Antibody Panel Based N-glycan Profiling by MALDI MS for N-Glycoprotein Biomarker Discovery from Patient Biofluids
Alyson P. Black, Peggi M. Angel, Richard R. Drake, Anand S. Mehta Medical University of South Carolina
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Alyson Black (Presenter) Medical University of South Carolina
Relevant Financial Disclosures
(within past 24 months)
No relevant financial relationship(s) to disclose.
Abstract
Introduction: The majority of biomarkers used in the detection of cancer are glycoproteins, and recent reports have indicated that the glycan component of the glycoprotein can act as a more powerful marker of cancer than the protein component. Described here is a novel platform for N-glycoprotein biomarker discovery from patient biofluid samples using MALDI mass spectrometry.
Objectives: The primary objective was to use a novel MS-based method for multiplexed analysis of glycoproteins from human serum in the context of cancer biomarker discovery.
Methods: Antibody Panel Based N-glycan profiling is adapted from MALDI MS imaging methodologies, and uses MALDI MS for 2D localization of N-glycans across immunocaptured glycoproteins. Antibodies are spotted to microscope slides in a panel and incubated with serum samples, requiring no further clean-up of the samples. N-glycans are enzymatically released from captured glycoproteins with PNGaseF, which is sprayed to retain localization of glycans. Slides are then sprayed with CHCA matrix and imaged with a MALDI FT-ICR MS for N-glycan detection corresponding to each protein. N-glycan intensities from each captured glycoprotein are quantified by area under monoisotopic peak and compared across patient groups.
Results: An antibody panel targeting 7 serum glycoproteins was created and found to reproducibly detect N-glycans from the captured proteins: alpha-1-antitrypsin, alpha-1-acid glycoprotein, haptoglobin, hemopexin, immunoglobulin G, low molecular weight kininogen, and transferrin. This method was used to analyze serum samples from a cohort of patients with hepatocellular carcinoma compared to a control group with liver cirrhosis. A pattern of increased sialylation was observed across the proteins in patients with hepatocellular carcinoma, as well as changes in fucosylation that varied by protein.
Conclusion: This novel mass spectrometry approach allows for multiplexed N-glycoprotein analysis from patient biofluids. APB N-glycan profiling can be expanded to include any glycoprotein for which a validated antibody exists - analyzing potentially 100s of individual glycoproteins in just one MALDI MS run. Additionally, this platform can be adapted for any biofluid or biological sample that can be analyzed by antibody arrays. This technique has exciting potential to be applied in the clinic as both a biomarker discovery tool as well as a screening tool for cancer using minimal amounts of readily available clinical samples.
= Discovery stage.
= Translation stage.
= Clinically available.
