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Imaging of N-linked glycans from formalin-fixed paraffin-embedded tissue sections using MALDI mass spectrometry.

ABSTRACT: Aberrant glycosylation is associated with most of the diseases. Direct imaging and profiling of N-glycans on tissue sections can reveal tissue-specific and/or disease-associated N-glycans, which not only could serve as molecular signatures for diagnosis but also shed light on the functional roles of these biomolecules. Mass spectrometry imaging (MSI) is a powerful tool that has been used to correlate peptides, proteins, lipids, and metabolites with their underlying histopathology in tissue sections. Here, we report an MSI technique for direct analysis of N-glycans from formalin-fixed paraffin-embedded (FFPE) tissues. This technique consists of sectioning FFPE tissues, deparaffinization, and rehydration of the sections, denaturing tissue proteins, releasing N-linked glycans from proteins by printing peptide-N-glycosidase F over the sections, spray-coating the tissue with matrix, and analyzing N-glycans by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Brain sections from a C57BL/6 mouse were imaged using this technique at a resolution of 100 ?m. Forty-two N-glycans were analyzed from the mouse brain section. The mass spectrometry images were used to study the relative abundance of oligomannose, nonfucosylated, and fucosylated complex N-glycans in different brain areas including isocortex, hippocampal formation, and brainstem and specific glycans associated with different areas of the brain were identified. Furthermore, glioblastoma tumor xenografts in a NOD/SCID mouse were imaged. Several glycans with differential expression in tumor versus normal brain tissues were identified. The MSI technique allows for imaging of N-glycans directly from FFPE sections. This method can potentially identify tissue-specific and/or disease-associated glycans coexpressed with other molecular signatures or within certain histological structures.

SUBMITTER: Toghi Eshghi S

PROVIDER: S-EPMC4168804 | biostudies-other | 2014 Sep

REPOSITORIES: biostudies-other

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Imaging of N-linked glycans from formalin-fixed paraffin-embedded tissue sections using MALDI mass spectrometry.

Toghi Eshghi Shadi S Yang Shuang S Wang Xiangchun X Shah Punit P Li Xingde X Zhang Hui H

ACS chemical biology 20140730 9

Aberrant glycosylation is associated with most of the diseases. Direct imaging and profiling of N-glycans on tissue sections can reveal tissue-specific and/or disease-associated N-glycans, which not only could serve as molecular signatures for diagnosis but also shed light on the functional roles of these biomolecules. Mass spectrometry imaging (MSI) is a powerful tool that has been used to correlate peptides, proteins, lipids, and metabolites with their underlying histopathology in tissue secti ...[more]

PMID: 25029481

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Project description:Ovarian cancer is a fatal gynaecological malignancy in adult women with a five-year overall survival rate of only 30%. Glycomic and glycoproteomic profiling studies have reported extensive protein glycosylation pattern alterations in ovarian cancer. Therefore, spatio-temporal investigation of these glycosylation changes may unearth tissue-specific changes that occur in the development and progression of ovarian cancer. A novel method for investigating tissue-specific N-linked glycans is using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) on formalin-fixed paraffin-embedded (FFPE) tissue sections that can spatially profile N-glycan compositions released from proteins in tissue-specific regions. In this study, tissue regions of interest (e.g. tumor, stroma, adipose tissue and necrotic areas) were isolated from FFPE tissue sections of advanced serous ovarian cancers (n = 3). PGC-LC-ESI-MS/MS and MALDI-MSI were used as complementary techniques to firstly generate structural information on the tissue-specific glycans in order to then obtain high resolution images of the glycan structure distribution in ovarian cancer tissue. The N-linked glycan repertoires carried by the proteins in these tissue regions were structurally characterized for the first time in FFPE ovarian cancer tissue regions, using enzymatic peptide-N-glycosidase F (PNGase F) release of N-glycans. The released glycans were analyzed by porous graphitized carbon liquid chromatography (PGC-LC) and collision induced electrospray negative mode MS fragmentation analysis. The N-glycan profiles identified by this analysis were then used to determine the location and distribution of each N-glycan on FFPE ovarian cancer sections that were treated with PNGase F using high resolution MALDI-MSI. A tissue-specific distribution of N-glycan structures identified particular regions of the ovarian cancer sections. For example, high mannose glycans were predominantly expressed in the tumor tissue region whereas complex/hybrid N-glycans were significantly abundant in the intervening stroma. Therefore, tumor and non-tumor tissue regions were clearly demarcated solely on their N-glycan structure distributions.

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Imaging of N-linked glycans from formalin-fixed paraffin-embedded tissue sections using MALDI mass spectrometry.

Publications

Imaging of N-linked glycans from formalin-fixed paraffin-embedded tissue sections using MALDI mass spectrometry.

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