DOI: 10.5061/dryad.b5mkkwhrk
title: Demonstrating voxel-by-voxel (v×v) single-point calibration in liver tissue by IR-MALDESI quantitative MSI

description: The quantification of glutathione (GSH) on a per-voxel basis was achieved
 by homogenously spraying a known amount of stable isotope labeled
 glutathione (SIL-GSH) on a microscope slide. Next, liver sections were
 mounted on top of the coated slides and imaged using IR-MALDESI MSI.
 Statistical analysis has shown high precision for V×V quantification over
 a wide concentration range, but the accuracy of this method is currently
 limited due to the sprayer configuration. Results support the feasibility
 of V×V quantification as evidence by concentration heatmaps. Additionally,
 voxel-based quantification provides benefits such as high specificity,
 straightforward sample preparation, and promising initial statistics
 analysis which will be advantageous for future qMSI studies. 
description: # Demonstrating voxel-by-voxel (v×v) single-point calibration in liver
 tissue by IR-MALDESI quantitative MSI Dataset DOI:
 [10.5061/dryad.b5mkkwhrk](10.5061/dryad.b5mkkwhrk) ## Description of the
 data and file structure Data collected for this manuscript. All files were
 converted to .mzML and then to .imzML format. ### Files and variables ####
 File: VxV_GSH_MS1_1.raw **Description:** Replicate 1 of VxV
 quantification. Image of mouse liver at 7 μm where stable isotope labeled
 version glutathione (SIL-GSH) was sprayed underneath the liver section and
 served as a single point calibration at each voxel within the image. The
 concentration of endogenous GSH (NAT-GSH) was calculated using the
 Voxel-by-Voxel quantification tool in MSiReader.  #### File:
 VxV_GSH_MS1_2.raw **Description:** Replicate 2 of VxV quantification. The
 same conditions from the first replicate of VxV quantification apply to
 replicate 2.  #### File: SIL_GSH_MS1_1.raw **Description:** Replicate 1 of
 spatial curve quantification. Image of mouse liver at 7 μm where the
 SIL-GSH was spotted on top of the liver section at concentrations of 0,
 0.03125, 0.625, 0.125, 0.25, 0.50, 1.0 mg/mL to output a calibration
 curve. The calibration curve was used to determine the average on tissue
 concentration of NAT-GSH for this liver section. The spatial calibration
 curve tool in MSiReader was utilized for the calibration curve output and
 calculations.  #### File: SIL_GSH_MS1_2.raw **Description:** Replicate 2
 of spatial curve quantification. The same conditions from the first
 replicate of the spatial calibration curve apply to replicate 2. ####
 File: 0.03125_2to1_Solution.raw **Description:** 2 to 1 solution with
 concentration of 0.03125 mg/mL. Image of sprayed glass slide with a 2 to 1
 solution of NAT-GSH and SIL-GSH at a total analyte concentration of
 0.03125 mg/mL. There were a total of 2704 scans in this image. There were
 6 bioinformatic ROIs employed where the abundance of NAT-GSH and SIL-GSH
 were exported. For each bioinformatic ROI, we calculated the average
 abundance of SIL-GSH, the average abundance ratio between NAT-GSH and
 SIL-GSH, as well as the statistical variance of those ratios.   #### File:
 0.0625_2to1_Solution.raw **Description:** 2 to 1 solution with
 concentration of 0.0625 mg/mL. Image of sprayed glass slide with a 2 to 1
 solution of NAT-GSH and SIL-GSH at a total analyte concentration of 0.0625
 mg/mL. There were a total of 2704 scans in this image. There were 6
 bioinformatic ROIs employed where the abundance of NAT-GSH and SIL-GSH
 were exported. For each bioinformatic ROI, we calculated the average
 abundance of SIL-GSH, the average abundance ratio between NAT-GSH and
 SIL-GSH, as well as the statistical variance of those ratios.   #### File:
 0.125_2to1_Solution.raw **Description:** 2 to 1 solution with
 concentration of 0.125 mg/mL. Image of sprayed glass slide with a 2 to 1
 solution of NAT-GSH and SIL-GSH at a total analyte concentration of 0.125
 mg/mL. There were a total of 2704 scans in this image. There were 6
 bioinformatic ROIs employed where the abundance of NAT-GSH and SIL-GSH
 were exported. For each bioinformatic ROI, we calculated the average
 abundance of SIL-GSH, the average abundance ratio between NAT-GSH and
 SIL-GSH, as well as the statistical variance of those ratios.   #### File:
 0.25_2to1_Solution.raw **Description:** 2 to 1 solution with concentration
 of 0.25 mg/mL. Image of sprayed glass slide with a 2 to 1 solution of
 NAT-GSH and SIL-GSH at a total analyte concentration of 0.25 mg/mL. There
 were a total of 2704 scans in this image. There were 6 bioinformatic ROIs
