Mass spectrometry (MS) is a method for characterizing and quantifying proteins based on mass differences. When proteins fail to resolve in the lab, researchers often resort to submitting bands cut out of a gel to specialized MS facilities for protein determination. However, this service comes at a price—$180 per 5 mm gel slice, for instance. It’s important then to note any guidelines or restrictions a facility may have for submission. To get the most bang for your buck, we’ve collected the following recommendations.
Avoiding keratin contamination
Keratin contamination is a common problem when working with 1D- or 2D-gel/MS and liquid chromatography (LC)/MS platforms. Keratin is a naturally occurring protein found commonly in fingerprints, hair, dead skin, wool clothing, DTT, ß-mercaptoethanol, buffers, and latex gloves. In low concentrations, keratin is not a problem. But when keratins are present in concentrations greater than the protein of interest, keratin overwhelms an MS system. To minimize contamination, always wear a lab coat and use non-latex gloves. Anything that touches the gel or sample is a possible source of contamination. Wash all plates thoroughly with 70% ethanol prior to casting. Avoid storing gels in plastic wrap and use new, clean plastic or glass gel trays. Destain the gel in a clean container that has been rinsed thoroughly with 70% ethanol or methanol/acetonitrile. Conduct the entire in-gel digestion in a laminar flow hood or another clean work area. There’s nothing worse than submitting a sample only to get worthless data—or no data—in return.
Lookout for trypsin
Although every sample has unique requirements in terms of preparation, ionization, and detection for various MS-platforms, there are some general restrictions that apply to reagents. Trypsin digestion yields peptides of varying molecular weights for MS analysis, but the specificity of trypsin is something to watch out for. Native trypsin is subject to autolysis, generating pseudotrypsin, which exhibits a broadened specificity. Autolysis products—present in a trypsin preparation—result in additional peptide fragments that can hinder proper protein identification.
Note staining restrictions
Requirements for staining are highly specific. Ensure that the proteins are not covalently modified or irreversibly fixed in the gel during staining. When individual components of protein complexes are separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by silver (or Coomassie) staining, it is important to use silver staining protocols without glutaraldehyde. Glutaraldehyde chemically modifies the protein and inhibits subsequent steps. For low-abundant proteins (often those requiring visualization by silver stain), to ensure your protein is present at a proper detection limit, it’s worth combining multiple gel bands for submission by running the sample in multiple lanes (10μl to 30 μl total). For an SDS-gel sample, if a band is visible with Coomassie blue stain (detection limit, 100 ng), there is probably enough material present for protein identification. The detection limits of stains are also dependent on variables like gel thickness and the width of the lanes.
Excise gel bands carefully
Before you cut any band it’s a good idea to take an image. Submit one copy with the sample and indicate where the bands were cut. When you cut a band of interest always use a sterile razor blade or scalpel for each sample. Acrylamide contains many interfering agents in the MS analysis so minimize its presence. Omit diffuse stained edges and excise only a clearly stained band. Capturing too little gel means loss of sample and too much gel may lower the yield of peptides. There’s a fine balance to discover! (MS facilities always try to introduce the minimum amount of material necessary to produce a useful spectrum. Too much sample means higher background signals, requiring downtime for cleaning.) Also, do not mince the gel, as small pieces are easily lost. Submit intact bands only. Put each gel band into a separate clean microcentrifuge tube. Do not use any tubes with O-rings or gaskets. Do not seal with parafilm or tape, as they are also common sources of keratin contamination. Always submit a blank gel slice for use as a control. Store the pieces without any liquids in the microcentrifuge tubes at -20°C or -80°C until you are ready to submit your bands.
The more information you can provide the MS facility upon submission of your gel slices the better. Remember a purified sample always has the best chance for positive identification. Analysis of pure, low-molecular weight organic compounds is easy; mixtures are harder; and comprehensive analysis of complex mixtures of high-molecular weight compounds can be very time-consuming. But a mass spec facility will work with you if you have a deadline. You’re the customer, after all! Good luck.