Simultaneous Detection of Zinc and Its Pathway Metabolites Using MALDI MS Imaging of Prostate Tissue

Abstract

Levels of zinc, along with its mechanistically related metabolites citrate and aspartate, are widely reported as reduced in prostate cancer compared to healthy tissue and are therefore pointed out as potential cancer biomarkers. Previously, it has only been possible to analyze zinc and metabolites by separate detection methods. Through matrix-assisted laser desorption/ionization mass spectrometry imaging (MSI), we were for the first time able to demonstrate, in two different sample sets ( n = 45 and n = 4), the simultaneous spatial detection of zinc, in the form of ZnCl 3 – , together with citrate, aspartate, and N -acetylaspartate on human prostate cancer tissues. The reliability of the ZnCl 3 – detection was validated by total zinc determination using laser ablation inductively coupled plasma MSI on adjacent serial tissue sections. Zinc, citrate, and aspartate were correlated with each other (range r = 0.46 to 0.74) and showed a significant reduction in cancer compared to non-cancer epithelium ( p < 0.05, log 2 fold change range: −0.423 to −0.987), while no significant difference between cancer and stroma tissue was found. Simultaneous spatial detection of zinc and its metabolites is not only a valuable tool for analyzing the role of zinc in prostate metabolism but might also provide a fast and simple method to detect zinc, citrate, and aspartate levels as a biomarker signature for prostate cancer diagnostics and prognostics.

Spatial differentiation of metabolism in prostate cancer tissue by MALDI-TOF MSI

“… *For ZnCl 3 - , the isotopes with m/z 174.83 were used to represent zinc due to an overlapping contaminant [ 17 ] **Ascorbate and glucurone have identical masses and MS/MS suggests a mix of the two metabolites, which both belongs to the same Ascorbate and aldarate metabolic pathway …”

“…*For ZnCl 3 - , the isotopes with m/z 174.83 were used to represent zinc due to an overlapping contaminant [ 17 ]…”

“…This step was repeated 1000 times and the reported Benjamini-Hochberg adjusted p value (< 0.05 were considered significant) is the average p value from all 1000 iterations. Further details and the full R script is provided in a previous publication [ 17 ]. Additionally, log 2 fold changes (log 2 FC) were calculated to show the differences in levels between the tissue types for all extracted masses.…”

“…All metabolites and lipids were identified by accurate mass from Orbitrap acquisition and by manually assessing the fragment pattern and comparing the averaged MS/MS spectra to those in the data bases METLIN [ 30 ], the Human Metabolome Database (HMDB) [ 31 ], and Alex 123 Lipid Calculator [ 32 ]. The metal zinc (in form of ZnCl 3 - ) was identified through isotopic peak pattern, accurate mass, and laser ablation inductively coupled plasma (LA-ICP) MSI as described in our previous publication [ 17 ].…”

“…Lower levels of citrate in prostate cancer compared to normal tissue have also been observed with MSI using desorption electrospray ionization (DESI) [ 15 , 16 ]. We have recently demonstrated reduced levels of citrate and aspartate, along with the metal zinc, in prostate cancer tissue using matrix-assisted laser desorption ionization (MALDI) MSI [ 17 ]. Lipid metabolism is another key metabolic alteration in prostate cancer, including both increased fatty acid synthesis (FAS) and energy utilization of lipids through β-oxidation.…”