I am facing one problem with identification carried out using Protein Pilot, I get many hits whose description shows reverse protein. Does anyone have any explanation for this?
Use of Mascot however solves the problem, I don't get such ids, its only the case with protein pilot.
These are results of using a (Forward+Reverse) decoy database to estimate the False Discovery Rate. This is by now the "gold standard method" of estimating the probability of having 1% / 5% / 10% false positives overall. Sort the protein list by unused score, truncate it at the number of proteins at e.g. 1% global FDR provided in the "xxxx_FDR.xlsx", and remove the hits labeled as reverse. What remains is the list of proteins you have identified at an FDR of 1%. Mind that FDR is a property of the dataset, not the individual protein identification.
There is an ample amount of published literature. MASCOT or MaxQuant use the same approach to FDR estimation, MASCOT if you search with the "decoy" option, MaxQuant by default.
And yes, please read more. All of this is refelcted in textbooks and scientific literature.
It is strongly recommended to read manuals of PARAGON algorithm and FDR analysis before. Reversed proteins are sequences genberated by algorithm to perform FDR analysis which allows to determine border of true positive hits.
I concur with what Sergey has said, read the manual and find out what the settings for the program actually do. You are clearly selecting FDR analysis in ProteinPilot, hence the reverse hits. If you have many hits, and they are high in the list, I can only assume the data you are searching is not great.
For Mascot, you need to select ‘Decoy’ to tell it to do a decoy database search. Again, I can only assume you are not selecting this option.
Ohh thanks both of you, I do get reverse hits, but they are coming only as the last few hits, suppose I am able to resolve 3000 odd proteins, last couple of hundred proteins come as reverse hits.
Thanks for your advice of reading out the manual of protein pilot and how the algorithm really works.
Which version of ProteinPilot do you use?
Check in the "results" folder of ProteinPilot if you have a file "xxxx_FDR.xlsx". In "Protein FDR summary", you will see the border of true positive hits for a FDR at 5% or 1%.
Alain, I already know that, those hits are not coming as True Hits, I just wanted know what does it really mean by reversed protein.
I am using Protein Pilot version 4.5
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