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Isolation of Complex Glycosphingolipids in Pancreatic Tissue Using Hydrophilic Interaction Liquid Chromatography-Electrospray Ionization Mass Spectrometry

Autoři: Veseli Vulnet | Hořejší Karel | Jirásko Robert | Peterka Ondřej | Holčapek Michal

Rok: 2024

Druh publikace: ostatní - přednáška nebo poster

Strana od-do: nestránkováno

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Jazyk	Název	Abstrakt	Klíčová slova
eng	Isolation of Complex Glycosphingolipids in Pancreatic Tissue Using Hydrophilic Interaction Liquid Chromatography-Electrospray Ionization Mass Spectrometry	Glycosphingolipids (GSL), predominant in higher animals, represent a highly heterogeneous class of lipids[1]. The study of these important molecules with many cellular functions could lead to new ways of diagnosing and treating human diseases[2]. GSLs are frequently present at extremely low levels, requiring the utilization of more sensitive strategies for their detection. This involves robust extraction and separation methods prior to performing mass spectrometry (MS) analysis[1]. In the biological matrix, GSLs coexist with dominant lipid subclasses, such as acylglycerols, phospholipids, and sphingomyelins (SM), along with biopolymers, such as proteins and nucleic acids. Consequently, they are relatively less abundant, and they compete for ionization[1]. In this study, non-lyophilized porcine pancreatic tissue was homogenized using a tissue homogenizer and an appropriate volume of solvent. The elimination of major lipids, such as acylglycerols and phospholipids was done by alkaline methanolysis. After cleaning and evaporating, acetylation using a combination of pyridine and acetyl chloride was used to change the polarity of GSL in order to separate high abundant sphingomyelins from low-abundant GSL. This approach involved silica chromatography, where sphingomyelins can be retained in the silica column, while modified (acetylated) GSL are eluted using varying percentages of methanol in chloroform. The fraction containing GSL are then evaporated, reconstituted in chloroform/methanol (1:1, v:v), and analyzed using Hydrophilic interaction liquid chromatography (HILIC) coupled to electrospray ionization QTOF mass spectrometry. The developed method can be used for further clinical and biological research of GSL in healthy or cancerous human pancreatic tissue.	Isolation; Glycosphingolipids; Pancreatic Tissue; Hydrophilic Interaction; Liquid Chromatography; Ionization Mass Spectrometry

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