I knocked down a protein and now looking for its substrates , so need to be high confident about my proteins ID.
I used the Mass-spectrometer to analyse the samples and got my protein sequences data . After doing the comparison against the right database I got many of the 'unnamed proteins', so I blasted those unknown AA sequences against various sequence search tools, and did compare results in search for the closest homologous sequence .The main issue that I got totally different results,proteins, for blasting the same sequence on different DBs .
what criteria/parameter should I look at or rely on the most in order to be certain about my proteins ID? what is the most reliable factor to select the best hit match that most represents my sequence?
Your help would be highly appreciated.
Hi,
When you BLAST against multiple databases it is expected that you would get different IDs for same sequence. Did you try ID mapping between different databases? tht would take care of multiple databases. secondly in your homolog search of unnamed proteins if you use one organism at a time as filter (ex: mouse homolog) should help you.
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Rachita
Dear Hajer,
First check the lenght of your assembled protein sequences. The smaller they are, the less informative would the blast be. Once you have considered that, I assume you are working with a non model organism, right? If that is the case, use databases such as RefSeq and restrict the species you want to blast against: use the one that is most closely related to the species you are working with.
As for which parameter you should trust the most, I'd go first with the E-value but for each case consider also the coverage: if your best hit has a good e-value but a poor coverage, then you can discard it. I would also recommend that you use a bidirectional best hit. Once you blast your query protein, select the best hit and blast it against a local database including all your protein IDs, which ideally will retrieve your original query as the best hit.
A final note: have you tried to look for protein domains instead? Using maybe superfam or any other protein domains databases. It may worth it depending on what analysis you want to conduct hereafter.
Best regards.
Use BLAST2Go, you will get all the multiple IDs in one column. It will even give the search results based on the presence of domains in your protein, i.e, InterproScan in BLAST2Go.
The tool is very easy to run and install in windows/linux. So long you don't have a interfering internet connect.
Thanks to all of you guys.
Rachita could you elaborate more how when I BLAST against multiple databases it is expected to get different IDs for same sequence . it supposed to give a range of matching , Right? as from my short experience on blasting, my list of hits that match the sequence may vary , long or short, or no matching at all ( as I get for some ). I understand that one sequence supposed to be a homologous of one protein at least from same family and SAME or at least simillar result I supposed to get on all DP.
how to do please ID mapping between different databases, have no idea ?
Also I tried first to find a a homologous protein in my organism database , but although my organism is a model organism due to lack of annotation I did not get any match. Hence, I tried to blast against the closest species, which is a model organism, and if I still did not succeed in my homolog search I go to a higher classification ,where my organism belongs to like the Phylum. The problem here all of that did not help with those unnammed proteins , as my blast resulted in proteins from totally different family!! so how I decide which one of these my protein sequence is belongs to!
Dear Gustavo
the length of my protein sequences is between 20-800 AA. Unfortunately, most of them are small, but they are valuable to me, so need to identify. Also I am working with a model organism , but it is not fully annotated, so did not get any result on its DB neither on the most closely related species which forced me to wide my search blast to get a hit. I did also restrict the search on some DP that offer this option , but some that give a match do not give the restriction option .
As for the parameters you mentioned, definitely I look at the E-value, but some DP that did give me a result did not show the E- value , most importantly. Also, as most of my AA sequences are short (20-150), so trusting a high coverage looks not possible with such short AA, and same challenge for looking for the protein domains. Is it enough to trust only the E- value if that was my only option?
I googled the bidirectional hit and how to use it, but could not get much help , as it is new to me. Could you send me the link please. with many thanks
Different databases will have different accessions for the same protein.
Hey Salem, you can consider evalue and bitscore for infereing homology. Here is paper would help you
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3820096/
If you have short proteins, you should use a similarity scoring matrix like PAM30 or BLOSUM80 with BLAST. Or you should use a program designed for peptides, such as FASTS (available at the European Bioinformatics Institute, http://www.ebi.ac.uk/Tools/sss/fastm/). In addition to searching your organism database, consider searching a better annotated organism that is close evolutionarily.
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