I am working on a project of sequencing a myoglobin protein. Well I managed to get my SDS page bands digested at a facility near me and mass spec'd and fingerprinted, and the results, while good, only resulted in 81% coverage of my entire sequence. Since they only do tryptic digests I'm faced with the task of doing an alternate digest myself to increase coverage. Based on my theoretical sequence derived from the DNA, I've chosen Glu-C endoproteinase as my enzyme (with Asp-N as a backup).
Does anyone have a simple and good protocol for a Glu-C in-gel digest? I am worried I won't have access to the kind of equipment I may need such as speed-vacs, agitated water baths, and the like although I've heard these things aren't absolutely necessary.
I've thought about getting a kit like such: http://www.piercenet.com/product/in-gel, estion-kit. But I don't know if that's necessarily worth it. I'd still need to get some Glu-C because it's a tryptic kit.
Do I really need to alkylate and/or reduce? I've read this is for cysteine residues and my sequence shouldn't have any cysteines I believe.
I have not used enzymes other than trypsin so I will only address some of your other questions. Evolutionarily, myoglobins do not have disulfide bonds and if your cDNA sequence does not show a Cys residue, there would be no point in doing the reduction and alkylation step. That step converts the disulfide bond to two Cys with free SH residues, thus making more of the protein more accessible to trypsin or other protease or cleaving reagent. The alkylation of the sulfhydryl group prevents the Cys residues from forming a disulfide bond again. As for equipment, any temperature-controlled device like an incubator/oven will do. Instead of a SpeedVac, you could use a lyophilizer. Both devices are used to evaporate solvent by setting up a vacuum so the evaporation can happen in a protected environment, not exposed to air that carries dust which will have contaminating keratins. The evaporation only takes a few hours and can process many samples at one time so it should be possible to request time and space from a lab that has the equipment. What you need to ask is whether mass spectrometry is the technique for your goal. If it is to get a complete sequence at the protein level, there is a chance that some peptides may not ionize easily. Generating different peptides with a different protease is one way, Using a different way to introduce your sample into the mass spec is another way. The two most common are nano spray and MALDI. Finding a facility that has the traditional automated Edman sequencing machine is a better guarantee that you will get your full sequence, but there are not as many such facilities in operation as before since we have come to rely on cDNA sequences to get the full protein sequence. Hope this helps.
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