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- De Novo Peptide Sequencing Tutorial
De Novo Sequencing Software
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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...."
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The Exercise: For this part of the tutorial we have MS/MS q-TOF data in .pkl format that you can download from IonSource.com. 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 webmaster@ionsource.com, there is no obligation or personal data required. The data file is 12MB so we will send it to you via a service called yousendit.com 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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