 employed where the abundance of NAT-GSH and SIL-GSH were exported. For
 each bioinformatic ROI, we calculated the average abundance of SIL-GSH,
 the average abundance ratio between NAT-GSH and SIL-GSH, as well as the
 statistical variance of those ratios.   #### File: 0.5_2to1_Solution.raw
 **Description:** 2 to 1 solution with concentration of 0.50 mg/mL. Image
 of sprayed glass slide with a 2 to 1 solution of NAT-GSH and SIL-GSH at a
 total analyte concentration of 0.50 mg/mL. There were a total of 2704
 scans in this image. There were 6 bioinformatic ROIs employed where the
 abundance of NAT-GSH and SIL-GSH were exported. For each bioinformatic
 ROI, we calculated the average abundance of SIL-GSH, the average abundance
 ratio between NAT-GSH and SIL-GSH, as well as the statistical variance of
 those ratios.   #### File: 1.0_2to1_Solution.raw **Description:** 2 to 1
 solution with concentration of 1.0 mg/mL. Image of sprayed glass slide
 with a 2 to 1 solution of NAT-GSH and SIL-GSH at a total analyte
 concentration of 1.0 mg/mL. There were a total of 2704 scans in this
 image. There were 6 bioinformatic ROIs employed where the abundance of
 NAT-GSH and SIL-GSH were exported. For each bioinformatic ROI, we
 calculated the average abundance of SIL-GSH, the average abundance ratio
 between NAT-GSH and SIL-GSH, as well as the statistical variance of those
 ratios.   #### File: PRM_of_GSH_1.raw **Description:** Replicate 1 of
 PRM-qMSI. Image of mouse liver at 7 μm where SIL-GSH was sprayed
 underneath the liver section and served as a single point calibration at
 each voxel within the image. The collision energy was set to 20% while the
 isolation width was set to 4 *m/z*. By applying the collision energy, a
 fragmentation pattern for NAT-GSH and SIL-GSH were observed. A peak at
 272.0888 was identified as the endogenous GSH quantifier peak while a peak
 at 275.0926 was classified as the SIL-GSH quantifier peak. Those *m/z*
 values were used in the VxV tool in MSiReader to propagate concentration
 heatmaps of our quantifier peak. #### File: PRM_of_GSH_2.raw
 **Description:** Replicate 2 of PRM-qMSI. The same conditions from the
 first replicate of the PRM imaging apply to replicate 2. #### File:
 Liver_031_3.raw **Description:** Replicate 3 of Liver and 0.03125 mg/mL
 sprayed SIL-GSH. The same conditions apply from File: Liver_031_1.raw
 apply to this data file.  #### File: Liver_031_2.raw
 **Description:** Replicate 2 of Liver and 0.03125 mg/mL sprayed
 SIL-GSH. The same conditions apply from File: Liver_031_1.raw apply to
 this data file.  #### File: Liver_031_1.raw **Description:** Replicate 1
 of Liver and 0.03125 mg/mL sprayed SIL-GSH.  Image of mouse liver at 7 μm
 where SIL-GSH was sprayed underneath the liver section at 0.03125 mg/mL
 and served as a single point calibration at each voxel within the image.
 There were 6 bioinformatic ROIs employed where the abundance of NAT-GSH
 and SIL-GSH were exported. For each bioinformatic ROI, we calculated the
 average abundance of SIL-GSH, the  average abundance ratio between NAT-GSH
 and SIL-GSH, as well as the statistical variance of those ratios. 
 Biological variability is introduced due to the liver section. #### File:
 Liver_062_3.raw **Description:** Replicate 3 of Liver and 0.0625 mg/mL
 sprayed SIL-GSH. The same conditions apply from File: Liver_062_1.raw
 apply to this data file.  #### File: Liver_062_1.raw
 **Description:** Replicate 1 of Liver and 0.0625 mg/mL sprayed
 SIL-GSH. Image of mouse liver at 7 μm where SIL-GSH was sprayed underneath
 the liver section at 0.0625 mg/mL and served as a single point calibration
 at each voxel within the image. There were 6 bioinformatic ROIs employed
 where the abundance of NAT-GSH and SIL-GSH were exported. For each
 bioinformatic ROI, we calculated the average abundance of SIL-GSH, the
 average abundance ratio between NAT-GSH and SIL-GSH, as well as the
 statistical variance of those ratios.  Biological variability is
 introduced due to the liver section. #### File: Liver_062_2.raw
 **Description:** Replicate 2 of Liver and 0.0625 mg/mL sprayed
 SIL-GSH. The same conditions apply from File: Liver_062_1.raw apply to
 this data file.  #### File: Liver_125_3.raw **Description:** Replicate 3
 of Liver and 0.125 mg/mL sprayed SIL-GSH. The same conditions apply from
 File: Liver_125_1.raw apply to this data file.  #### File: Liver_125_2.raw
 **Description:** Replicate 2 of Liver and 0.125 mg/mL sprayed SIL-GSH. The
 same conditions apply from File: Liver_125_1.raw apply to this data file. 
