title: HILIC-UPLC Analysis of Brain Tissue N-Glycans.
abstract: Extraction of N-glycans from intact tissue presents a unique set of challenges which makes it a relatively laborious and time-consuming process in comparison to other sample types, such as plasma. Here we present an approach designed for the extraction, purification, and labeling of free N-glycans from brain tissue. Using this method, up to 16 samples can be processed at once which translates to an output of 48 samples per week when rounds of extraction are staggered. Moreover, although intended for brain tissue, the method could easily be adapted to other tissue types as well. The protocol involves several stages. First, the tissue is homogenized and total proteins are isolated using chloroform-methanol extraction. The proteins are then deglycosylated using the Peptide N-Glycosidase F (PNGase F) enzyme in a reaction lasting two days. The released N-glycans are subsequently cleaned up from the reaction mixture using a centrifugal filter device and dried overnight. Next, the N-glycans are resuspended, labeled with 2-aminobenzamide (2-AB) and once again cleaned up using a filter plate. The purified N-glycans are released from the filter using ultrapure water and are then ready for analysis by for hydrophilic interaction ultra performance liquid chromatography (HILIC-UPLC).
authors.affiliation: Genos Glycoscience Research Laboratory, Hondlova 2/11, HR-10000, Zagreb, Croatia.
authors.forename: Thomas
authors.initials: T
authors.lastname: Klarić
authors.affiliation: Genos Glycoscience Research Laboratory, Hondlova 2/11, HR-10000, Zagreb, Croatia. igudelj@genos.hr.
authors.forename: Ivan
authors.initials: I
authors.lastname: Gudelj
mesh_terms: D000818:Animals; D001923:Brain Chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D054794:Glycomics / Q000379:methods*; D006023:Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification; D006031:Glycosylation; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D011134:Polysaccharides / Q000032:analysis* / Q000302:isolation & purification; D013194:Staining and Labeling / Q000379:methods; D062367:ortho-Aminobenzoates / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D006023:Glycoproteins; D011134:Polysaccharides; D062367:ortho-Aminobenzoates; C000219:anthranilamide
keywords: 2-aminobenzamide; Brain tissue; Glycomics; Glycosylation; HILIC-UPLC; N-glycans