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A feature for Identifying intact glycopeptides from MS/MS scans has been an integral part of MASSAI for years, but sometimes it is practical to separate the glycans from the peptide chemically, for instange with a PNGase prior to search, in order to establish a glycan population.
In the new version, you can load peaklists from both MS/MS analyses of released glycans, and from intact glycopeptides, AND combine the two types of setup for higher confidence.
In the sample below, we have chosen to import 14 mgf files as one batch.
13 of these are MS/MS runs of the intact glyco-peptides, fragmented in CID and HCD modes.
The last mgf file contains MS/MS scans of released glycans (separated with PNGase and then modified with PMP).
We use the "CID/HCD" and "RELEASED GLYCANS" buttons to specify the type of file. The specification for each file is also shown in the
"FRAGMENT MODE" column.
Then we define the search space. In this case we have a FASTA file handy, containing the sequences of two proteins in the sample.
We select the proteases used (three in our case) and allow for two missed cleavage sites per peptide.
Next we select the peptide modification(s) to include in our search. (The PMP modification on the glycan is selected later).
Our protein sample has been isolated from pork, and so we restrict our glycan search to the types of glycosylation commonly associated with mammals.
The released glycans have been modified with PMP to facilitate fragmentation. This is selected from the list.
We expect some degree of sodium adduct to the glycan moiety.
The search returns a list of identified released glycans.
Notice the large number of high-scoring glycans with sodium adducts.
As always, any result can be clicked in order to view the annotated scan(s).
Here I have highlighted an identified high-mannose structure with five mannoses and one Na adduct.
The annotated scan shows predominantly fragments from the core 5 structure along with the attached PMP molecule, and also a handful of oxonium ions. This pattern is very common when we use HCD fragmentation.
If we then turn our attention to the intact glycopeptides, we also find a mannose 5 glycosylation on a specific residue in a number of scans.
Notice that the results from the released glycans are used to annotate that the released glycan has indeed been observed on its own. Both unmodified and sodiated glycans count as an observation.