Monitoring the degree of in-source phospholipid fragmentation during MALDI mass spectrometry imaging

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a powerful tool for tissue lipid analysis. Yet, the validity of fatty acid (FA) signals is often questioned due to the potential of in-source lipid fragmentation. This study investigates the extent of in-source phospholipid fragmentation by examining different phospholipid headgroups, specifically phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS) and phosphatidylcholine (PC), containing a mix of saturated and unsaturated tails, comparing in-source fragmentation of FA tails. These standards evaluated phospholipid fragmentation during laser intensity optimization for MALDI-MS and MSI. Fragmentation was assessed in negative ion mode using four MALDI matrices: norharmane (NRM), 9-aminoacridine (9AA), 1,5-diaminonaphthalene (DAN), and 1,6-diphenyl-1,3,5-hexatriene (DPH). By examining the extent of in-source fragmentation during MALDI MSI, an exogenous standard can be chosen to monitor in-source fragmentation during experiments. Various techniques for standard deposition were evaluated, including manual spotting versus automated spraying and pre-depositing standards beneath tissue sections compared to applying them directly onto tissue surfaces. The results demonstrate the ability of these phospholipid standards to detect in-source fragmentation using different matrices, as demonstrated by the detection of the exogenous FA 17:0 tail signal. Furthermore, the study highlights the importance of selecting a standard that closely matches the lipid of interest to optimize laser energy for MSI, enhancing endogenous FA signals while minimizing in-source fragmentation. This work provides a workflow for mitigating in-source fragmentation in MALDI-MSI experiments, enabling more reliable lipid analysis in complex biological tissues.