Guidelines for In-Gel Digestion Sample Preparation
In-gel digestion is a fundamental technique in proteomics used to identify proteins separated by polyacrylamide gel electrophoresis (PAGE). To ensure high-quality mass spectrometry (MS) results, rigorous attention must be paid to sample preparation. Contamination is the primary cause of poor data quality, and following standardized procedures is essential for success.
Gel Staining Considerations
The choice of staining method significantly impacts downstream MS analysis. Mass spectrometry is highly sensitive to chemical interference.
- Coomassie Brilliant Blue: The preferred method for MS analysis. It is compatible with most digestion protocols and does not interfere significantly with peptide recovery.
- Silver Staining: While sensitive, standard silver staining often requires glutaraldehyde for fixation, which cross-links proteins and makes digestion difficult. If using silver, ensure the use of "MS-compatible" silver stain kits that do not contain glutaraldehyde.
- Fluorescent Stains: Stains like SYPRO Ruby are generally MS-compatible, provided the gel is properly washed and processed.
Excision of Protein Bands
The physical extraction of the protein band from the gel must be performed in a clean environment to avoid keratin contamination, which is ubiquitous in laboratory settings.
- Use a clean, sterile scalpel blade for each individual band.
- Cut the gel piece as close to the visible protein band as possible to minimize the amount of background polyacrylamide.
- Place the gel pieces into high-quality, low-protein-binding microcentrifuge tubes.
Preventing Contamination
Keratin from skin, hair, and dust is the most common contaminant in proteomics. Because MS is extremely sensitive, even microscopic amounts can overwhelm the signal from your target protein.
- Always wear powder-free nitrile gloves. Change them frequently.
- Wear a clean lab coat and avoid touching your face or hair while handling samples.
- Work in a clean area, ideally under a laminar flow hood or in a space cleared of dust.
- Use only HPLC-grade or MS-grade reagents and ultrapure water (18.2 Mcm) for all buffers and wash steps.
Sample Quantity and Storage
The sensitivity of modern mass spectrometers has greatly reduced the amount of protein required, but sufficient material is still necessary for robust identification.
- Protein Amount: For standard bands, 50100 nanograms of protein is typically sufficient for identification. Low-abundance proteins may require more, while highly abundant proteins can lead to ion suppression.
- Storage: If the digestion cannot be performed immediately, store the excised gel pieces in 5% acetic acid or ultrapure water at 4C. Avoid storing them in buffers containing high concentrations of salts or detergents for extended periods.
- Shipping: If sending samples to an external facility, ship gel pieces in a small volume of liquid to prevent dehydration, and use appropriate secondary containment to avoid leakage.
Critical Reagents
The reagents used during the destaining, reduction, and alkylation steps must be prepared freshly. Old buffers, particularly those containing DTT (dithiothreitol) or iodoacetamide, can lose efficacy. Furthermore, ensuring that the trypsin used for digestion is of high quality and free from autolysis products is vital for clean spectra.
Final Checklist Before Submission
Before proceeding with digestion or sending your samples for analysis, ensure you have documented the gel type (SDS-PAGE gradient or percentage), the staining method used, and any specific concerns regarding the protein of interest (e.g., if it is known to be difficult to solubilize).
