Abstract
Simple, robust and versatile LC–MS based methods add to the rapid assessment of the lipidome of biological cells. Here we present a versatile RP-UHPLC-MS method using 1-butanol as the eluent, specifically designed to separate different highly hydrophobic lipids. This method is capable of separating different lipid classes of glycerophospholipid standards, in addition to phospholipids of the same class with a different acyl chain composition. The versatility of this method was demonstrated through analysis of lipid extracts of the bacterium Escherichia coli and the archaeon Sulfolobus acidocaldarius. In contrast to 2-propanol-based methods, the 1-butanol-based mobile phase is capable of eluting highly hydrophobic analytes such as cardiolipins, tetraether lipids and mycolic acids during the gradient instead of the isocratic purge phase, resulting in an enhanced separation of cardiolipins and extending the analytical range for RPLC.
Highlights
Cellular membranes fulfil the essential function of compartmentali zation, separating cellular contents from the extracellular environment and enable the formation and maintenance of specialized reaction compartments
To provide sufficient retention for less hydro phobic analytes, such as lyso-phospholipids, up to 40 % of MQ was added. This was sufficient to allow for separation of dodecyl maltoside (DDM) and lyso-phosphatidic acid (LPA) and lyso-phosphatidyl glycerol (LPG) without causing miscibility issues when combined with eluent B
Mobile phase performance was compared to the same mobile phase with 2-PrOH instead of 1-BuOH and two 2-PrOH-based mobile phases described in literature (Damen et al, 2014; Criscuolo et al, 2019)
Summary
Cellular membranes fulfil the essential function of compartmentali zation, separating cellular contents from the extracellular environment and enable the formation and maintenance of specialized reaction compartments. NPLC is often used to analyze envi ronmental samples and generally uses relatively apolar mobile phase solvent mixtures consisting of 2-propanol (2-PrOH), hexane, chloroform and small amounts of water This is used in combination with bare silica or diol-columns to separate lipids into different lipid classes, mostly based on polar head group hydrophilicity (Sollai et al, 2019; Sturt et al, 2004; Van Mooy et al, 2009; Scherer et al, 2010; Van Mooy et al, 2006). In contrast to NPLC-based techniques, RPLC uses hydrophobic sta tionary phases, most commonly C18-modified silica combined with more polar solvent mixtures such as water, MeCN and 2-PrOH This difference in solvents and column material shifts the selectivity of RPLC methods to separate lipids mostly based on the hydrophobic properties of the lipid tail. We present a versatile RP-UHPLC-ESI-MS method capable of separating complex biological lipid mixtures, including especially hydrophobic molecules such as the isoprene-based cardiolipin mole cules and tetraether lipid species that are often found in archaeal lipid membranes
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