Robust analytical methods for bis(monoacylglycero)phosphate profiling in health and disease

Bis(monoacylglycero)phosphate (BMP), a distinct anionic phospholipid predominantly found in late endosomes and lysosomes, plays a pivotal role in supporting lysosomal functions and maintaining metabolic homeostasis. Its impaired function is associated with an array of disorders, notably neurodegenerative diseases. However, the identification and quantitation of BMP remains difficult due to its structural similarity to isomer phosphatidylglycerol (PG), thus necessitating robust analytical methods for accurate and reliable BMP profiling. In this study, we present comprehensive liquid chromatography – tandem mass spectrometry (MS2) methodologies for the precise and systematic analysis of BMP species in biological samples. We detail LC/MS methods for both an untargeted Orbitrap mass spectrometer and a targeted triple quadrupole (QQQ) mass spectrometer. We utilize differences in polarity and structure to annotate BMPs and PGs based on retention time and positive mode MS2 fragmentation patterns, respectively. Further, we propose a new approach for overcoming common challenges in BMP profiling by leveraging the newly discovered biochemical function of CLN5 as the BMP synthase. Since genetic ablation of CLN5 leads to specific depletion of BMPs but not PGs, we use lipid extracts from *CLN5* knockout (KO) and wild-type (WT) cells as biological standards to confidently annotate BMPs as targets with significantly low BMP Identification Index (BMPII), defined as BMPII = *CLN5* KO / WT. We additionally propose the BMP enrichment score (BMPES) as a secondary validation metric, defined as lysosomal abundance of BMP / whole-cell abundance. Altogether, this approach constitutes a robust method for BMP profiling and annotation, furthering research into health and disease.