Dear Michael,

if you convert your raw LC-MS file to .mpg format, upload it and do a MASCOT search it will show you based on the peptides identified in your proteomic experiment (highlighted in red) and use that to retrieve how much of your protein sequence is covered. Will that suit your purpose? 

Hope it helps, Ana

Thanks Ana. In fact I can do the same or similar with MaxQuant, which is where the data originate. But I was looking for a means to do it with a specific list of peptides that I can manually edit and create a simple figure for sharing with collaborators etc. Also it's useful to know where the enzyme has cut the protein to generate the detected peptides, which I think MaxQunt and Mascot output don't simply show.

Maybe I should use this as an excuse to write a 'simple' script.

sounds like a very good excuse!! 

Hi Mike,

Have you tried Jalview? If the protein is not too big you can do it manually, otherwise I'd stitch all your peptides into one sequence and do a pairwise alignment to the whole protein and then tweak it to suit.

Oh wait, you'd first need to know what order the peptides appear in the whole protein. Hmm...

How about this? Again in Jalview try a multiple sequence alignment of all the peptides as separate sequences relative to the whole. That'll give you the order in which to stitch them in and then do the above. It's a bit fiddly, but should work.

Nice idea Chris. I'd thought about doing the same with Clustal (sorry to swear but it's the one I know), but worried it would be almost as fiddly as a manual edit. Let's see.

Hmmm. Well with Clustal at least It seems to want to treat each entry as a protein (which makes sense for its usual application) and align them with one-another. So no matter how relaxed I make the alignment parameters, it tries to stack them one on top of the other rather then match them to the whole protein sequence. I'm guessing Jalview will be the same.

Ironically, I can do what I want with a word macro that performs multiple find and replaces using the protein sequence as the word file and the peptide list as 'find' items. But to be honest it is still a bit of a fiddly route to what seems a simple task.

Ah, yes. True. Clustal will try to do that. Not so helpful...

Maybe a script is best, but a Word macro...!? Questions can be really simple, but answering sometimes isn't.

Thanks Chris. Just let me know once it's ready ;-)

Like I said, maybe a good excuse to try to write one myself. It can't be that difficult.....

Whenever I've tried to do something similar I've used clustal with the order parameter set to 'input' and ensured that the target peptide sequence is first in any alignment file.  Something like this;

http://www.ebi.ac.uk/Tools/services/web/toolresult.ebi?jobId=clustalo-I20141105-135507-0040-71360819-oy&tool=clustalo

Obviously, you have to have already extracted just those peptides which should align and the order will be random. You can't mix and match parameters to keep the target sequence at the top and order hits by coordinates. 

The old school method of doing this is via a text representation of HSPs in a blastp job. In a former life I used to use MSPcrunch from Erik Sonnhammer to do just this.

Why don’t you use a current protein sequence aligner first (e.g. ClustalW, T-coffee) to align your peptides and the protein sequences? You can then examine the resulting multiple sequence alignment in your preferred sequence alignment viewer (e.g. Gendoc, Jalview).

I can't seem to get the alignment parameters that allow each peptide to be aligned only against the whole protein, at least with clustalW. It just stacks them up, one on top of the other thinking they are similar proteins to be aligned. But I'm not expert in sequence alignment packages so maybe you have a suggestion?

Thanks Daniel. I've just tried Clustal Omega and while in principle it does excatly what I want (thanks), the output files are very cumbersome, especially as I have over 70 peptides to align to a protein with over 1000 resudues. At this scale, adjusting the output files to make a simple representation of the protein with all the identified peptides, say, in a different colour, takes almost as long as multiple find and replace rounds in MS Word.

Surely there must be a simple algorithm somewhere where one inputs the protein sequence and all identified peptides, and it creates a simple graphic or text file showing sequence coverage......?

Probably, but I expect that they are all embedded into something else. Most blast interfaces have this functionality. As  I said MSPcrunch does this but is very old now. You could write something simple using Lincoln Stein's GD perl module based on parsing the results of a pairwise blast/blat alignment. I don't know what your scripting skills are like. If your feeling masochistic you can even do it in HTML!

Ha ha. Thanks for your time Daniel. My scripting skills ended with basic on a ZX Spectrum in 1987. It's such a simple problem, I could probably write a basic program for it. But I've been meaning to learn some form of modern programming, so maybe now is the perfect time. Thanks again. Mike

try mmass..it's a free software too!

If you are able to generate a .mgf file, why not use a mascot to generate your representation? Once you have been searching for a protein, you may click in the proteins name to get a nice alignment of all identified peptides in a ceratein protein (as long as your protein is available ind the databases that is..)

Hey guys,

was there a solution to this problem? I'm struggling with exactly the same thing now. 

Don't know if you're still struggling with this problem, but here's the solution:

PepExplorer, a software within the PatternLab suite of proteomics tools (http://patternlabforproteomics.org/) can read any list of peptides derived from de novo sequencing approaches and align these identified peptides to a protein sequence in a database in FASTA format. It will give you the coverage you expect, the number of alignments and redundancies, if any.

Another (fairly straightforward, manual) way to do this is via ncbi blastp:

1. go to

https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins&PROGRAM=blastp

2. check "Align two or more sequences"

3. Enter your template/protein sequence in the upper window

e.g.

>prot

QVQLVESGGGVVQPGGSLRLSCAASGFAFNNYGMHWVRQAPGKGLEWVAVIIFDGRNQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDLYSIYSDYKINDSWGQGTLVIVSSASTKGPSVFPL

4. Peptide sequences with fasta headers go in the lower window

e.g.

>pep1

QVQLVESGGGVVQPGGSLRLSCAAS

>pep2

MHWVRQAPGKGLEWVAV

etc..

5 Go.

Most useful output: Graphical summary, or the Alignment view (Query-anchored)