Robust liquid chromatography – tandem mass spectrometry approaches for bis(monoacylglycero)phosphate profiling in health and disease

Introduction:: Bis(monoacylglycero)phosphate (BMP) is a unique class of anionic phospholipid enriched in late endosomes and lysosomes. BMP supports lysosomal functions and metabolic homeostasis, and its dysfunction leads to a multitude of diseases including neurodegeneration. Despite its critical function, it has been challenging to reliably identify and quantify BMP due to its relatively low abundance at the whole-cell level. Additionally, BMP and phosphatidylglycerol (PG) are isomers and only differ in the connectivity of glycerol, phosphate, and fatty acyl chains. Therefore, robust analytical techniques are required to distinguish BMP and PG. Here, we developed robust liquid chromatography – tandem mass spectrometry (LC/MS/MS) approaches that rely on ion mobility, MS/MS fragmentation, subcellular enrichment pattern and biochemical perturbation for unbiased and targeted profiling of BMP in a variety of biological samples in health and disease.

Materials and Methods:: HEK 293 cells, mouse tissues from LysoTag mouse and human clinical samples were used in this work.

Lysosome purification by LysoIP was adapted from the method previously described (DOI: dx.doi.org/10.17504/protocols.io.bybjpskn). Briefly, cells were homogenized by glass Douncers and prepared for binding with anti-HA beads in the presence of KPBS. Lipids were extracted chloroform:methanol at ratio of 2:1 (v/v) containing SPLASH LipidoMix internal standard mix, respectively.

Untargeted lipidomics was performed on an Orbitrap ID-X Tribrid mass spectrometer with a heated ESI probe. An Ascentis Express C18 150-mm by 2.1-mm column was used to carry out C18-based lipid separation.

Targeted lipid quantitation was performed on a Triple-Quadrupole mass spectrometer. An Ascentis Express C18 50-mm by 4.6-mm column was used to carry out this short C18-based lipid separation.

Results, Conclusions, and Discussions:: Our LC/MS/MS-based BMP profiling methods are widely applicable to a variety of biological systems. Using an Orbitrap ID-X mass spectrometer, we curated high-resolution tandem mass spectra for common BMP and PG species. Quantitation parameters are also reported on a Triple-Quadrupole mass spectrometer. We also established BMP mass list libraries for both the unbiased and targeted approaches. Additionally, we proposed the use of BMP identification indices to further validate BMP against its isomeric PG counterpart. Through our LC/MS/MS BMP methods, we profiled BMP level in several neurodegenerative disease models and uncovered a few BMP modulatory mechanisms within lysosomes.

Our work establishes a set of robust LC/MS/MS techniques for identifying and quantifying BMP in different biological systems. We improved the capability to distinguish BMP and PG by comparing retention time, analyzing MS/MS fragmentation and benchmarking BMP identification indices. Our clinical sample studies revealed that abnormal levels of BMP are implicated in Parkinson’s, Alzheimer’s and several lysosomal storage diseases. Our methods pave the way for us and others to investigate BMP metabolism and characterize its pathological changes in neurodegeneration and other diseases where lysosomal dysfunctions are implicated.


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