Spatial Localization of Vitamin D metabolites in Mouse Kidney by Mass Spectrometry Imaging

Introduction
Vitamin D has become a main focus of research worldwide. It is a vital component for several biological processes such as calcium homeostasis and immune function. Vitamin D metabolites are primarily measured in biomatrices by LC-MS/MS. However, these quantitative approaches, do not offer any spatial information. MSI has been proven to be a powerful tool to image the spatial distribution of molecules from the surface of biological tissue sections. Steroidal class compounds are known to have poor ionization efficiency in mass spectrometry due to a lack of ionizable moieties. Here, a method for monitoring the spatial distribution of vitamin D metabolites in murine kidney tissue section using an on-tissue chemical derivatization (OTCD) procedure with MALDI-MSI is presented
Methods
In this study, several derivatization reagents, deposition techniques and reaction conditions were evaluated using different ionization techniques including MALDI and desorption electrospray ionization (DESI). Murine kidney tissue sections were used at 12 µm thickness. OTCD was performed using the Bruker ImagePrep and matrix (CHCA) was applied using a modified 3D printer. MALDI-MSI was performed using a 9.4 T SolariX with continuous accumulation of selected ions (CASI) at 4M time domain. Images were acquired at 100 × 100 µm, using 400 laser shots/pixel images using FlexImaging. DESI-MSI experiments were performed on a DESI 2D source and a Waters Xevo G2-XS mass spectrometer. Confirmatory LC-MS/MS analysis was performed by a triple-quadrupole linear ion trap mass spectrometer coupled with an ACQUITY UPLC.
Preliminary Data or Plenary Speakers Abstract
Here we show the successful development of a MSI method of vitamin D metabolites in a mouse kidney by OTCD. Amplifex, 4-Phenyl-1,2,4-triazoline-3,5-dione (PTAD) and DMEQ-TAD derivatives were assessed for MALDI ionization efficiency using a stable isotope vitamin D metabolite (d6-25-OH-D3). Amplifex and DMEQ-TAD were further assessed for on-tissue ion suppression, with Amplifex outperforming DMEQ-TAD in terms of ion production yields. Amplifex reaction conditions (off- and on-tissue) optimization was performed with optimal conditions observed at 1 hour at room temperature.
MALDI and DESI were evaluated as MSI platforms with best results achieved by MALDI regarding sensitivity and reproducibility. Further development was carried out in derivatization reagent deposition using a commercial airbrush and the Bruker ImagePrep to assess analyte detection, diffusion and reproducibility. The ImagePrep was selected as the optimal deposition application as it outperformed the manual airbrush. Ion signal intensity was increased in the ImagePrep MS images, as better S/N ratios were obtained than with the airbrush method. Moreover, tissue-to-tissue reproducibility and analyte diffusion were improved by using the automated Bruker ImagePrep deposition method. For the first time, vitamin D metabolites were successfully detected, and their spatial distribution assessed in murine kidney tissue sections using the optimized automated OTCD-MALDI-MSI platform. Two key endogenous vitamin D metabolites 25-(OH)-D and 1, 25-(OH) 2-D3 were detected as Amplifex derivatives with a mass accuracy of 5.0 ppm. 1, 25-(OH) 2-D3 was mainly distributed across the cortex gathering near the renal vein and 25-(OH)-D3 was mainly detected across the medulla and inner cortex. Identification and spatial distribution were further confirmed by on-tissue collision induced dissociation (CID) experiments and tissue homogenate LC/MS/MS analysis
Novel Aspect
First time spatial distribution of vitamin D metabolites achieved at tissue level by OTCD-MALDI-MSI using the ImagePrep as OTCD chamber.