I want to identify a particular protein from yeast. I tried to do MSMS. But it's never giving any hit with any protein from Saccharomyces cerevisiae. For your information I have only the SDS PAGE band of the protein and I can only do the gel extraction.
Dear Sayan,
Can you please give us more information about the approach you attempted (1-D or 2-D gel? How did you obtain proteins from the gel? How did you run MS/MS? What was your readout?). I know that 2-D PAGE followed by MS/MS is a classic method, so you should be able to use that. The more info you give us about what you tried, the more we can help you find out how to improve!
Best Regards
The gel was 1D SDS PAGE. I used thermo trypsin gigestion kit to gel extract and digest the protein. I used MALDI TOF TOF for MSMS. The spectra was good. But the hit was never Saccharomyces cerevisiae. It always showed some other organism. Might be some problem with the data bases. I tried to use the raw data to run on mascot. But then also i didn't get significant result. May be the parameters in mascot as I am not so used to I have given wrong. Any guidance on how to match the raw data with specific yeast database would be highly appeciated. We tried same MSMS from 2D SDS PAGE also resulting in some hypothetical protein from other fungi (not yeast). Any other suggestion on how to identify this protein would be great. Thanks Nathaniel E Wiest for your reply. Hope to have a way out soon.
If you went directly to Maldi TOF you may be in presence of an abundant protein that hide your protein of interest. Was there trypsin in your search database? This is probably the source of your good signal. I would recommend LC-MSMS instead it will be more sensitive and more specific.
@Hugo Gagon- I didn't find trypsin in my result. there's no hit for that. LC-MSMS I didn't try. So that's another option. thanks for your answer. Is there any other way?
You should have at least sequenced Trypsin. A good MS signal doesn't explicitly that the signal you observed are peptides, may be contaminants. Other techniques: Affinity purification combined with Lc-MS/MS. If you can achieve very good purity, N-terminal sequencing. But in your case you only have 1D SDS page. Normally you should see 10s-100s of protein in a single band. If you are not detecting any yeast protein there: Consider contamination, improper system calibration, improper database search parameters. I hope this could help!
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