An improved evanescent fluorescence scanner suitable for high-resolution glycome mapping of formalin-fixed paraffin-embedded tissue sections.

Lectin microarray (LMA) is a high-throughput platform that enables the rapid and sensitive analysis of N- and O-glycans attached to glycoproteins in biological samples, including formalin-fixed paraffin-embedded (FFPE) tissue sections. Here, we evaluated the sensitivity of the advanced scanner based on the evanescent-field fluorescence principle, which is equipped with a 1× infinity correction optical system and a high-end complementary metal-oxide semiconductor (CMOS) image sensor in digital binning mode. Using various glycoprotein samples, we estimated that the mGSR1200-CMOS scanner has at least fourfold higher sensitivity for the lower limit of linearity range than that of a previous charge-coupled device scanner (mGSR1200). A subsequent sensitivity test using HEK293T cell lysates demonstrated that cell glycomic profiling could be performed with only three cells, which has the potential for the glycomic profiling of cell subpopulations. Thus, we examined its application in tissue glycome mapping, as indicated in the online LM-GlycomeAtlas database. To achieve fine glycome mapping, we refined the laser microdissection-assisted LMA procedure to analyze FFPE tissue sections. In this protocol, it was sufficient to collect 0.1 mm2 of each of the tissue fragments from 5-µm-thick sections, which differentiated the glycomic profile between the glomerulus and renal tubules of a normal mouse kidney. In conclusion, the improved LMA enables high-resolution spatial analysis, which expands the possibilities of its application classifying cell subpopulations in clinical FFPE tissue specimens. This will be used in the discovery phase for the development of novel glyco-biomarkers and therapeutic targets, and to expand the range of target diseases.

LM-GlycomeAtlas Ver. 2.0: An Integrated Visualization for Lectin Microarray-based Mouse Tissue Glycome Mapping Data with Lectin Histochemistry.

Laser microdissection-assisted lectin microarray has been used to obtain quantitative and qualitative information on glycans on proteins expressed in microscopic regions of formalin-fixed paraffin-embedded tissue sections. For the effective visualization of this "tissue glycome mapping" data, a novel online tool, LM-GlycomeAtlas (https://glycosmos.org/lm_glycomeatlas/index), was launched in the freely available glycoscience portal, the GlyCosmos Portal (https://glycosmos.org). In LM-GlycomeAtlas Version 1.0, nine tissues from normal mice were used to provide one data set of glycomic profiles. Here we introduce an updated version of LM-GlycomeAtlas, which includes more spatial information. We designed it to deposit multiple data sets of glycomic profiles with high-resolution histological images, which included staining images with multiple lectins on the array. The additionally implemented interfaces allow users to display multiple histological images of interest (e.g., diseased and normal mice), thereby facilitating the evaluation of tissue glycomic profiling and glyco-pathological analysis. Using these updated interfaces, 451 glycomic profiling data and 42 histological images obtained from 14 tissues of normal and diseased mice were successfully visualized. By easy integration with other tools for glycoproteomic data and protein glycosylation machinery, LM-GlycomeAtlas will be one of the most valuable open resources that contribute to both glycoscience and proteomics communities.

N-glycan structures of Wisteria floribunda agglutinin-positive Mac2 binding protein in the serum of patients with liver fibrosis†.

The extent of liver fibrosis predicts prognosis and is important for determining treatment strategies for chronic hepatitis. During the fibrosis progression, serum levels of Mac2 binding protein (M2BP) increase and the N-glycan structure changes to enable binding to Wisteria floribunda agglutinin (WFA) lectin. As a novel diagnostic marker, glycosylation isomer of M2BP (M2BPGi) has been developed. However, its glycan structures recognized by WFA are unclear. In this study, we analyzed site-specific N-glycan structures of serum M2BP using Glyco-RIDGE (Glycan heterogeneity-based Relational IDentification of Glycopeptide signals on Elution profile) method. We evaluated five sample types: (1) M2BP immunoprecipitated from normal healthy sera (NHS-IP(+)), (2) M2BP immunoprecipitated from sera of patients with liver cirrhosis (stage 4; F4-IP(+)), (3) M2BP captured with WFA from serum of patients with liver cirrhosis (stage 4; F4-WFA(+)), (4) recombinant M2BP produced by HEK293 cells (rM2BP) and (5) WFA-captured rM2BP (rM2BP-WFA(+)). In NHS-IP(+) M2BP, bi-antennary N-glycan was the main structure, and LacNAc extended to its branches. In F4-IP(+) M2BP, many branched structures, including tri-antennary and tetra-antennary N-glycans, were found. F4-WFA(+) showed a remarkable increase in branched structures relative to the quantity before enrichment. In recombinant M2BP, both no sialylated-LacdiNAc and -branched LacNAc structures were emerged. The LacdiNAc structure was not found in serum M2BP. Glycosidase-assisted HISCL assays suggest that reactivity with WFA of both serum and recombinant M2BP depends on unsialylated and branched LacNAc and in part of recombinant depends on LacdiNAc. On M2BPGi, the highly branched LacNAc, probably dense cluster of LacNAc, would be recognized by WFA.

