Keck Mass Spec & Proteomics: FAQ

​The Decoy Score: is the lower the better? What is the range?​

Yes, the lower the decoy score the better.  Find additional detailed calculations and information here. Typically, you want this to be less than 5% (false discovery rate).  Many proteomics people use a more stringent number (i.e., 1-2% or less).​

Can I calculate the protein abundance in the sample based on the results? And how?​

Yes, you can calculate the relative protein abundance between the protein within the same data analysis/search results. You would need to export out the protein list in .csv from the export feature found at the end of the protein list/table (bottom right and side). Once exported, the last column is the emPAI Score, which is a unitless measurement that estimates the relative amount/abundance of a protein that was ID in relative to the other proteins. More detailed information can be found here.​

Could I utilize other enzymes to digest my sample besides trypsin?​

Having said the above, other enzymes that you can use are Lys C, Asp-N, Glu C, and chymotrypsin. Note that if we use non-trypsin enzymes, there will be additional costs to the basic protein ID costs which will account for the much higher cost to us to purchase these other enzymes. To decide on the best enzyme to use, you should do a predictive digestion of the protein sequence that you are interested in using to ensure that theoretical digested peptides have MW between 600-3000 Da for optimum MS/MS fragmentation to ID the peptide.​

Is the expectation value a measure of the confidence on the protein/peptide ID. For example, if the number is 0.05, are we 95% confident the protein is present? If it's something like "8.00E-25," are we 100% sure that the protein is there?​

Yes, the expectation value is roughly equivalent to probability, with 0.05 to be 95% confident. Here is more information on Mascot scoring and the expectation score.​

% coverage: what does that mean?​

The % coverage is the coverage of the primary amino acids sequence for the protein that is IDd.  This accounts for all the peptides that are listed in the “peptide table”.​

What is your suggestion of protease inhibitor for lysing cells and tissues?​

We must always add protease inhibitor to the lysis buffer when doing cell lysis. We must add phosphatase inhibitor when the goal is to enrich phosphopeptides. From Thermo Fisher Scientific, the cost of 1 mL protease inhibitor (100X) is $86.00 and the cost of 1 mL of phosphatase inhibitor (100X) is $176.00. Protease inhibitor cocktail (100X) and phosphatase inhibitor cocktail (100X)- store at 4C when it arrives (super convenient! No freeze-thaw necessary). 

For 1 ml lysis buffer: add 990 ul lysis buffer + 10 ul of protease inhibitor. For phosphopeptide enrichment, for 1 mL lysis buffer: add 965 ul lysis buffer + 10 ul Protease inhibitor + 25 ul phosphatase inhibitor. 

When you mix the inhibitors and the lysis buffer, just mix the amount you need and use it within a week. If not, make it fresh next time.​