Rapid N-Glycan Profiling of Serum and Plasma by a Novel Slide-Based Imaging Mass Spectrometry Workflow.

Abstract

Changes in the levels and compositions of N-glycans released from serum and plasma glycoproteins have been assessed in many diseases across many large clinical sample cohorts. Assays used for N-glycan profiling in these fluids currently require multiple processing steps and have limited throughput, thus diminishing their potential for use as standard clinical diagnostic assays. A novel slide-based N-glycan profiling method was evaluated for sensitivity and reproducibility using a pooled serum standard. Serum was spotted on to an amine-reactive slide, delipidated and desalted with a series of washes, sprayed with peptide N-glycosidase F and matrix, and analyzed by MALDI-FTICR or MALDI-Q-TOF mass spectrometry. Routinely, over 75 N-glycan species can be detected from one microliter of serum in less than 6.5 h. Additionally, endoglycosidase F3 was applied to this workflow to identify core-fucosylated N-glycans and displayed the adaptability of this method for the determination of structural information. This method was applied to a small pooled serum set from either obese or nonobese patients that had breast cancer or a benign lesion. This study confirms the reproducibility, sensitivity, and adaptability of a novel method for N-glycan profiling of serum and plasma for potential application to clinical diagnostics.