Targeted MultiNotch MS(3) 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 Y(1) ion triggered, targeted MultiNotch MS(3) 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 Y(1) 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 Y(1) ion triggered, targeted MultiNotch MS(3) approach offers improved accuracy, precision, and sensitivity for relative quantification compared to traditional data-dependent MS(2) and Y(1) ion MS(3) quantification methods.