An Improved Method for Cell Type-Selective Glycomic Analysis of Tissue Sections Assisted by Fluorescence Laser Microdissection.

Lectin microarray (LMA) is a highly sensitive technology used to obtain the global glycomic profiles of endogenous glycoproteins in biological samples including formalin-fixed paraffin-embedded tissue sections. Here, we describe an effective method for cell type-selective glycomic profiling of tissue fragments collected by laser microdissection (LMD) under fluorescent histochemical visualization. We optimized each step of histochemical staining and confirmed the reliability and validity of glycomic profiling. Using the optimized procedure, glycomic profiles were obtained with 0.5 mm² of stained thymic sections (5-µm-thick) from 8-week-old C57BL/6J male mice. The glycomic profiles of Ulex europaeus agglutinin-I (UEA-I)-stained medullary regions showed higher UEA-I signals than those of the morphologically determined medulla regions, indicating the utility of this method for UEA-I(+) cell-selective analysis. To further evaluate this method, tissue fragments was serially collected from stained and unstained areas of medullary epithelial cell probes (UEA-I and anti-cytokeratin 5 antibody) and a cortex-staining probe (peanut agglutinin). The medullary regions assigned by the three probes showed significantly different glycomic profiles, highlighting the difference in subpopulation recognition among the three probes, which was consistent with previous reports. In conclusion, our fluorescence LMD-LMA method enabled cell type-selective tissue glycomic analysis of pathological specimens and animal models, especially for glyco-biomarker discovery.

LM-GlycomeAtlas Ver. 1.0: A Novel Visualization Tool for Lectin Microarray-Based Glycomic Profiles of Mouse Tissue Sections.

For the effective discovery of the biological roles and disease-specific alterations concerning protein glycosylation in tissue samples, it is important to know beforehand the quantitative and qualitative variations of glycan structures expressed in various types of cells, sites, and tissues. To this end, we used laser microdissection-assisted lectin microarray (LMA) to establish a simple and reproducible method for high-throughput and in-depth glycomic profiling of formalin-fixed paraffin-embedded tissue sections. Using this "tissue glycome mapping" approach, we present 234 glycomic profiling data obtained from nine tissue sections (pancreas, heart, lung, thymus, gallbladder, stomach, small intestine, colon, and skin) of two 8-week-old male C57BL/6J mice. We provided this LMA-based dataset in the similar interface as that of GlycomeAtlas, a previously developed tool for mass spectrometry-based tissue glycomic profiling, allowing easy comparison of the two types of data. This online tool, called "LM-GlycomeAtlas", allows users to visualize the LMA-based tissue glycomic profiling data associated with the sample information as an atlas. Since the present dataset allows the comparison of glycomic profiles, it will facilitate the evaluation of site- and tissue-specific glycosylation patterns. Taking advantage of its extensibility, this tool will continue to be updated with the expansion of deposited data.

Differential Glycoform Analysis of MUC1 Derived from Biological Specimens Using an Antibody-Overlay Lectin Microarray.

The association between altered glycosylation of MUC1 and various disease events has sparked significant interest. However, analytical technologies to investigate the disease-related glycoforms of endogenous MUC1 in blood and tissue specimens are limited. Therefore, we devised a reliable technique for differential analysis of endogenous MUC1 glycoforms based on an antibody-assisted lectin microarray. Its highly sensitive detection aids in analyzing soluble MUC1 from relatively small amounts of serum via a simple enrichment process. Micro-/macro-dissection of the MUC1-positive region is combined with glycoform analysis of the membrane-tethered MUC1. Thus, we have optimized the protocol for sample qualification using immunohistochemistry, sample pretreatment for tissue sections, protein extraction, purification via immunoprecipitation, and the antibody-overlay lectin microarray, which are sequentially essential for differential glycoform analysis of endogenous MUC1.