I have separated two peptide using SDS-PAGE. The desired band in the PAGE-gel was sliced, peptide was eluted and treated with the trypsin enzyme. The peptide fragments obtained after trypsin treatment was analysed with MALDI-TOF-MS and a peptide mass fingerprint (PMF) was obtained. I selected certain peaks of peptide and proceeded for second step of MS/MS for the selectec peptide peak. The second step MS/MS ion fingerprint was obtained. The difference in the ion peaks of same series gives an idea about the sequence of amino acids.How to identify which ion peak is 'y' ion, 'a' ion or 'b' ion.???? Please suggest me how to go for de novo sequencing of amino acids using this raw data (MS/MS ion fingerprint data).
Hi,
In order to completely de novo sequence a peptide, you'll need quite comprehensive MS/MS data. The first thing you can do is look for two fragments for which the sum of the masses equals that of the precursor. This means that you've identified a b-ion and the corresponding y-ion. From those ions on, you can look for additional ions that differ by the residual mass of the amino acids, in order to build the consecutive series of b- ions and y-ions that will eventually give you the peptide sequence. It can help to look for the immonium ions (if any are present..), which will tell you what amino acids are in the peptide: then you will know what mass differences to expect between the fragment ions.
Good luck, it could help posting the MS/MS spectrum here, if that's possible.
I am trained in this technique in (ICGEB- Delhi) India. on 2012.
I attach file , you found work plan
In order to completely de novo sequence a peptide, you'll need quite comprehensive MS/MS data.
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