The gtIsoDetect tool performs three major functions:

• Lists MSⁿ fragmentation pathways and assigns compositions consistent with a proposed structure.
• Identifies MSⁿ pathways and product ions whose compositions could not have come from a set of expected structures, indicating the possibility that additional structural isomers are present.
• Determines what fragmentation pathways are diagnostic to a specific structural isomer.

The screenshot below exhibits the gtIsoDetect window in gtSuite 2.0. To invoke gtIsoDetect, the analyst first uses gtAnnotate to assign compositions to the peaks in the MSⁿ data set, and then selects the gtIsoDetect tab.

The tiles of the gtIsoDetect windows are as follows:

• The linear code of the expected structure(s) is displayed in the top tile.
• A graphical representation of the selected expected structure is displayed directly below the linear code entry box. Circles represent hexoses (H) and squares represent HexNAcs (N). The reducing-end residue (n) is on the right.
• The full list of MSⁿ fragmentation pathways extracted from the previously-defined gtAnnotate data set is displayed in the bottom left tile. This tile is called the Compatibility Report. A checkmark in the S1 column means the pathway is compatible with expected structure number one. A checkmark in the S2 column means the pathway is compatible with expected structure two, and so forth. A disassembly pathway is compatible with a structure if some sequential disassembly of the structure yields the observed MSⁿ ion sequence. If a pathway is not marked, then that pathway is not compatible with any of the entered structure candidates.
• A representation of how the selected MSⁿ fragmentation pathway is or is not compatible with each expected structure is displayed in the bottom right tile. The selected MSⁿ fragmentation pathway is interactive and when an m/z value is selected the origin of the fragment within the compatible structure is highlighted in the graphical representation of the compatible proposed structure.

There are two documented structural isomers with the composition of Hex³HexNAc4 reported for chicken egg ovalbumin (Reference). The checkmarks in the Compatibility Report tile denote MSⁿ fragmentation pathways that are consistent with a proposed structure. For example, the pathway 1677.78_1384.55_1125.55_866.36_648.27_444.18 has a series of assigned compositions that are consistent with the second structure having a bisecting HexNAc residue. In the selected pathway, the terminal ion has a possible composition of HN-(ene)(OH3)--that is, a HN fragment with four cleavages required to remove it from the intact glycan. This fragment within the context of the MSⁿ fragmentation pathway is compatible with structure two and not compatible with structure one.

The Compatibility Report, also, contains MSⁿ fragmentation pathways that are not consistent with the expected two structures indicating the possible presence of additional structural isomers. For example, the 3rd pathway from the top m/z 1677.78_1384.55_1125.55_866.36_676.27_458.27_268.0 has a series of assigned compositions that is not compatible with the two expected structures. There is no stepwise disassembly of the two expected structures that match this observed pathway. After further investigation, the analyst may propose a third structure, which is compatible with this pathway, and enters it into the upper left tile using the linear code. The result is shown in the gtIsoDetect screenshot below and demonstrates the pathway m/z 1677.78_1384.55_1125.55_866.36_676.27_458.27_268.0 is compatible with the third proposed structural isomer. The execution of a full compatibility report for three proposed structures is generated in approximately 0.1 seconds. To our knowledge, this third structure, having two terminal N-acetylhexosamine residues extending from 1,3-linked hexose of the pentasaccharide core, has not been reported in the chicken egg ovalbumin. This finding illustrates the power of combining permethylated MSⁿ and the gtIsoDetect tool for the interpretation of mass spectral data and accelerating the discovery of additional glycan structures.