Rapid Automated Annotation and Analysis of N-Glycan Mass Spectrometry Imaging Data Sets Using NGlycDB in METASPACE

Abstract

Imaging N-glycan spatial distribution in tissues using mass spectrometry imaging (MSI) is emerging as a promising tool in biological and clinical applications. However, there is currently no high-throughput tool for visualization and molecular annotation of N-glycans in MSI data, which significantly slows down data processing and hampers the applicability of this approach. Here, we present how METASPACE, an open-source cloud engine for molecular annotation of MSI data, can be used to automatically annotate, visualize, analyze, and interpret highresolution mass spectrometry-based spatial N-glycomics data. METASPACE is an emerging tool in spatial metabolomics, but the lack of compatible glycan databases has limited its application for comprehensive N-glycan annotations from MSI data sets. We created NGlycDB, a public database of N-glycans, by adapting available glycan databases. We demonstrate the applicability of NGlycDB in METASPACE by analyzing MALDI-MSI data from formalin-fixed paraffin-embedded (FFPE) human kidney and mouse lung tissue sections. We added NGlycDB to METASPACE for public use, thus, facilitating applications of MSI in glycobiology.

Spatial Mapping of Plant N-Glycosylation Cellular Heterogeneity Inside Soybean Root Nodules Provided Insights Into Legume-Rhizobia Symbiosis

“…To obtain maximal coverage of N -glycans from the soybean root nodules, we tested and compared commercially available recombinant PNGaseF PRIME that has been widely used in N -glycan MALDI-MSI of mammalian tissues (Powers et al, 2014 ; Carter et al, 2020 ; Blaschke et al, 2021 ; Mcdowell et al, 2021 ) and acidic PNGase H+ that has previously shown high specificity in bulk plant N -glycan profiling (Du et al, 2015 ). For assessing their efficiency, we concurrently sprayed these enzymes over the cross-section of wild type and nifH - FFPE soybean root nodule, analyzed released glycans by MALDI-MSI, and automatedly annotated N -glycans using the NGlycDB (Velickovic et al, 2021 ) in METASPACE. METASPACE is an open cloud software platform that performs molecular annotations for MSI data—based not only on the accurate mass information but also on a comprehensive bioinformatics framework that considers the relative intensities and spatial colocalization of isotopic peaks, as well as quantifies spatial information with a measure of spatial chaos followed by the estimation of the False Discovery Rate (FDR) (Palmer et al, 2017 ).…”

“…METASPACE is an open cloud software platform that performs molecular annotations for MSI data—based not only on the accurate mass information but also on a comprehensive bioinformatics framework that considers the relative intensities and spatial colocalization of isotopic peaks, as well as quantifies spatial information with a measure of spatial chaos followed by the estimation of the False Discovery Rate (FDR) (Palmer et al, 2017 ). Recently, we introduced NGlycDB which contains naturally occurring N -linked glycans, including all reported plant glycans, for researchers worldwide to annotate their spatial N -glycomics using METASPACE (Velickovic et al, 2021 ).…”

“…Imaging data files were imported into the SCiLS lab software, exported without normalization to imzML, and the resulting .imzML and .ibd files were then submitted to METASPACE for data processing and annotation. The NGlycDB (Velickovic et al, 2021 ) was chosen as the database, with positive polarity and [M+Na] + selected as the ionization adduct. The default METASPACE m/z tolerance of ± 3 ppm was used.…”

“…The method relies on spraying the enzyme peptide N -glycanase (PNGase) over formalin-fixed paraffin-embedded (FFPE) tissue sections to in-situ release the N -linked carbohydrates from their carrier proteins. Released glycans are then spatially probed by MALDI-MSI, which uses a focused laser to ablate and ionize molecules for MS analysis (Drake et al, 2018 ; Velickovic et al, 2021 ). In this work, we adapted this approach, by using a unique and novel plant-specific PNGase, to demonstrate its validity, robustness, and depth of coverage for characterizing plant N -glycans' spatial patterns and cell-type-specific profile.…”

Controlled Humidity Levels for Fine Spatial Detail Information in Enzyme-Assisted N-Glycan MALDI MSI

“…This problem is even more emphasized in highly heterogeneous tissues, composed from the spatially close but different cell types (e.g., kidney). For example, reported ion images of released N -glycans in kidney tissue sections can often look “smeared” without distinct, clear, spatial patterns that colocalize with glomeruli or tubular structures. ,, …”