Mass spectrometric characterization of aminophospholipids containing N-(2-hydroxyethyl)glycine in kombu algae extracts

Rationale: 1,2-Diacyl-sn-glycero-3-phospho-O-[N-(2-hydroxyethyl)glycines] (PHEGs) are a class of rare aminophospholipids found specifically in brown algae, including kombu seaweed. Despite their potential importance in algal physiology, a comprehensive mass spectrometry (MS) characterization, useful to understand their biological behaviour, is still lacking. Methods: To establish the structural regiochemical features of PHEGs, we employed hydrophilic interaction liquid chromatography (HILIC). Following separation, the isolated band of PHEGs was analyzed using MS techniques. This included multistage tandem MS experiments, performed in both positive and negative electrospray ionization modes at low and high resolution. Results: By comparing MS/MS and MS3 spectra acquired in negative ion mode, the regiochemical rules for PHEG identification were established. The most abundant PHEG species in kombu seaweed, from both Laminaria ochroleuca (European Atlantic) and Laminaria longissima (Japan), was identified as PHEG 20:4/20:4. Less abundant species included PHEG 20:4/20:5 and hydroxylated forms of both PHEG 20:4/20:4 (i.e. 40:8;O) and 20:4/20:5 (40:9;O). The presence of a lyso PHEG 20:4 was consistently detected but at very low levels. Conclusions: This study employed MS analysis to elucidate the regiochemical patterns of PHEGs in kombu seaweed. We identified PHEG 20:4/20:4 as the dominant species, along with several less abundant variants, including hydroxylated forms. These findings provide valuable insights into the potential roles and metabolism of PHEGs in brown algae, paving the way for further investigation into their biological functions.