MSACL 2023 Abstract

INTRODUCTION: Alterations in the glycosylation of circulating and cell surface glycoproteins is a hallmark of cancer, inflammation and most disease processes. High throughput and reproducible analysis of glycosylation in liquid biopsy and tissue samples at the clinical diagnostic level has long been challenging, due to lengthy processing, derivatization and processing workflows. Our group has collectively adapted an N-glycan MALDI imaging mass spectrometry method to multiple approaches for analysis of biofluids, cultured cells and immune cells using direct slide-based capture approaches.

OBJECTIVES: Develop a platform technology to rapidly and reproducibly detect glycan changes by MALDI-QTOF MS profiling in liquid biopsy clinical specimens.

METHODS: Serum cohorts related to early detection of breast and liver cancers were evaluated, as well as human and mouse peripheral blood mononuclear cells (PBMC). For biofluid glycan profiling, diluted aliquots of biofluids are spotted directly on amine reactive glass slides, rinsed, and sprayed (HTX imaging, Durham, NC) with a molecular coating of PNGase F to release N-glycans. For antibody array capture, antibodies to major serum glycoproteins and immune cell markers are spotted on amine reactive slides, and incubated with serum or immune cell samples. Released N-glycans are detected using a timsTOF fleX MALDI-QTOF (Bruker, Billerica, MA). Data analysis is done using SCiLS Lab software (Bruker, Billerica, MA)

RESULTS: The standard N-glycan MALDI IMS workflow for analysis of clinical FFPE tissues involves an antigen retrieval step followed by molecular spraying of PNGase F to release N-glycans. After application of matrix, the N-glycans are detected by MALDI MS. The enzyme spraying and detection steps have proven to be robust and reproducible across thousands of tissue samples. We have subsequently found that applying any biological sample that contains N-glycans to a slide can be assayed with essentially the same digestion and detection workflow developed for tissue slides. Four main approaches have been adapted to on-slide N-glycan analysis of non-tissue samples: cultured cells grown directly on slides; blood and urine spotted directly on amine-reactive slides; individual glycoproteins captured on antibody arrays; and individual immune cell types captured by anti-CD marker antibody arrays. The cultured cell and direct biofluid methods are established and published. Profiling of total N-glycan content in large cohorts of breast cancer and benign serum samples (n = 298), and liver cancer and cirrhosis serum cohorts, have been recently completed Analysis of N-glycans in lupus nephritis plasma, urine, and urine exosomes are ongoing. Using specific antibodies spotted onto slides to capture and N-glycan profile individual serum glycoproteins has been developed initially by targeting immunoglobulin G (IgG) and IgG sub-types. Serum or plasma samples are incubated in wells containing the specific antibody, and following rinsing, N-glycans attached to the captured glycoprotein are released by sprayed PNGase F and detected at each antibody-antigen spot by MALDI MS. Termed GlycoTyper, this approach is being optimized for commercial diagnostic applications for early detection of fatty liver disease, cirrhosis and liver cancers. In an ongoing approach using a serum cohort (n =200) representing subjects with cirrhosis or early liver cancers, a slide array that includes 15 additional antibodies to acute phase reactant serum glycoproteins was evaluated. The newest assay developed involves use of antibodies to immune cell markers like CD4 and CD8 to capture and glycoprofile individual immune cell subtypes directly on a glass slide. Different mouse and human immune cell mixtures have been used to optimize capture and processing details. A unique aspect of this assay is the visualization and counting of captured immune cells by light microscopy prior to glycan release and MS analysis. For each of the four assay formats, example applications and initial results in clinical samples, experimental parameter optimizations, data analysis and quality control issues will be discussed.

CONCLUSIONS: The method workflow originally created for N-glycan tissue imaging MS has been adapted to be an extensible and rapid glycan analysis platform. Glycoproteins in clinical biofluids and cellular samples captured on antibody array slides can be efficiently evaluated for N-glycan content using a timsTOF fleX MALDI QTOF. This clinical biospecimen glycan analysis platform technology is amenable to analysis of large clinical sample cohorts and can be significantly expanded to include additional antibodies of interest for glycoprotein and cell capture applications.