How can you ensure protein sample purity for mass spectrometry?

1 Sample preparation

The first step to ensure protein sample purity for MS is to optimize the sample preparation process, which involves extraction, solubilization, quantification, and storage of the proteins. Depending on the source and type of the proteins, you may need to use different methods and reagents to extract and solubilize them efficiently and avoid protein loss or damage. For example, you may need to use mechanical or enzymatic methods to break down the cell walls or membranes, and buffer solutions with appropriate pH, ionic strength, and detergents to dissolve the proteins. You should also quantify the protein concentration using a suitable method, such as the Bradford assay or the bicinchoninic acid (BCA) assay, and store the samples at low temperatures or freeze them to prevent degradation.

2 Sample fractionation

The next step to ensure protein sample purity for MS is to separate the proteins based on their properties, such as size, charge, hydrophobicity, or affinity. This can help to reduce the complexity and dynamic range of the protein mixture, and to enrich the proteins of interest or remove the unwanted proteins. There are various techniques for protein fractionation, such as gel electrophoresis, liquid chromatography, or immunoprecipitation. You should choose the technique that best suits your research question and sample type, and optimize the conditions and parameters to achieve high resolution and recovery.

3 Sample digestion

The third step to ensure protein sample purity for MS is to digest the proteins into smaller peptides, which can be more easily detected and analyzed by the MS instrument. The most common method for protein digestion is to use proteolytic enzymes, such as trypsin, that cleave the proteins at specific amino acid residues. You should consider the specificity, activity, and stability of the enzyme, and the optimal temperature, pH, and time for the digestion reaction. You should also avoid using buffers or reagents that contain contaminants or incompatible components, such as salts, metals, or detergents, that can interfere with the MS analysis.

5 Sample quality control

The fifth step to ensure protein sample purity for MS is to perform quality control (QC) tests on the sample before loading it onto the MS instrument. This can help to verify the purity, concentration, and integrity of the sample, and to identify any potential problems or errors in the previous steps. Some of the QC tests that you can perform include measuring the absorbance or fluorescence of the sample, running a small aliquot of the sample on a gel or a chromatogram, or analyzing the sample with a low-resolution MS instrument. You should compare the QC results with the expected values or standards, and troubleshoot any discrepancies or anomalies.

6 Sample optimization

The final step to ensure protein sample purity for MS is to optimize the sample for the specific MS instrument and method that you are using. This involves adjusting the sample volume, concentration, and composition to match the requirements and specifications of the MS instrument and the ionization and detection modes. For example, you may need to dilute or concentrate the sample, add or remove certain additives or modifiers, or change the solvent or buffer composition. You should follow the manufacturer's instructions and recommendations, and perform calibration and validation experiments to ensure the optimal performance and accuracy of the MS instrument and method.

7 Here’s what else to consider

This is a space to share examples, stories, or insights that don’t fit into any of the previous sections. What else would you like to add?