 #### File: Liver_125_1.raw **Description:** Replicate 1 of Liver and 0.125
 mg/mL sprayed SIL-GSH. Image of mouse liver at 7 μm where SIL-GSH was
 sprayed underneath the liver section at 0.125 mg/mL and served as a single
 point calibration at each voxel within the image. There were 6
 bioinformatic ROIs employed where the abundance of NAT-GSH and SIL-GSH
 were exported. For each bioinformatic ROI, we calculated the average
 abundance of SIL-GSH, the average abundance ratio between NAT-GSH and
 SIL-GSH, as well as the statistical variance of those ratios.  Biological
 variability is introduced due to the liver section. #### File:
 Liver_250_3.raw **Description:** Replicate 3 of Liver and 0.25 mg/mL
 sprayed SIL-GSH. The same conditions apply from File: Liver_250_1.raw
 apply to this data file.  #### File: Liver_500_3.raw
 **Description:** Replicate 3 of Liver and 0.50 mg/mL sprayed SIL-GSH. The
 same conditions apply from File: Liver_500_1.raw apply to this data file. 
 #### File: Liver_250_2.raw **Description:** Replicate 2 of Liver and 0.25
 mg/mL sprayed SIL-GSH. The same conditions apply from File:
 Liver_250_1.raw apply to this data file.  #### File: Liver_1000_3.raw
 **Description:** Replicate 3 of Liver and 1.0 mg/mL sprayed SIL-GSH. The
 same conditions apply from File: Liver_1000_1.raw apply to this data
 file.  #### File: Liver_250_1.raw **Description:** Replicate 1 of Liver
 and 0.25 mg/mL sprayed SIL-GSH. Image of mouse liver at 7 μm where SIL-GSH
 was sprayed underneath the liver section at 0.25 mg/mL and served as a
 single point calibration at each voxel within the image. There were 6
 bioinformatic ROIs employed where the abundance of NAT-GSH and SIL-GSH
 were exported. For each bioinformatic ROI, we calculated the average
 abundance of SIL-GSH, the average abundance ratio between NAT-GSH and
 SIL-GSH, as well as the statistical variance of those ratios.  Biological
 variability is introduced due to the liver section. #### File:
 Liver_500_2.raw **Description:** Replicate 2 of Liver and 0.50 mg/mL
 sprayed SIL-GSH. The same conditions apply from File: Liver_500_1.raw
 apply to this data file.  #### File: Liver_500_1.raw
 **Description:** Replicate 1 of Liver and 0.50 mg/mL sprayed
 SIL-GSH. Image of mouse liver at 7 μm where SIL-GSH was sprayed underneath
 the liver section at 0.5 mg/mL and served as a single point calibration at
 each voxel within the image. There were 6 bioinformatic ROIs employed
 where the abundance of NAT-GSH and SIL-GSH were exported. For each
 bioinformatic ROI, we calculated the average abundance of SIL-GSH, the
 average abundance ratio between NAT-GSH and SIL-GSH, as well as the
 statistical variance of those ratios.  Biological variability is
 introduced due to the liver section. #### File: Liver_1000_2.raw
 **Description:** Replicate 2 of Liver and 1.0 mg/mL sprayed SIL-GSH. The
 same conditions apply from File: Liver_1000_1.raw apply to this data
 file.  #### File: Liver_1000_1.raw **Description:** Replicate 1 of Liver
 and 1.0 mg/mL sprayed SIL-GSH.  Image of mouse liver at 7 μm where SIL-GSH
 was sprayed underneath the liver section at 1.0 mg/mL and served as a
 single point calibration at each voxel within the image. There were 6
 bioinformatic ROIs employed where the abundance of NAT-GSH and SIL-GSH
 were exported. For each bioinformatic ROI, we calculated the average
 abundance of SIL-GSH, the average abundance ratio between NAT-GSH and
 SIL-GSH, as well as the statistical variance of those ratios.  Biological
 variability is introduced due to the liver section. ## Code/software *
 Freestyle 1.8 SP 2 - MSiReader v3.10 (QMSI Spatial Calibration Curve Tool
 & QMSI Voxel-by-Voxel Quantification Tool) ## Access information Other
 publicly accessible locations of the data: * Data was derived from the
 following sources: *