible, avoiding the use of strong physical or chemical methods to prevent protein denaturation or degradation.

 

Sample Compatibility

Mass spectrometry compatibility: The reagents and buffers used in the sample treatment process should be compatible with subsequent mass spectrometry analysis, avoiding the use of reagents that may interfere with mass spectrometry detection, such as those containing high concentrations of salt, detergents, or certain preservatives.

 

Special Samples

For gel strips, protein samples separated by SDS-PAGE, protein bands stained with KOMAS and SYPRO Ruby have good compatibility with mass spectrometry identification. However, for silver-stained proteins, glutaraldehyde should not be used as a fixative, as it will affect subsequent mass spectrometry analysis.