Q: What is the sequence coverage obtained in a typical protein identification?
A: Generally, the sequence coverage is between 10% and 90% per protein, which is dependent on the protein abundance and protein nature.
Q: What are the most common reasons for obtaining poor mass spectrometric data?
1. Use of a staining method that is not compatible for mass spectrometry detection;
2. The presence of ion suppressing reagents, such as polymers, detergents, and glycerol in the sample;
3. Overloading with contaminating proteins, such as antibodies in affinity purifications, and high abundance proteins in serum samples;
4. Protein specific properties; e.g. heavily modified proteins, very few lysine and arginine residues in a particular protein.
Q: How do I avoid contamination with keratin?
A: Keratins are the most common protein contaminants derived primarily from human skin and hair, or sometimes from clothes like wool sweaters. These are tips for you to avoid keratin contamination of your sample:
1. Work in a clean lab with clean surfaces;
2. Use pipette tips and microcentrifuge tubes from closed boxes;
3. Rinse equipment in clean water before use (bottles, glass plates, electrophoresis equipment, staining trays);
4. Always wear gloves during your sample preparation and digestion. Never touch the gel with your fingers. Do not touch your hair or skin with gloved hands.
Q: When can I expect my results back?
A: The typical turn around time is 2 weeks-2 months.