Two-Phase and Graph-Based Clustering Methods for Accurate and Efficient Segmentation of Large Mass Spectrometry Images

Clustering is widely used in MSI to segment anatomical features and differentiate tissue types, but existing approaches are both CPU and memory-intensive, limiting their application to small, single data sets. We propose a new approach that uses a graph-based algorithm with a two-phase sampling method that overcomes this limitation. We demonstrate the algorithm on a range of sample types and show that it can segment anatomical features that are not identified using commonly employed algorithms in MSI, and we validate our results on synthetic MSI data. We show that the algorithm is robust to fluctuations in data quality by successfully clustering data with a designed-in variance using data acquired with varying laser fluence. Finally, we show that this method is capable of generating accurate segmentations of large MSI data sets acquired on the newest generation of MSI instruments and evaluate these results by comparison with histopathology

Mass Spectrometry Imaging for Dissecting Steroid Intracrinology within Target Tissues

Steroid concentrations within tissues are modulated by intracellular enzymes. Such “steroid intracrinology” influences hormone-dependent cancers and obesity and provides targets for pharmacological inhibition. However, no high resolution methods exist to quantify steroids within target tissues. We developed mass spectrometry imaging (MSI), combining matrix assisted laser desorption ionization with on-tissue derivatization with Girard T and Fourier transform ion cyclotron resonance mass spectrometry, to quantify substrate and product (11-dehydrocorticosterone and corticosterone) of the glucocorticoid-amplifying enzyme 11β-HSD1. Regional steroid distribution was imaged at 150–200 μm resolution in rat adrenal gland and mouse brain sections and confirmed with collision induced dissociation/liquid extraction surface analysis. In brains of mice with 11β-HSD1 deficiency or inhibition, MSI quantified changes in subregional corticosterone/11-dehydrocorticosterone ratio, distribution of inhibitor, and accumulation of the alternative 11β-HSD1 substrate, 7-ketocholesterol. MSI data correlated well with LC-MS/MS in whole brain homogenates. MSI with derivatization is a powerful new tool to investigate steroid biology within tissues

Deuterated Matrix-Assisted Laser Desorption Ionization Matrix Uncovers Masked Mass Spectrometry Imaging Signals of Small Molecules

D⁴-α-Cyano-4-hydroxycinnamic acid (D⁴-CHCA) has been synthesized for use as a matrix for matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) and MALDI-MS imaging (MSI) of small molecule drugs and endogenous compounds. MALDI-MS analysis of small molecules has historically been hindered by interference from matrix ion clusters and fragment peaks that mask signals of low molecular weight compounds of interest. By using D⁴-CHCA, the cluster and fragment peaks of CHCA, the most common matrix for analysis of small molecules, are shifted by + 4, + 8 and + 12 Da, which expose signals across areas of the previously concealed low mass range. Here, obscured MALDI-MS signals of a synthetic small molecule pharmaceutical, a naturally occurring isoquinoline alkaloid, and endogenous compounds including the neurotransmitter acetylcholine have been unmasked and imaged directly from biological tissue sections

Investigating Nephrotoxicity of Polymyxin Derivatives by Mapping Renal Distribution Using Mass Spectrometry Imaging

Colistin and polymyxin B are effective treatment options for Gram-negative resistant bacteria but are used as last-line therapy due to their dose-limiting nephrotoxicity. A critical factor in developing safer polymyxin analogues is understanding accumulation of the drugs and their metabolites, which is currently limited due to the lack of effective techniques for analysis of these challenging molecules. Mass spectrometry imaging (MSI) allows direct detection of targets (drugs, metabolites, and endogenous compounds) from tissue sections. The presented study exemplifies the utility of MSI by measuring the distribution of polymyxin B1, colistin, and polymyxin B nonapeptide (PMBN) within dosed rat kidney tissue sections. The label-free MSI analysis revealed that the nephrotoxic compounds (polymyxin B1 and colistin) preferentially accumulated in the renal cortical region. The less nephrotoxic analogue, polymyxin B nonapeptide, was more uniformly distributed throughout the kidney. In addition, metabolites of the dosed compounds were detected by MSI. Kidney homogenates were analyzed using LC/MS/MS to determine total drug exposure and for metabolite identification. To our knowledge, this is the first time such techniques have been utilized to measure the distribution of polymyxin drugs and their metabolites. By simultaneously detecting the distribution of drug and drug metabolites, MSI offers a powerful alternative to tissue homogenization analysis and label or antibody-based imaging

Results from an untargeted analysis of kidney tissue from drug treated animals and vehicle controls.

