A 5-Da isotope labeling strategy combined with a novel binary matrix 2-aminoisophthalic acid/α-cyano-4-hydroxycinnamic acid for accurate fruit N-glycome quantification in MALDI-TOF-MS

Universal 5-Da labeling creates internal standards for accurate N-glycome analysis. • Novel matrix 2-AIA/CHCA ensures uniform co-crystallization and high reproducibility. • H3N2F1X1 is a cross-species candidate biomarker for fruit cold stress. N-glycosylation is a crucial post-translational modification regulating protein function in plants. However, its accurate quantification by matrix-assisted laser desorption/ionization-mass spectrometry with time-of-flight is challenged by low glycoprotein abundance, poor glycan ionization, uneven matrix crystallization, and insufficient internal standards. Here, we introduce a novel 5-Da stable isotope labeling method, combining enzymatic 18 O-labeling (+2-Da) and reduction with NaBD 4 (+3-Da) on a portion of the sample itself to universally generate internal standards that eliminate isobaric interference. Furthermore, a binary matrix of 2-aminoisophthalic acid (2-AIA) plus α-cyano-4-hydroxycinnamic acid (CHCA) was developed, providing high sensitivity and homogeneous co-crystals. The integration of isotope labeling with 2-AIA/CHCA allows linear quantification ( R 2  > 0.99) over a two orders of magnitude. Applied to tomato and peach during cold storage, the method revealed that H3N2F1X1, the most abundant N-glycan in both fruits, was significantly downregulated, suggesting its role as a candidate biomarker for cold stress response and fruit quality control. Download: Download high-res image (204KB) Download: Download full-size image