Highly sensitive analysis of N-glycans via derivatization with quinoline hydrazide reagents

Abnormal serum N-glycans are linked to various diseases, offering potential for early diagnosis. However, glycans lack readily ionizable groups and their high hydrophilicity further hinders ion formation, resulting in low detection sensitivity in mass spectrometry. To overcome this, we synthesized five hydrophobic quinoline hydrazide reagents to derivative N-glycans, aiming to enhance MS performance. This study evaluates these reagents for derivatization efficiency and sensitivity improvement, focusing on their application in glycomic analysis of human serum for disease biomarker discovery.

All five quinoline hydrazide reagents increased the detection sensitivity of maltotetraose (DP7) by over 20-fold in LC-ESI-MS compared to underivatized glycans. The most effective reagent, 2-methyl-6-methoxy-4-quinoline hydrazide (MMQCH), enhanced sensitivity by 32-fold. When applied to human serum N-glycans, MMQCH derivatization significantly expanded the number of detectable glycan species from 19 to 58. Comparative analysis of serum from lung cancer patients and healthy controls revealed several differentially expressed glycan biomarkers, such as H4N3F1, with high statistical significance (p < 0.0001). These findings demonstrate that quinoline hydrazide-based derivatization substantially improves glycan detection sensitivity and enables the identification of potential diagnostic markers in clinical samples.

This study introduces an effective derivatization strategy using quinoline hydrazide reagents to significantly enhance N-glycan detection sensitivity in MS. The improved method facilitates the discovery of clinically relevant glycan biomarkers, supporting early disease diagnosis. These findings advance glycomic analysis and offer a practical tool for developing non-invasive diagnostic assays based on serum N-glycan profiling.