Analytical Chemistry

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

Authors

  • Kevin Zemaitis Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory ,
  • Dušan Veličković Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory ,
  • William Kew Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory ,
  • Kyle Fort Thermo Fisher Scientific (Bremen) GmbH ,
  • Maria Reinhardt-Szyba Thermo Fisher Scientific (Bremen) GmbH ,
  • Annapurna Pamreddy Center for Renal Precision Medicine, Department of Medicine, University of Texas Health, San Antonio ,
  • Yanli Ding Department of Pathology and Laboratory Medicine, University of Texas Health, San Antonio ,
  • Dharam Kaushik Department of Urology, University of Texas Health, San Antonio ,
  • Kumar Sharma Center for Renal Precision Medicine, Department of Medicine, University of Texas Health, San Antonio & Audie L. Murphy Memorial VA Hospital, South Texas Veterans Health Care System ,
  • Alexander Makarov Thermo Fisher Scientific (Bremen) GmbH & Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht ,
  • Mowei Zhou Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory ,
  • Ljiljana Paša-Tolić Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory

Abstract

Core histones including H2A, H2B, H3, and H4 are key modulators of cellular repair, transcription, and replication within eukaryotic cells, playing vital roles within 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 ultra-high 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 tissue 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 considerable increase in duty cycle over other Fourier trans-form 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 map-ping demonstrates several steps towards high-throughput accurate identification of proteoforms and provides a new tool for mapping biomolecule distributions throughout tissue sections in extended mass ranges.

Version notes

The manuscript underwent editing and polishing with details added for clarity of several points, some further Supplementary Information was added as well including two new Supplementary Figures.

Content

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Supplementary material

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SI for Enhanced Spatial Mapping of Histone Proteoforms in Human Kidney Through MALDI-MSI by High-Field UHMR Orbitrap Detection
Various supplementary information, supplementary figures, and a supplementary table. Outlining extended instrument parameters; S1: Digital photograph of the EP-MALDI Q Exactive HF Orbitrap-MS; S2: Demonstration of high and low mass transmission within CsI clusters; S3: Representation of direct tissue acidification effects on proteoform signal; S4: Exemplary serial line scans showing the effect of IST desolvation voltage; S5: Simulation of isotopic distributions of histone H4 N-Ac me2 on UHMR Orbitraps; S6: Simulation of isotopic distributions of histone H4 N-Ac me2 on a 15T FTICR MS for various detection periods; S7: Experimental data showing an average of scans from 0.512 s transients and 1.024 s transients on tissue; S8: Additional ion images of proteoforms from nephrectomy containing the RCC tumor; Table S1: Extracted resolutions from the simulations within Figure S3 and S4; and references.