Imaging of Phospholipids in Formalin Fixed Rat Brain Sections by Matrix Assisted Laser Desorption/Ionization Mass Spectrometry

Abstract

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a valuable tool for the analysis of molecules directly from tissue. Imaging of phospholipids is gaining widespread interest, particularly as these lipids have been implicated in a variety of pathologic processes. Formalin fixation (FF) is the standard protocol used in histology laboratories worldwide to preserve tissue for analysis, in order to aid in the diagnosis and prognosis of diseases. This study assesses MALDI imaging of phospholipids directly in formalin fixed tissue, with a view to future analysis of archival tissue. This investigation proves the viability of MALDI-MSI for studying the distribution of lipids directly in formalin fixed tissue, without any pretreatment protocols. High quality molecular images for several phosphatidylcholine (PC) and sphingomyelin (SM) species are presented. Images correspond well with previously published data for the analysis of lipids directly from freshly prepared tissue. Different ionization pathways are observed when analyzing fixed tissue compared with fresh, and this change was found to be associated with formalin buffers employed in fixation protocols. The ability to analyze lipids directly from formalin fixed tissue opens up new doors in the investigation of disease profiles. Pathologic specimens taken for histologic investigation can be analyzed by MALDI-MS to provide greater information on the involvement of lipids in diseased tissue.

Global changes of phospholipids identified by MALDI imaging mass spectrometry in a mouse model of Alzheimer's disease

“…The increase in LPC and decrease in PC in the AD brains detected in our positive ion mode analysis ( Fig. 5 ) directly resulted from the activation of PLA 2 , which is reported to be hyperactivated in various disease conditions, including AD, Parkinson's disease, ischemia, spinal cord trauma, and head injury (34)(35)(36)(37)(38)(39)(40). It was also reported that increased neuronal activity of PLA 2 converts PC to LPC, which stimulates glial cells and subsequently induces neuroinfl ammation ( 34 ).…”

Sample preparation for mass spectrometry imaging: Small mistakes can lead to big consequences

“…Recently reported phospholipidomic MSI has been achieved that showed no signs of delocalization or interference from the formalin in formalin-fixed tissue. However, an increase in sodium adduct ions was reported as a result of the sodium buffers associated with the formalin fixation protocol [56]. MSI has also been used to identify drugs in formalin-fixed tissue samples, for example confirming the identification of microcrystalline material in histopathologic tissue sections [57].…”

MALDI Imaging Mass Spectrometry—A Mini Review of Methods and Recent Developments

“…12,13) For the analysis of lipids, some species may be analyzed with no special pre-treatment of the samples. 14) One should remember that heat stabilization, formalin fixation and microwave irradiation permanently protects samples from enzymatic changes, whereas freezing only preserves the sample state until the temperature is risen and enzymatic processes re-occur.…”