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. 2022 Sep 20;94(37):12604-12613.
doi: 10.1021/acs.analchem.2c01034. Epub 2022 Sep 6.

Enhanced Spatial Mapping of Histone Proteoforms in Human Kidney Through MALDI-MSI by High-Field UHMR-Orbitrap Detection

Kevin J Zemaitis  1 , Dušan Veličković  1 , William Kew  1 , Kyle L Fort  2 , Maria Reinhardt-Szyba  2 , Annapurna Pamreddy  3 , Yanli Ding  4 , Dharam Kaushik  5 , Kumar Sharma  3   6 , Alexander A Makarov  2   7 , Mowei Zhou  1 , Ljiljana Paša-Tolić  1
Affiliations

Enhanced Spatial Mapping of Histone Proteoforms in Human Kidney Through MALDI-MSI by High-Field UHMR-Orbitrap Detection

Kevin J Zemaitis et al. Anal Chem. .

Abstract

Core histones including H2A, H2B, H3, and H4 are key modulators of cellular repair, transcription, and replication within eukaryotic cells, playing vital roles in the pathogenesis of disease and cellular responses to environmental stimuli. Traditional mass spectrometry (MS)-based bottom-up and top-down proteomics allows for the comprehensive identification of proteins and of post-translational modification (PTM) harboring proteoforms. However, these methodologies have difficulties preserving near-cellular spatial distributions because they typically require laser capture microdissection (LCM) and advanced sample preparation techniques. Herein, we coupled a matrix-assisted laser desorption/ionization (MALDI) source with a Thermo Scientific Q Exactive HF Orbitrap MS upgraded with ultrahigh mass range (UHMR) boards for the first demonstration of complementary high-resolution accurate mass (HR/AM) measurements of proteoforms up to 16.5 kDa directly from tissues using this benchtop mass spectrometer. The platform achieved isotopic resolution throughout the detected mass range, providing confident assignments of proteoforms with low ppm mass error and a considerable increase in duty cycle over other Fourier transform mass analyzers. Proteoform mapping of core histones was demonstrated on sections of human kidney at near-cellular spatial resolution, with several key distributions of histone and other proteoforms noted within both healthy biopsy and a section from a renal cell carcinoma (RCC) containing nephrectomy. The use of MALDI-MS imaging (MSI) for proteoform mapping demonstrates several steps toward high-throughput accurate identification of proteoforms and provides a new tool for mapping biomolecule distributions throughout tissue sections in extended mass ranges.

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Conflict of interest statement

The authors declare the following competing financial interest(s): K.L.F, M.R.S., and A.A.M. declare the following competing financial interest(s): these authors are employees of Thermo Fisher Scientific, manufacturer of Orbitrap mass spectrometers.

Figures

Figure 1.
Figure 1.
Full mass spectrum from the proteoform mapping of human kidney at 40 μm obtained by averaging a total of 86,189 pixels highlighting both the singly and doubly charged core histone proteoform ion clusters. Expanded views of each singly charged histone (H2A, H2B, H3, H4) proteoform family are also shown with isotopic resolution throughout the entire m/z range. The only other near isobaric species other than histone proteoforms are located within the histone H3 proteoform envelope, where several hemoglobin subunits are also presently detected.
Figure 2.
Figure 2.
Ion images produced from an expanded view of the MALDI-MSI analysis of a tissue section from a cortical nephrectomy block. The tissue was imaged at 20 μm and ion images were obtained by averaging 25 ppm windows for an acetylated and dimethylated histone H4 proteoform at m/z 11,306.3939 (−1.12 ppm) and a diacetylated histone H4 proteoform at m/z 11,320.3661 (−1.74 ppm). Images are all scaled to 200 μm each with separate relative ion abundance. The corresponding microscopic image from the PAS staining post MALDI-MSI is shown at the bottom to highlight atrophic tubular regions with no significant enhancement in signal intensity (orange) and areas of tubular atrophy with the enhancement of the signal (white). Glomeruli are highlighted in yellow.
Figure 3.
Figure 3.
Microscopy image of the PAS-stained section of the healthy nephrectomy post MALDI-MSI. Highlighted within the tissue section are tubular regions identified from MALDI-MSI with differential levels of histone proteoforms, showing higher abundance (ET/yellow) and lower abundance (DT/teal) for an acetylated and dimethylated histone H4 proteoform at m/z 11,306.3939 (−1.12 ppm) and a diacetylated histone H4 proteoform at m/z 11,320.3661 (−1.74 ppm). The bar chart shows extracted mean intensities normalized to the area (mm2), with the calculated ratio for each region averaged below. Error bars represent one standard deviation.
Figure 4.
Figure 4.
(A) Bright microscopy image of the RCC section prior to MALDI-MSI analysis. (B) PAS-stained section post MALDI-MSI. Ion images are produced from singular isotopic peak distributions and show an expanded view of the MALDI-MSI analysis at 30 μm spatial resolution. Spatial distribution of (C) acetylated and dimethylated histone H4 proteoforms at m/z 11,306.4130 (0.57 ppm); (D) diacetylated histone H4 proteoform at m/z 11,320.3657 (0.42 ppm); and (E) diacetylated and dimethylated histone H4 proteoforms at m/z 11,348.4344 (1.78 ppm). Scale bars within the ion images are 600 μm, and each image has a separate ion intensity scale.
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