Thank you Mr. Malchow. I am sorry, but is there any other methods beside MS & Edman for peptide sequencing? Edman Peptide Sequencer is not available to me at all, and only GC-MS may be managed.

I was wondering if ionizer of the MS causes significant fragmentation (e.g. electron impact), is sequencing possible? I read a few articles where MALDI-TOF has been used for fingerprinting, but not de novo sequencing. Is in-source dissociation not sufficient here?

Of course, in the end, I quess I can't just make up methods without any validation.

Hi Shadid

yes, you can use MS only, but of course it's harder. You can use accurate mass, because that in itself will help to disambiguate, especially below 3ppm mass accuracy - but from your note it looks like you have a nominal mass instrument?

You also don't say whether this is a known protein: if it is known, or there are analogs, you can compare intact mass and tryptic digest information against a database search and narrow down the options. (standard proteomics technique). Then if you want to know more you should use the chromatography as an indicator of likely sequence and make sure your separations are good, because you can use the orthogonal information. In LC you should also use a UV in-line because you can tune to a wavelength that is specific to peptides (and ignore non peptides). Also, as Sebastian pointed out, you can use cone voltage to do the fragmentation to a certain degree and therefore get b/y fragments to provide information. If you combine with GC, then you can perform derivatization for the GC experiments that are specific to amino acids (there is decades of literature on this; kits are easy to get, e.g. Sigma) and therefore combine your information. That's why we write software to automate all of that process, because it's tedious by hand!

So it's a combination of techniques, especially if you need every single AA in the sequence and you don't know the protein. But of course you may find it easier to speak to a friend with a high res mass spec .... 

good luck!

St John

http://www.proteinmetrics.com/supernovo/

https://www.ncbi.nlm.nih.gov/pubmed/22709442

Thank you Mr. Skilton.

I am interested in identifying unknown antimicrobial peptides.

Regarding the instrument, honestly the GC-MS is located in another lab. I don't have much specifics about it yet, all I know is that it is a GC-MS. I'll have to go through various channels to access the instrument. That is why I asked this question. If a GC-MS can't do the job, I won't make any approach. But since now I know that it may be possible, I'll try to find out if it a HRMS or LRMS.

Thanks again.

Dear Sir,

We done our ESI-MS of our novel recombinant protein sample and found varying color (green,red,yellow) of peptide when matching to our known protein sequence (isolated and purified from plant source its ms spectra have identified 7 peptide and expressed it recombinantly). I have found all the peptide matching in our ESI-MS of recombinant protein with native protein ms (out of 7 peptide we have found 3 green 1 yellow and 3 red peptide). please clarify should i consider it my recombinant protein is have same sequence as native plant protein. Secondly what could be the reason of getting varying colour peptide.

Please clarify the significant of peptide deduced in sequence represented with varying color like green,Red and yellow. Could you please elaborate the percentage of similarity level of green, yellow and green peptides????

Thanks

Regards