Balkan Journal of Medical Genetics

RENOPATHOLOGICAL MICROSTRUCTURE VISUALIZATION FROM FORMALIN FIXED KIDNEY TISSUE BY MATRIXASSISTED LASER/DESORPTION IONIZATION-TIME-OFFLIGHT MASS SPECTROMETRY IMAGING
Fröhlich S1, Putz B1, Schachner H2, Kerjaschki D2,Allmaier G1, Marchetti-Deschmann M1,*
*Corresponding Author: Dr. Martina Marchetti-Deschmann, Vienna University of Technology, Institute of Chemical Technologies and Analytics, Getreidemarkt 9/164-IAC, 1060 Vienna, Austria; Tel.: +43-1- 58801-15152; Mobile: +43+664-605887663; Fax: +43-1-58801-915162; E-mail: martina.marchettideschmann@ tuwien.ac.at
page: 13

RESULTS AND DISCUSSION

Histology and Mass Spectrometry Imaging. Integrating MSI in histopathology necessitates the combination of different requirements from pathology and MS. The primary demands for histology are the preservation of high spatial resolution for tissue structures, long lasting analyte preservation and low sample consumption. For MS, spatial resolution can usually be disregarded and analytes have to be mobilized instead of preserved. For MSI, additionally, histological information has to be retained and tissue disintegration has to be limited to a minimum. We present an approach arranging histological staining prior to MSI using ultra-thin tissue sections showing good molecular results comparable to microscopic images. It was found that MALDI-TOF MSI analysis of H/E-stained tissue showed no limiting aspects concerning proteomic approaches. Protein digestion directly from the tissue and measuring peptides by MS could be achieved. Stain-related signals, however, were observed below m/z signals of 500, eventually leading to ion suppression effects for neutral lipids. Phospholipid classes were obtained unaffected from ultra-thin sucrose samples. Histological images of OCT embedded samples were often difficult to correlate to MSI results because of structural changes after thawing. Sucrose embedding in combination with cutting thickness of 1 mm was sufficient for IMS analysis, even reducing signal background and enhancing signal quality. The selected ultrathin samples considerably improved signal quality whilst preserving spatial resolution. Lipid Differentiation. Lipid-based histological differentiation was performed using unfixed rat kidney samples embedded in sucrose and formalinfixed tissue embedded in OCT Low embedding medium related signals and intensive species-related signals were obtained from both OCT and sucrose embedded samples. Several lipid classes could be localized, identified and characterized by fragmentation experiments. Unfixed tissue species revealed different distribution pattern phosphoatidylcholine species with varying alkyl chain lengths. The 1-tetradecanoyl-2-sn-glycero-3-phosphocholine, 1-hexadecanolyl-2-sn-glycero-3-phosphocholine and 1-octadecanoyl-2-sn-glycero-3-phosphocholine showed specifiable areas related to kidney structures, i.e., cortex, medulla and pelvis (Figure 1). The 1-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine and 1-hexadecanoyl-sn-glycero-3-phosphocholine exhibited different localization as well as 1-O-(1Z-tetradecenyl)-2-(9Z-octadecenoyl)-snglycerol and its oxidized co-species. Besides 16 different phospholipids, diacylglycerophosphoethanolamine, N-(tetracosanoyl)-sphing-4-enine- 1-phospho-choline and two glomeruli specific metabolites, cholesterol and squalene, were also discovered. Histological correlation revealed the potential to identify pathologically modified membrane structures in glomerular associated diseases. Protein Identification. To detect most of the proteins available in an ultra-thin sample, several washing steps were required for complete removal of analyte species which are much easier to ionize than proteins, e.g., lipids [7]. Our study showed that mass spectra quality for tissue analysis was enhanced by heat treatment before MSI experiments. Several tissue-related peptides could be identified by PSD and CID, revealing the possibility to distinguish pelvis and cortex, both tissue macrostructures. For all investigated embedding materials as well as fixation methods, it was possible to obtain sufficient signals up to m/z values of 10000 (Figure 2). The application of danaturation agents and heat treatment supports subsequent on-tissue enzymatic treatment for protein identification on fixed and embedded rat kidney. Highly cross-linked proteins (formalin) showed improved signal intensities for tryptic peptides after denaturation, which significantly supported protein identification based on peptide fragmentation. For protein identification in MSI approaches, peptide sequencing was indispensable because of protein co-localization and the non applicable approach of peptide mass fingerprinting. The presented protocol on ultra-thin tissue sections allowed the identification of Tubulin and Claudin-4, besides seven other peptide fragments on differentiated, but so far unspecific, tissue regions. Perspectives. Combining histopathology and MSI for investigating structural membrane modifications associated with glomerular related nephropathology looks promising. Structural modifications and molecular changes can be detected and investigated very early in progression

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