2-Nitro-4-Carboxyphenylhydrazine and 2,4-Dicarboxylphenylhydrazine as a Pair of Novel Reactive MALDI Matrices for Rapid and Accurate Profiling of N-Glycome in Dual Ion Modes

N-glycosylation is closely linked to a wide range of biological functions in organisms. Owing to the constriction of awful crystals formed by conventional MALDI matrices and the extremely inferior ionization efficiency of N-glycans, the traditional direct detection of N-glycans by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been gradually replaced by postderivatization detection using reactive matrices. Nevertheless, the laborious identification of complex spectral peaks remains the major difficulty in N-glycan profiling. Hence, we logically designed and synthesized two novel reactive matrices, 2-nitro-4-carboxylphenylhydrazine (NCPH) and 2,4-dicarboxylphenylhydrazine (DCPH), and separately combined them with the acidic matrix 2,5-dihydroxybenzoic acid (DHB) to constitute two composite matrices with high on-target derivatization efficiency and significant promotion of N-glycan ionization for productive MALDI analysis in dual ion modes. Using both composite matrices, we can actualize MALDI-MS and MS² mass calibration in dual ion modes by postderivatization detection and fragmentation of dextrans and selectively enhance the ionization effect of oligosaccharides in mixed systems. Quite homogeneous cocrystals can ensure N-glycan quantification with decent linearity and reproducibility. A fixed mass difference derived from the identical N-glycan in two ion modes is available for rapid identification in complex biological samples. Ultimately, the developed strategy was triumphantly employed to identify and quantify the relative content and alteration tendency of peach N-glycans, which can be referable to the latent correlation between N-glycan expression and peach ripening.