Spatial localization of β-unsaturated aldehyde markers in murine diabetic kidney tissue by mass spectrometry imaging

Abstract

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease. Limitations in current diagnosis and screening methods have sparked a search for more specific and conclusive biomarkers. Hyperglycemic conditions generate a plethora of harmful molecules in circulation and within tissues. Oxidative stress generates reactive α-dicarbonyls and β-unsaturated hydroxyhexenals, which react with proteins to form advanced glycation end products. Mass spectrometry imaging (MSI) enables the detection and spatial localization of molecules in biological tissue sections. Here, for the first time, the localization and semiquantitative analysis of “reactive aldehydes” (RAs) 4-hydroxyhexenal (4-HHE), 4-hydroxynonenal (4-HNE), and 4-oxo-2-nonenal (4-ONE) in the kidney tissues of a diabetic mouse model is presented. Ionization efficiency was enhanced through on-tissue chemical derivatization (OTCD) using Girard’s reagent T (GT), forming positively charged hydrazone derivatives. MSI analysis was performed using matrix-assisted laser desorption ionization (MALDI) coupled with Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR). RA levels were elevated in diabetic kidney tissues compared to lean controls and localized throughout the kidney sections at a spatial resolution of 100 µm. This was confirmed by liquid extraction surface analysis–MSI (LESA-MSI) and liquid chromatography–mass spectrometry (LC–MS). This method identified β-unsaturated aldehydes as “potential” biomarkers of DN and demonstrated the capability of OTCD-MSI for detection and localization of poorly ionizable molecules by adapting existing chemical derivatization methods. Untargeted exploratory distribution analysis of some precursor lipids was also assessed using MALDI-FT-ICR-MSI. Graphical abstract

Analysis of reactive aldehydes in urine and plasma of type-2 diabetes mellitus patients through liquid chromatography-mass spectrometry: Reactive aldehydes as potential markers of diabetic nephropathy

“…The autosampler was kept at 5°C and injection volume was 20 μl. LC-MS chromatograph-spectrum of the determined compounds have been previous published ( 33 ).…”

“…All samples were analysed in duplicate and linear regression analysis for all standards [ratio against internal standard (ISTD)] was used to calculate the content of reactive aldehyde in the samples using an y = ax + b equation. The methodology employed for the LC-MS is as previously described ( 33 ). Data were reported as the average of the two injections.…”

“…Data were reported as the average of the two injections. The method was fully validated as per International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines on bioanalytical method validation ( Supplementary Data Sheet 1 ; 33 ).…”

“… Schematic diagram of the reaction between aldehydes and Girard’s reagent T forming hydrazone derivatives (top) , and the hydrazone formed through reaction with three reactive aldehydes; 4-hydroxyhexenal (4-HHE), 4-oxo-2-nonenal (4-ONE), and 4-hydroxynonenal (4-HNE) (below) . Image taken from Harkin, et al ( 33 ) and reproduced under the Creative Commons License ( http://creativecommons.org/licenses/by/4.0/ ). …”

Ion-to-Image, i2i, a Mass Spectrometry Imaging Data Analysis Platform for Continuous Ionization Techniques

“…Pioneering work in biological mass spectrometry imaging (MSI) introduced in the late 1990s and early 2000s enabled the visualization of molecular distributions in thin tissue sections without any chemical labeling or tagging. , Recent advances in MSI techniques facilitates the simultaneous generation of hundreds to thousands of ion images showing molecular distributions of small and large molecules, including lipids, metabolites, proteins, and drugs. − Compared to traditional methods, where the tissue is homogenized prior to analysis, the additional dimension of localization contributed by MSI enhances biological understanding by revealing the chemical microenvironments. Some recent examples utilizing the spatial distribution to study in situ tissue molecular aberrations include animal models for cancer, , diabetes, , and ischemic stroke. , …”