How to use Bio Tool Kit for interpreting peptide MS/MS data acquired with CID and EAD fragmentation?

Peptide MS/MS spectra acquired using either collision induced dissociation (CID) or electron activated dissociation (EAD) can be analyzed using the Peptide Fragments pane in Bio Tool Kit in SCIEX OS Software. Modification can be added to the peptide sequence as a mass shift. The Bio Tool Kit is a very manual and time consuming options compared to more automated software such as Biologics Explorer (BE). It can be used strategically for specific synthetic or modified peptide sequences. This is why a we demonstrate it for a glycopeptide G1F example.
This community post explains how to used the Bio Tool Kit for EAD obtained M/MD spectra.   https://community.sciex.com/2021/08/07/using-bio-tool-kit-for-interpreting-peptide-ms-ms-data-obtained-using-electron-activated-dissociation-ead/  The SCIEX OS software help for TOF instruments describes the Bio Tool Kit Peptide Fragment for CID MS/MS data. It can be found here: C:\Program Files\Sciex\SCIEX OS\Documentation.

How to interpret MS/MS glycopeptide data using the Bio Tool Kit for CID:

Step 1: Open the CID MS/MS spectrum that you want to interpret. Then, launch the Peptide Fragments pane from the Bio Tool Kit menu.
Step 2:  Open the Options window from the top, left corner of the fragments pane to adjust the fragment ions computed for CID. For CID MS/MS of peptides use a,b and y-ions.



Matched fragments will display as bold red. You need to drag the blue arrow on the y-axis downward to label all the MS/MS fragments on the spectrum and for them to appear as matches in the fragments pane.
This glycopeptide G1F will only show the mass shift for the N-glycan portion. CID (Collision-Induced Dissociation) tend to produce abundant glycosidic bond cleavages for glycopeptides so called B- and Y-ions (Ref. Domon, Costello, Glycoconj. J. 5 (1988) 397-409).  Unfortunately,  the Bio Tool Kit reaches its limitation and cannot  annotated B-and Y ions. The CID spectrum of the Glycopeptide G1F shows predominantly the oxonium ions of monosaccharides from the glycan portion as shown with BE software.

However, the Bio Tool kit will give you a hint for a possible glycan composition for this attached N-glycan.

How to interpret MS/MS glycopeptide data using the Bio Tool Kit for EAD:

Step 1: Open the EAD MS/MS spectrum that you want to interpret. Then, launch the Peptide Fragments pane from the Bio Tool Kit menu.
Step 2:  Open the Options window from the top, left corner of the fragments pane to adjust the fragment ions computed for EAD. For EAD MS/MS of peptides, we recommend turning on the C, z, z+1, and z+2 ions.

Matched fragments will display as bold red. You need to drag the blue arrow on the y-axis downward to label all the MS/MS fragments on the spectrum and for them to appear as matches in the fragments pane.
The charge state computed for both the precursor and the fragment ions can be changed, then click compute.  A red italic number in the pane indicates a match to a fragment in a different charge state than that displayed.
In contrast to CID MS/MS spectra, EAD MS/MS spectra show the fragmentation scheme for production of c- and z-type ions and the reduced number of glycopeptide and its charge reduced species. Second, it efficiently dissociates the peptide backbone while leaving the labile glycan mostly intact. The Bio Tool Kit annotate  the  cleavage of affects  N‐C bond, so that the resulting fragment ion spectra typically show c or z type ions. See Table match. It  cannot annotate the charge reduced species. Typically this occurs by capturing an electron, [M+nH]^•(n-1)+ is produced, where charge is reduced and this has an unpaired electron (•).