Profiling and quantification of aminophospholipids based on chemical derivatization coupled with HPLC-MS

Abstract

and metabolic roles (1). PLs consist of a glycerol backbone esterified with two fatty acids at the sn-1 and sn-2 positions. The backbone's sn-3 position is occupied by a phosphate group attached to a polar head of different nature. Aminophospholipids [phosphatidylethanolamine (PE) and phosphatidylserine (PS)] belong to the category of PLs. PE and PS account for approximately 20% and 3-15% of total PLs in eukaryotic cells, respectively. They play a critical role in regulating various biological processes, including intraand inter-cellular signaling, phagocytic recognition, apoptotic cell clearance, cell division, angiogenesis, and vascular remodeling under normal physiological conditions in eukaryotic cells (2). Furthermore, aminophospholipids are associated with various human diseases, including cancer, diabetes, obesity, and neurodegeneration (3, 4). Hence, profiling of the individual aminophospholipid molecular species in cellular components is critical to link physiological or pathophysiological changes to the underlying biochemistry and thus target therapeutic interventions. However, precise profiling of aminophospholipids is not easy because of the sheer number and the high complexities of their structures. Direct infusion MS equipped with an ESI source holds much promise for the characterization of PLs (5). Through effective intra-source separation of predetermined groups of lipid classes according to their intrinsic electrical propensities, lipid extracts can be analyzed directly without preseparation. However, the disadvantages of the direct infusion MS method are ion suppression for lipid species with low proton affinities, negative impact of unavoidable matrix effects on the analysis of minor content of lipid species in biological samples, and serious contamination to Abstract In this study, a novel strategy based on acetone stable-isotope derivatization coupled with HPLC-MS for profiling and accurate quantification of aminophospholipids (phosphatidylethanolamine and phosphatidylserine) in biological samples was developed. Acetone derivatization leads to alkylation of the primary amino groups of aminophospholipids with an isopropyl moiety; the use of deuterium-labeled acetone (d6-acetone) introduced a 6 Da mass shift that was ideally suited for profiling and quantification analysis with high selectivity and accuracy. After derivatization, significantly increased column efficiency for chromatographic separation and detection sensitivity for MS analysis of aminophospholipids was observed. Furthermore, an accuracy quantification method was developed. Aminophospholipids in biological samples were derivatized with d0-acetone; while more than two aminophospholipid standards were selected for each class of aminophospholipid and derivatized with d6-acetone, which were then used as the internal standards to typically construct a calibration curve for each class to normalize the nonuniformity response caused by the differential fragmentation kinetics resulting from the distinct chemical constitution of individual...

Glycosphingolipids and Infection. Potential New Therapeutic Avenues

“…Improvements have been made in lipid extraction methods (Cruz et al, 2016; Löfgren et al, 2016) and internal standards, such as isotope encoded analogs, become increasingly available (Wisse et al, 2015; Mirzaian et al, 2016; Wang et al, 2017). Derivatization or deacylation of specific lipids may assist their quantitative detection (Mirzaian et al, 2017; Ma et al, 2019). An important new development is the availability of techniques to study the biology of lipids in living cells.…”

Deep Profiling of Aminophospholipids Reveals a Dysregulated Desaturation Pattern in Breast Cancer Cell Lines

“…Other methods such as fluorenylmethoxylcarbonyl chloride and 4-(dimethylamino)­benzoic acid NHS ester and trimethylation enhancement using diazomethane (TrEnDi) have been demonstrated for improved analysis of APLs from biological samples. Recently, Ma et al coupled reductive alkylation via d 0 / d 6 -acetone with reversed-phase LC–MS for profiling of APLs . The derivatization was found effective in reducing peak tailing of PS on the column, another common cause for poor separation and low detection sensitivity of PSs by LC–MS…”

“…Recently, Ma et alkylation via d 0 /d 6 -acetone with reversed-phase LC−MS for profiling of APLs. 15 The derivatization was found effective in reducing peak tailing of PS on the column, another common cause for poor separation and low detection sensitivity of PSs by LC−MS. 16 Despite these improvements, detailed structural information of APLs, such as the location of carbon−carbon double bonds (CCs), cannot be obtained by only derivatizing the amine function group.…”