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Mass Spectrometry Educational Resource




- De Novo Peptide Sequencing Tutorial 

De Novo Sequencing Software


If you have been following along with the tutorial, by now you have been through several manual de novo sequencing exercises.  The one un-blinded, and two blinded sequences have been fairly complete with abundant fragmentation.  Just to ground you in reality, this is not always the case, and  more often than not the abundance of fragment ions tends to thin near the fringes of the spectrum making it difficult to determine a complete peptide sequence.  It also makes it difficult to start a sequence, as your first jump will often be a combination of 2 or 3 amino acids.  In addition to this complication, triply charged ions or ions of higher charge states can give fragments of doubly, singly, and triply charge states, making the problem so much more complicated.  The de novo problem would seem to lend itself well to a computational solution.  Amazingly, until just recently, few if any de novo programs have given satisfactory results leading most experts in the field to say, "I can do better by hand."  Well, you've seen "first hand" the effort and the time that it takes to determine a sequence by hand.  A computational solution should be possible.  The human brain is our most powerful computer, after looking at CID spectra for many years one begins to be able to glance at a spectrum and say, "Ah, this is a peptide fragmentation." Translating this type of insight to machine language has apparently been a challenge.  Many of the earlier de novo software packages gave less than satisfactory results, leaving many of us to conclude, "hmm...." 




The Exercise:

For this part of the tutorial we have MS/MS q-TOF data in .pkl format that you can download from  The data has been "zipped" so you will need to decompress it once it is downloaded.  To download the .pkl file, right click on the download link and choose "Save Target As..." and save to a location on your computer. 

Well, you have made it through the tutorial and you are nearly a de novo expert.  Practice makes perfect.  You can request the native q-TOF data from IonSource if you want more practice, just e-mail, there is no obligation or personal data required.  The data file is 12MB so we will send it to you via a service called so that you can download it from a server, without clogging up your e-mail.

We have included a reading list, if you would like to become a true expert, just click on the next cube.  Thanks for following along.  I





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Last updated:  Monday, February 01, 2016 11:04:07 AM














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