Analysis of Neuronal Cardiolipin and Monolysocardiolipin from Biological Samples with Cyclic Ion Mobility Mass Spectrometry

Katlynn Joy Emaus^1,2Carmen A. Dunbar^3,4Joseph Caruso⁵Brandon T. Ruotolo⁴Joseph M Wider^1,2,6Thomas H Sanderson^1,2,6,7*

• ¹Neuroscience Graduate Program, University of Michigan, Ann Arbor, Michigan, United States
• ²Department of Emergency Medicine, School of Medicine, Michigan Medicine, University of Michigan, Ann Arbor, Michigan, United States
• ³Biological Mass Spectrometry Core, University of Michigan, Ann Arbor, Michigan, United States
• ⁴Department of Chemistry, College of Literature, Science and the Arts, University of Michigan, Ann Arbor, Michigan, United States
• ⁵Olink, Thermo Fisher Scientific, Waltham, Massachusetts, United States
• ⁶The Max Harry Weil Institute for Critical Care Research, University of Michgian, Ann Arbor, Michigan, United States
• ⁷Department of Molecular and Integrative Physiology, School of Medicine, University of Michigan, Ann Arbor, Michigan, United States

The mitochondrial phospholipid cardiolipin is essential for proper mitochondrial function and energy production. Cardiolipin has four distinct fatty acid tails with varying expression composition, resulting in a highly variable tissue specific distribution of isomer expression. Neuronal cardiolipin has a remarkable variety of subspecies and has recently been used as a biomarker to predict brain injury severity following cardiac arrest and traumatic brain injury.Alzheimer's Disease, Parkinson's Disease, Barth Syndrome, and astrocytoma. The clinical relevance of cardiolipin as a biomarker and the importance of the mechanistic role of cardiolipin remodeling in disease emphasizes the demand for a reliable and accurate means of identification and quantification of cardiolipin. In this study we outline the use of a novel method of cardiolipin analysis using cyclic ion mobility mass spectrometry (cIMS-MS) to isolate and identify cardiolipin subspecies in several biological samples. Further cIMS-MS established the composition of the cardiolipin profile by individual subspecies across biological samples under basal conditions. Identification of monolysocardiolipin, the precursor mature cardiolipin, and a primary diagnostic biomarker of Barth Syndrome, was isolated from cardiolipin and identified. The monolysocardiolipin : cardiolipin ratio was quantified in brain samples from Tafazzin knockout mice, demonstrating accumulation of MLCL, providing direct evidence of the validity of this cIMS-MS methodology through genetic loss-of-function. The novel, multiple pass feature of cIMS-MS provided isolation and amplification of less abundant cardiolipin subspecies, in standards and in biological samples. This protocol allows rapid analysis of biological samples allowing researchers to further dissect the mechanistic role of cardiolipin in injury pathology with simplified sample preparation and a reduced potential introduction of artifact.