Graphene Oxide/TiO<sub>2</sub> Nanocomposite-Assisted\nTwo-Step Ambient Liquid Extraction Mass Spectrometry Imaging for Comprehensively\nEnhancing Lipid Coverage in Spatial Lipidomics

It is challenging to have comprehensive spatial lipidomic\nanalysis\nby mass spectrometry imaging (MSI) due to the strong ion suppression\nand peak interference from high-abundance polar lipids to low-abundance\npoorly ionizable lipids. In this work, we proposed a new MSI approach\nvia ambient liquid extraction techniques assisted by a new mixed-mode\nadsorptive material, graphene oxide (GO)/TiO<sub>2</sub> nanocomposite.\nThe material combines chelation affinity from TiO<sub>2</sub> and\nhydrophobic interaction from GO. By finely tuning the adsorption solvent\nas 9% H<sub>2</sub>O–6% ammonia–85% methanol/acetonitrile\n(1:1, v/v), simultaneous enrichment of poorly ionizable glycolipids\nand glycerides with separation from high-abundance phospholipids was\nachieved on the material. In 10 mg/mL glucose–6% ammonia–94%\nmethanol, all of the adsorbed glycolipids and glycerides could be\ndesorbed from the material efficiently. Then, GO/TiO<sub>2</sub> nanocomposite\nwas coated onto the sample plate for thaw-mounting the tissue section,\nand ambient liquid extraction probe was used to have pixel-to-pixel\ndesorption of the lipids on the section in two steps with the above\ntwo solvents. The results show that most of the phospholipids were\nimaged in the first step MSI, and glycolipids and glycerides were\nselectively imaged in the second step MSI, largely reducing ion suppression\nand peak interference. Compared with direct ambient liquid extraction\nMSI, much more glycolipid species (22 vs 9), glyceride species (10\nvs 5), phosphatidylethanolamines (11 vs 3), and lysophospholipids\n(12 vs 2) were detected via GO/TiO<sub>2</sub> nanocomposite-assisted\ntwo-step MSI. The ion images of most lipids show much higher signals\nand imaging quality with the new method than with the traditional\nmethod. Thus, comprehensive enhancement of lipid coverage in MSI by\non-tissue separation was achieved here for the first time, providing\nmore information about spatial lipidomics studies.