Targeted MultiNotch MS³ Approach for Relative Quantification of N-Glycans Using Multiplexed Carbonyl-Reactive Isobaric Tags

The recently developed and commercially available carbonyl-reactive tandem mass tags (aminoxyTMT) enable multiplexed quantification of glycans through comparison of reporter ion intensities. However, challenges still exist for collision activated dissociation (CAD) MS/MS based quantification of aminoxyTMT due to the relatively low reporter ion yield especially for glycans with labile structures. To circumvent this limitation, we utilized the unique structural features of N-glycan molecules, the common core sugar sequence (HexNAc)₂(Man)₃, and common m/z of Y_n ions generated from different types of precursors by MS/MS and designed a Y₁ ion triggered, targeted MultiNotch MS³ relative quantification approach based on aminoxyTMT labeling. This approach was implemented on a nanoHILIC-Tribrid quadrupole-ion trap-Orbitrap platform, which enables prescreening of aminoxyTMT labeled N-glycan precursor ions by Y₁ ion fragment ion mass in a higher-energy collisional dissociation (HCD) MS/MS scan and coisolation and cofragmentation of multiple Y_n fragment ions that carry the isobaric tags from the inclusion list in the MS/MS/MS scan. Through systematical optimization and evaluation using N-glycans released from several glycoprotein standards and human serum proteins, we demonstrated that the Y₁ ion triggered, targeted MultiNotch MS³ approach offers improved accuracy, precision, and sensitivity for relative quantification compared to traditional data-dependent MS² and Y₁ ion MS³ quantification methods.