Identification of remnant OCT media artifacts after tissue washing by MALDI mass spectrometry imaging

RATIONALE Universally, the most common method of embedding dissected or resected fresh frozen (FF) tissues is within optimal cutting temperature (OCT) media. Unfortunately, OCT is not ideal for mass spectrometry (MS) assays. The media perfuses through tissues, resulting in persistent polymer contamination causing issues for sensitive analyses. Complete removal of all OCT media background would enable techniques such as MS imaging (MSI) to be bridged to clinically relevant samples stored in biobanks which possess extensive medical histories. Even though methods have been outlined for spatial lipidomics and proteomics, many still avoid these tissues despite vast potential for biological discoveries applying MS or MSI. METHODS Reference FF human pancreas was prepared within OCT media and cryosectioned for matrix-assisted laser desorption/ionization (MALDI)-MSI. Sample preparation protocols using aqueous ammonium formate for spatial lipidomics, and a variety of solutions for fixation, delipidation, and salt removal enabled spatial proteomics as well. MSI was performed on a UHMR HF Orbitrap MS detected broad classes of lipids and intact proteins, enabling an assessment of polymer perfusion and signal delocalization over a broad mass range to evaluate any deleterious artifacts. RESULTS We observe lower polymer contamination from OCT tissues with ammonium formate washes as expected, with several caveats. This was most effective in negative ion mode, where in tandem with the boosted lipid signal positive results were achieved for phospholipids (600-900 Da) and higher mass lipids (>1250 Da). Positive ion mode analyses can be completed for phospholipids, but the condensation of alkali adducts to protonated forms not only increased lipid signals, but also increased signals of larger, persistent PEG oligomers (>1250 Da) which were not previously reported. Even with extensive tissue washing completing a spatial proteomics protocol, the removal of all remnant artifacts of OCT media was not feasible. CONCLUSIONS The finding of these persistent remnant polymers from the OCT embedding process has several consequences for MSI, directly effecting primary performance of the MALDI source and lipid and protein analyses. This does not mean OCT tissues should not be or cannot be used for spatial omics by MSI, but several quality control and quality assurance measures should be implemented, and we discuss paths forward and several considerations.