General Sampling Advice for Researchers new to Proteomics and Metabolomics
For those not experienced with proteomics, one of the major problems is contamination from exogenous proteins. This is most commonly due to either keratin (from skin and/or hair) or bovine proteins, such as BSA (bovine serum albumin) from glassware that has not been cleaned appropriately. Some advice from various sources:
From the University of Toledo’s Proteomics Facility
Sample Handling: (Something to think about: Dogma in Biology - garbage in, garbage out. Dogma in Proteomics - garbage in, MORE garbage out).
The sensitivity of mass spectrometers is both a boon and a bane in the demanding world of biological research. It is a boon because of its ability to characterize vanishingly small amounts of biological materials. The flip side of this advantage is that even small amounts of contaminants can spoil a perfectly planned, well executed experiment. The most common contaminant in the field of protein mass spectrometry is Keratin. Cytokeratin, an abundant protein in skin cell and hair follicles, is everywhere. Since keratins are present as contaminants on the surface of the improperly an handled gel, they get digested more easily than the protein of interest that is inside the gel. Also, keratin peptides ionize more efficiently than the peptides from the protein of interest, thereby, "drowning" their signal. It is almost impossible to avoid keratin, but some "good laboratory practices" can minimize it.
- Wear gloves all the time and change them often.
- Consider precast gels where applicable.
- Handle the gel as little as possible.
- Keep separate dishes (staining boxes, measuring cylinders, etc.) for gels intended for MS analysis.
- Run a parallel gel for documentation purposes, whenever possible. The surfaces of the light boxes, scanners, etc., are generally not “MS clean”.
- Clean the work area with Methanol or Ethanol.
From the Proteomics Facility at The University of Texas MD Anderson Cancer Center
My samples contain beta-lactoglobulin and other milk proteins. What can I do? Your lab probably does a lot of western blots using non-fat milk, so mush of the glass- and plastic-ware are probably contaminated with milk proteins. Although this may help you with your WBs, it makes it difficult to do mass spectrometry because the mass spectrometer will “see” peptides from all of these proteins. It is difficult to get glassware clean enough after it has been heavily saturated with protein, a simple solution is to acquire a few key pieces of equipment and reserve them for this purpose ONLY. Then NEVER expose them to milk protein!!
Protein Identification from Gel Band or Spot
We recommend that you use either Coomassie Blue or Sypro Ruby to stain your gels. These produce the best results. Stains with colloidal Coomassie G-250 generally produce “better stains”, most common Coomassie blue stains are capable with mass spectrometry. Silver stained gels generally produce less robust results because they modify the proteins and crosslink the peptides to the gels. If you need to use silver stains, they must be mass spec compatible. Most commercial suppliers will indicate this on their product insert.
Co-Immunoprecipitation (Co-IP) for Proteomics (Finding Binding Partners)
Many investigators are attempting to use proteomics to identify binding partners. We offer the following recommendations:
- Develop and verify that your Co-IP works prior to submitting any samples to the proteomics core. This is the hardest part of the process and is very dependent of the antibody being used.
- Tagged proteins (e.g. his tagged proteins) work the best because:
- There are usually excellent antibodies available.
- Transfections of vectors expressing only tag is an excellent control. If you are not familiar with this, there are many protocols and advice available on-line.
General Mass Spectrometry Links and Resources