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Mass Spectrometry Imaging of N-Linked Glycans in FFPE Human Prostate by Infrared Matrix-Assisted Laser Desorption Electrospray Ionization

ABSTRACT: N-linked glycans are structurally diverse polysaccharides that represent significant biological relevance due to their involvement in disease progression and cancer. Due to their complex nature, N-linked glycans pose many analytical challenges requiring the continued development of analytical technologies. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is a hybrid ionization technique commonly used for mass spectrometry imaging (MSI) applications. Previous work demonstrated IR-MALDESI to significantly preserve sialic acid containing N-linked glycans that otherwise require chemical derivatization prior to detection. Here we demonstrate the first analysis of N-linked glycans in situ by IR-MALDESI MSI. Formalin-fixed paraffin-embedded (FFPE) human prostate tissue was analyzed in negative ionization mode after tissue washing, antigen retrieval, and pneumatic application of PNGase F for enzymatic digestion of N-linked glycans. 53 N-linked glycans were confidently identified in the prostate sample where more than 60% contained sialic acid residues. This work demonstrates the first steps in N-linked glycan imaging of biological tissues by IR-MALDESI MSI.

INSTRUMENT(S): Orbitrap Exploris 240

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: David C. Muddiman

PROVIDER: MSV000088414 | MassIVE | Fri Nov 19 05:32:00 GMT 2021

REPOSITORIES: MassIVE

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Project description:Aberrant protease activity has been implicated in the etiology of various prevalent diseases including neurodegeneration and cancer, in particular metastasis. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) has recently been established as a key technology for bioanalysis of multiple biomolecular classes such as proteins, lipids, and glycans. However, it has not yet been systematically explored for investigation of a tissue’s endogenous protease activity. In this study, we demonstrate that different tissues, spray-coated with substance P as a tracer, digest this peptide with different time-course profiles. Furthermore, we reveal that distinct cleavage products originating from substance P are generated transiently and that proteolysis can be attenuated by protease inhibitors in a concentration-dependent manner. To show the translational potential of the method, we analyzed protease activity of gastric carcinoma in mice. Our MSI and quantitative proteomics results reveal differential distribution of protease activity – with strongest activity being observed in mouse tumor tissue, suggesting the general applicability of the workflow in animal pharmacology and clinical studies.Aberrant protease activity has been implicated in the etiology of various prevalent diseases including neurodegeneration and cancer, in particular metastasis. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) has recently been established as a key technology for bioanalysis of multiple biomolecular classes such as proteins, lipids, and glycans. However, it has not yet been systematically explored for investigation of a tissue’s endogenous protease activity. In this study, we demonstrate that different tissues, spray-coated with substance P as a tracer, digest this peptide with different time-course profiles. Furthermore, we reveal that distinct cleavage products originating from substance P are generated transiently and that proteolysis can be attenuated by protease inhibitors in a concentration-dependent manner. To show the translational potential of the method, we analyzed protease activity of gastric carcinoma in mice. Our MSI and quantitative proteomics results reveal differential distribution of protease activity – with strongest activity being observed in mouse tumor tissue, suggesting the general applicability of the workflow in animal pharmacology and clinical studies.

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Mass Spectrometry Imaging of N-Linked Glycans in FFPE Human Prostate by Infrared Matrix-Assisted Laser Desorption Electrospray Ionization

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