Key Methods in Proteomics: Protein Extraction Techniques for Animal Tissues

Sample preprocessing is a critical step in proteomics, directly affecting data quality and the accuracy of downstream analysis. The main steps include sample preparation, homogenization, protein extraction, reduction and alkylation, enzymatic digestion, and desalting. Among these, sample preparation, homogenization, and protein extraction largely determine the success of preprocessing. The following sections focus on these key aspects.

1. Preprocessing of Animal Tissues for Proteomics

Preprocessing involves collecting and preserving samples to ensure experimental success by removing non-target components like blood, fat, and connective tissue. Steps include:

1. Rapidly dissect and collect the target organ, carefully trimming away fat and connective tissue, then cutting the sample into small pieces (~soybean size).
2. Rinse the sample with pre-chilled saline or 1x PBS to remove blood and debris.
3. Transfer to cryovials, snap-freeze in liquid nitrogen, and store at -80°C or ship on dry ice.

2. Effective Grinding Methods of Animal Tissues

Once the organ tissue is prepped, it must be ground into fine particles or homogenized to release proteins and other biomolecules. Methods include:

• Liquid Nitrogen Grinding: Use a mortar and pestle to grind the frozen tissue.
• Homogenization: Use a homogenizer for tissue grinding.
• Mechanical Disruption: Employ a tissue grinder or cell disruptor for mechanical breakdown.

To maintain sample integrity during grinding, follow these effective practices:

• Pre-cooling the Grinder: Use liquid nitrogen or refrigeration to pre-cool the grinder, preventing heat-induced sample degradation and ensuring stability.
• Proper Grinding Speed: Avoid overheating and structural damage by maintaining an appropriate grinding speed. Too fast may cause overheating, while too slow reduces efficiency.
• Cleaning and Sterilization: After each use, clean the grinder with mild detergent and 70% ethanol, then rinse with sterile water and dry to prevent cross-contamination.

3. Protein Extraction Methods of Animal Tissues

Protein extraction involves isolating proteins from tissue homogenates by using lysis buffers to break down cell structures. After centrifugation to remove debris, the proteins are collected from the supernatant. Common lysis buffers for animal tissues include:

1. Sodium Dodecyl Sulfate (SDS): SDS is a detergent that solubilizes a wide range of proteins, including membrane proteins. However, it inhibits protease activity and interferes with peptide ionization in mass spectrometry, affecting accuracy .
2. Sodium Deoxycholate (SDC): SDC enhances trypsin activity, improving protein identification and hydrophobic peptide recovery. It is acid-insoluble and can be removed via acidification and centrifugation, avoiding interference with mass spectrometry. However, high-temperature heating is needed for membrane protein solubilization, which may affect post-translational modification studies .
3. 8M Urea: Urea denatures proteins effectively, but it degrades with heat, potentially altering protein properties and digestion .
4. N-Dodecyl β-D-Maltoside (DDM): DDM is a mild detergent that preserves enzyme activity and solubilizes hydrophobic proteins without altering their structure, especially useful for low-quantity samples .
5. Radio Immunoprecipitation Assay (RIPA): RIPA buffer effectively extracts soluble, membrane, and nuclear proteins, but may cause loss of high-molecular-weight proteins and extracellular matrix proteins .

Each extraction method has its strengths and weaknesses. Combining techniques, such as liquid nitrogen grinding with strong denaturing agents and sonication, can improve protein yield. Additional reagents like EDTA or DTT are often included to assist in protein extraction and release.

Reference

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