The gtAnnotate tool provides interactive composition analysis and spectrum annotation across a hierarchy of MSn spectra.
The tiles of the gtAnnotate windows are as follows:
- A hierarchy tree of MSn spectra loaded for analysis is displayed in the top left tile.
- A list of possible compositions for the selected precursor ion is displayed in upper right tile. In this view, H represents a hexose, N a HexNAc, D a deoxyhexose, h a reduced hexose, and n a reduced HexNAc. Other residue types are supported but not shown. The notation "(ene)" represents a double-bond "scar" left behind by a glycosidic bond cleavage of a permethylated structure, and "(OH)" represents an open hydroxyl scar. Roughly, "(ene)" corresponds to the Costello/Domon B- and Z-type ions, and "(OH)" corresponds to C- and Y-type ions (Domon and Costello).
- List of possible compositions for each product ion peak within the precursor ion spectrum is displayed in the middle right tile.
- An interactive spectrum display is located in the bottom tile or wherever the analyst chooses to locate the additional tile. The analyst can zoom and scroll within this area to examine peaks in detail.
The screenshot exhibits the gtAnnotate analysis window, as hosted by gtSuite 2.0. In this example, a set of 18 MSn spectra from an enzymatically released, reduced, permethylated glycan sample derived from chicken egg ovalbumin has been loaded for analysis. The root spectrum is the MS2 spectrum for ion m/z 1677.78. The indentation represents product spectra, so the first 662.30 entry represents the MS6 spectrum generated by fragmenting ion m/z 1677.78, then 1384.55, 1125.55, 866.36, and finally 662.30. The user can select any spectrum from this list and the remaining gtAnnotate tiles will update accordingly. As displayed there are 17 possible compositions for ion m/z 1677.78. Importantly, most of the composition possibilities may be ruled out via selection of the appropriate user constraintss (as shown in the subsequent screenshot), or by simply checking the Exclude ("Excl.") checkbox next to any composition.
User Options include:
- Apply precursor/product constraints: This constraint eliminates product composition assignments that cannot have come from the available precursor compositions. This allows the conversion of an m/z MSn fragmentation pathway into a feasible glycan composition pathway.
- Require N-linked core composition: This option eliminates all compositions and pathways that are not compatible with the conserved N-linked pentasaccharide core. If this constraint is not selected, gtAnnotate is free to propose any structure as well as perform O-linked glycan and glycosphingolipid analysis.
- Root ion is unfragmented glycan: The gtSuite 2.0 tool performs data analysis on fragment ions as well as intact glycan structures. When this constraint is selected, all possible compositions originate from an intact fully permethylated glycan composition at the initial MS stage.
- Hide excluded possibilities: When a composition has been ruled out, either by option or via the "Excl." exclusion checkmark, the entry is normally still displayed, but grayed out. This option hides excluded compositions from view.
- Show spectrum statistics: Displays information in the spectrum display, about the number of possible and excluded compositions for each spectrum.
- Exclude relative intensity below X%: This option excludes excludes fragment ion peaks that fall below an assigned peak intensity threshold.
- Apply: Updates the display to reflect any changed user constraint selections.
The screenshot below illustrates gtAnnotate's view of the same data set after the analyst has applied several constraints. Note that there is now exactly one possible composition for the root ion m/z 1677.78 (H3N3n), and only 11 compositions can be assigned to product ion peaks on the root spectrum. User constraint selection greatly reduces the dataset's complexity and gives the analyst much better insight across the full set of MSn spectra.
Applying these user constraints changes across all 18 spectra executes in less than 0.1 seconds.