A new matrix assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) method to spatially profile the location and distribution of multiple N-linked glycan species in tissues is ...described. Application of an endoglycosidase, peptide N-glycosidase F (PNGaseF), directly on tissues followed by incubation releases N-linked glycan species amenable to detection by MALDI-IMS. The method has been designed to simultaneously profile the multiple glycan species released from intracellular organelle and cell surface glycoproteins, while maintaining histopathology compatible preparation workflows. A recombinant PNGaseF enzyme was sprayed uniformly across mouse brain tissue slides, incubated for 2 h, then sprayed with 2,5-dihydroxybenzoic acid matrix for MALDI-IMS analysis. Using this basic approach, global snapshots of major cellular N-linked glycoforms were detected, including their tissue localization and distribution, structure, and relative abundance. Off-tissue extraction and modification of glycans from similarly processed tissues and further mass spectrometry or HPLC analysis was done to assign structural designations. MALDI-IMS has primarily been utilized to spatially profile proteins, lipids, drug, and small molecule metabolites in tissues, but it has not been previously applied to N-linked glycan analysis. The translatable MALDI-IMS glycan profiling workflow described herein can readily be applied to any tissue type of interest. From a clinical diagnostics perspective, the ability to differentially profile N-glycans and correlate their molecular expression to histopathological changes can offer new approaches to identifying novel disease related targets for biomarker and therapeutic applications.
Using prostatic fluids rich in glycoproteins like prostate-specific antigen and prostatic acid phosphatase (PAP), the goal of this study was to identify the structural types and relative abundance of ...glycans associated with prostate cancer status for subsequent use in emerging MS-based glycopeptide analysis platforms.
A series of pooled samples of expressed prostatic secretions (EPS) and exosomes reflecting different stages of prostate cancer disease were used for N-linked glycan profiling by three complementary methods, MALDI-TOF profiling, normal-phase HPLC separation, and triple quadropole MS analysis of PAP glycopeptides.
Glycan profiling of N-linked glycans from different EPS fluids indicated a global decrease in larger branched tri- and tetra-antennary glycans. Differential exoglycosidase treatments indicated a substantial increase in bisecting N-acetylglucosamines correlated with disease severity. A triple quadrupole MS analysis of the N-linked glycopeptides sites from PAP in aggressive prostate cancer pools was done to cross-reference with the glycan profiling data.
Changes in glycosylation as detected in EPS fluids reflect the clinical status of prostate cancer. Defining these molecular signatures at the glycopeptide level in individual samples could improve current approaches of diagnosis and prognosis.
A new Matrix Assisted Laser Desorption Ionization Imaging Mass Spectrometry (MALDI-IMS) method to spatially profile the location and distribution of multiple N-linked glycan species in tissues is ...described. Application of an endoglycosidase, peptide N-glycosidase F (PNGaseF), directly on tissues followed by incubation releases N-linked glycan species amenable to detection by MALDI-IMS. The method has been designed to simultaneously profile the multiple glycan species released from intracellular organelle and cell surface glycoproteins, while maintaining histopathology compatible preparation workflows. A recombinant PNGaseF enzyme was sprayed uniformly across mouse brain tissue slides, incubated for two hours, then sprayed with 2,5-dihydroxybenzoic acid matrix for MALDI-IMS analysis. Using this basic approach, global snapshots of major cellular N-linked glycoforms were detected, including their tissue localization and distribution, structure and relative abundance. Off-tissue extraction and modification of glycans from similarly processed tissues and further mass spectrometry or HPLC analysis was done to assign structural designations. MALDI-IMS has primarily been utilized to spatially profile proteins, lipids, drug and small molecule metabolites in tissues, but it has not been previously applied to N-linked glycan analysis. The translatable MALDI-IMS glycan profiling workflow described herein can readily be applied to any tissue type of interest. From a clinical diagnostics perspective, the ability to differentially profile N glycans and correlate their molecular expression to histopathological changes can offer new approaches to identifying novel disease related targets for biomarker and therapeutic applications.
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