Citation: Yadav, M.; Chaudhary, P.P.; D’Souza, B.N.; Spathies, J.; Myles, I.A. Impact of Skin Tissue Collection Method on Downstream MALDI-Imaging. Metabolites 2022, 12, 497. https://doi.org/10.3390/ metabo12060497 Academic Editors: Fidele Tugizimana, Michele Costanzo, Marianna Caterino and Lucia Santorelli Received: 23 March 2022 Accepted: 26 May 2022 Published: 30 May 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). metabolites H OH OH Article Impact of Skin Tissue Collection Method on Downstream MALDI-Imaging Manoj Yadav * ,† , Prem Prashant Chaudhary † , Brandon N. D’Souza, Jacquelyn Spathies and Ian A. Myles Epithelial Therapeutics Unit, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD 20892, USA; premprashant.chaudhary@nih.gov (P.P.C.); dsouza.80@buckeyemail.osu.edu (B.N.D.); jacquelyn.spathies@nih.gov (J.S.); mylesi@niaid.nih.gov (I.A.M.) * Correspondence: manoj.yadav@nih.gov † These authors contributed equally to this work. Abstract: MALDI imaging is a novel technique with which to study the pathophysiologies of diseases. Advancements in the field of metabolomics and lipidomics have been instrumental in mapping the signaling pathways involved in various diseases, such as cancer and neurodegenerative diseases (Parkinson’s). MALDI imaging is flexible and can handle many sample types. Researchers primarily use either formalin-fixed paraffin-embedded (FFPE) or fresh frozen tissue samples to answer their scientific questions. FFPE samples allow for easy long-term storage, but the requirement for extensive sample processing may limit the ability to provide a clear picture of metabolite distribution in biological tissue. Frozen samples require less handling, but present logistical challenges for collection and storage. A few studies, mostly focused on cancer cell lines, have directly compared the results of MALDI imaging using these two tissue fixation approaches. Herein, we directly compared FFPE and fresh frozen sample preparation for murine skin samples, and performed detailed pathway analysis to understand how differences in processing impact MALDI results from otherwise identical tissues. Our results indicate that FFPE and fresh frozen methods differ significantly in the putative identified metabolite content and distribution. The fixation methods shared only 2037 metabolites in positive mode and only 4079 metabolites in negative ion mode. However, both fixation approaches allowed for downstream fluorescent staining, which may save time and resources for samples that are clinically precious. This work represents a direct comparison of the impacts of the two main tissue processing methods on subsequent MALDI-MSI. While our results are similar to previous work in cancer tissue, they provide novel insights for those using MALDI-MSI in skin. Keywords: MALDI-MSI; FFPE; IHC; metabolites 1. Introduction Matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI) is an expanding tool which aims to understand the presence and distribution of different biomarkers, such as peptides, lipids, and other small metabolites. Unlike traditional MS, the spatial distribution of metabolites in tissue section can be studied using the MALDI- MSI. However, tissue samples must first be preserved prior to analysis. Two types of preservation methods are widely used: formalin-fixed paraffin-embedded (FFPE) and fresh frozen. FFPE preservation allows for retrospective analysis of tissue samples even after long term storage, but requires the samples to pass through enzymatic digestion, dehydrations, rehydrations, and antigen retrieval protocols. Frozen samples do not require enzymatic processing, but are reliant on immediate freezing and proper long term storage to preserve sample integrity [1–3]. A limited number of reports have directly compared FFPE and fresh frozen preserva- tion techniques on subsequent putative metabolite identification. These reports are from prostate cancer, colon cancer, and mouse models of Parkinson’s disease [1–5]. Specifi- cally, prior studies have evaluated prostate cancer cell lines (LNCaP and LNCaP-Abl [1], Metabolites 2022, 12, 497. https://doi.org/10.3390/metabo12060497 https://www.mdpi.com/journal/metabolites