Matrix assisted laser desorption ionization imaging mass spectrometry workflow for spatial profiling analysis of N-linked glycan expression in tissues.
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.
Department of Cell and Molecular Pharmacology and Experimental Therapeutics and MUSC Proteomics Center, Medical University of South Carolina , 173 Ashley Avenue, Charleston, South Carolina, 29425 United States.
Thomas W
TW
Powers
E Ellen
EE
Jones
Lucy R
LR
Betesh
Patrick R
PR
Romano
Peng
P
Gao
John A
JA
Copland
Anand S
AS
Mehta
Richard R
RR
Drake
D000818:Animals; D001921:Brain / Q000378:metabolism*; D005786:Gene Expression Regulation; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D051379:Mice; D011134:Polysaccharides / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D011134:Polysaccharides; D006026:Glycoside Hydrolases