Abstract
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.
Highlights
As one of the most important posttranslational modifications, protein glycosylation has crucial biological and physiological roles, from contributions to protein folding and quality control to involvement in various types of biological recognition events [1]
In the course of optimization, we compared two membrane glass slides (i.e., polyethylene naphthalate (PEN) and polyphenylene sulfide (PPS)) used for laser microdissection (LMD)-Lectin microarray (LMA) analysis of Ulex europaeus agglutinin-I (UEA-I)-stained thymic sections and found that similar glycomic profiles were obtained with these membrane glass slides (Figure S1 and Table S2)
The previously developed LMD-LMA method is a powerful tool for detecting tissue- and site-sTpheecipfircevpirooutesilny dgleyvceolosypleadtioLnMiDn-FLFMPAE tmisestuheodseicstiaonpsow[1e4r]f;uhlotwooelvfeorr, dtheitsecmtientghotidssiusen- oatnhdigsihtelyscpelelc-tiyfipcepsrpoetceiifincg
Summary
As one of the most important posttranslational modifications, protein glycosylation has crucial biological and physiological roles, from contributions to protein folding and quality control to involvement in various types of biological recognition events [1]. Disease-related alterations in protein glycosylation are promising targets for the discovery of glyco-biomarkers and development of therapeutic agents [2,3]. Lectin microarray (LMA) is a lectin-assisted glycomic technology used to obtain glycomic profiles of N- and O-glycans in glycoprotein samples without glycan liberation steps, enabling evaluation of the characteristics of glycan structures using simple and rapid procedures [4,5]. In LMA, glycoprotein samples are fluorescently labeled at their primary amine groups, allowing for glycans attached to glycoproteins to be selectively analyzed [5]. LMA facilitates mass spectrometry-based glycoproteomics for detailed analyses of glycosylation sites and their glycan structures [6,7], and this technology is useful for the most upper phases of glyco-biomarker discovery [3,8]. LMA is applicable for glycomic profiling formalin-fixed paraffin-embedded (FFPE)
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