Electron Transfer Higher-Energy Collisional Dissociation Can Distinguish Cobalt-Adducted Isomers of Human Milk Oligosaccharides

The ability to identify isomers of human milk oligosaccharides (HMOs) remains a challenge. Typically, HMOs are analyzed using a combination of liquid chromatography and tandem mass spectrometry (LC-MS/MS). Yet, separation methods have been unable to resolve several HMO isomers, necessitating the need for fragmentation methods that can differentiate these structures within mixtures. Common fragmentation methods, such as collision-induced dissociation (CID), often fail to form fragments that can differentiate the linkage and branching isomers. Previously, we determined that electron transfer dissociation (ETD) yields distinct fragmentation products when using certain metal-charge carriers, such as Co²⁺. Here, we compare fragmentation methods, including CID, higher-energy collisional dissociation (HCD), ETD, and electron transfer, higher-energy collisional dissociation (EThcD) for Co²⁺- and Na⁺-adducted HMO isomers. The formation of several fragments using only ETD and EThcD, but not CID and HCD, indicates that certain fragmentation products only form from electron dissociation. EThcD enabled differentiation of Co²⁺-adducted tri-, tetra-, and pentasaccharide isomers, while CID, HCD, and ETD were incapable of differentiating all the examined HMO isomers as Co²⁺, Na⁺, and 2Na²⁺ adducts. We also show that Co²⁺ adduction combined with EThcD generates fragments that are related to the core structure and fucose branching location, allowing for further validation of the HMO structures. This work establishes a general method using fragmentation that can be used to differentiate HMOs, which should enable improved identification and structural validation of various carbohydrate linkage and branching isomers.