<p>A) Total ion chromatogram of kidney extract from the LC-MS analysis of vehicle control (upper) and animal dosed with compound 1 (low crystal load) (lower), run on an HILIC column. B) Mass spectrum from the peak marked in red on panel A, showing a PEG distribution with <i>m/z</i> 44 distance between peaks. C) The distribution of PEG on the tissue is represented by the ion distribution image of <i>m/z</i> 437. This distribution pattern is overlaid on the tissue sections from (i) vehicle control, (ii) animal dosed with compound 1 (low crystal load), (iii) animal dosed with compound 1 (high crystal load), and (iv) animal dosed with compound 2 (not formulated in PEG400). The MSI samples were coated with CHCA and analyzed in positive mode on the UltraFlex II. The data was normalized by total ion count.</p

The structures of the two administered compounds (compound 1; 4-(phenylethynyl)-N-[(2-sulfamoylphenyl)sulfonyl]benzamide, compound 2; 4-(cyclobutylethynyl)-N-[(2-sulfamoylphenyl)sulfonyl] benzamide), and their common metabolite bisulphonamide (benzene-1,2-disulphonamide).

<p>The structures of the two administered compounds (compound 1; 4-(phenylethynyl)-N-[(2-sulfamoylphenyl)sulfonyl]benzamide, compound 2; 4-(cyclobutylethynyl)-N-[(2-sulfamoylphenyl)sulfonyl] benzamide), and their common metabolite bisulphonamide (benzene-1,2-disulphonamide).</p

Identification and mapping of renal crystalline deposits.

<p>A) i; Mass spectrum of crystals isolated from kidney tissue analyzed by LC-MS. Retention time and mass-to-charge ratio matches that of bisulphonamide standard. ii; MS/MS spectrum of 234.98 observed in the crystal isolate. The fragmentation pattern matches that of bisulphonamide standard. B)¹H-NMR spectra of the aromatic proton region of crystals from kidney tissue dissolved in DMSO. C) Optical images of analyzed tissue sections. i) vehicle control, ii) bisulphonamide standard on vehicle control, iii) compound 1 dosed tissue with a low crystal load, iv) compound 1 dosed tissue with a high density of crystals, v) compound 2 dosed tissue with a high crystal load. All samples were coated with sinapinic acid and analyzed in negative mode on the G2 Synapt. The data was normalized by total ion count. D) Ion distribution of bisulphonamide (m/z 235) on the tissue sections in panel C. The color intensity scale is adjusted to 2% of the maximum intensity on tissue v in order to visualize the distribution patterns on all tissues using the same color intensity scale. This means that pixels on the tissues in panel D above this value appear saturated. Data was acquired at a spatial resolution of 100 µm. E) Ion distribution of bisulphonamide overlaid on the tissue sections from the animal with a low crystal load following administration of compound 1. Left; ion distribution image of <i>m/z</i> 235 overlaid on scanned image of tissue section. Crystals are marked with arrows. Right; scanned image following H&E staining of the same tissue with example of a crystalline deposit in the kidney surrounded by a slight mononuclear cell reaction. Crystals are marked with arrows and circled in green. The size of the majority of the crystals ranged between 50 and 100 µm.</p

Ion distribution images of administered drug candidate compounds and some detected metabolites.

<p>A) Optical images of analyzed tissue sections. i; vehicle control, ii; compound 1 dosed animal tissue section with low crystal load, iii; compound 1 dosed animal tissue section with high density of crystals. B) Ion distribution of compound 1 (<i>m/z</i> 439) on the tissue sections in panel A. C) Optical images of analyzed tissue sections. i; vehicle control, ii; compound 2 dosed animal tissue section. D) Ion distribution image of compound 2 on the tissue sections in panel C. E) Ion distribution image of a compound 2 metabolite (doubly oxidized) on the tissue sections in panel C. F) Ion distribution image of compound 2 metabolite (triply oxidized) on the tissue sections in panel C. The samples were coated with sinapinic acid and analyzed in negative mode on the Synapt G2. The data was normalized by total ion count.</p