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

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.

Spatially resolved top-down proteomics of tissue sections based on a microfluidic nanodroplet sample preparation platform

“…The flow rate of matrix was 150 µL/min with a nozzle temperature of 30.0 o C; velocity was set to 1300 mm/min with 10 PSI of nitrogen gas. Matrix was then recrystallized with 5% acetic acid solution in water at 38.5 o C and dried for 3.5 minutes and then immediately analyzed using an elevated pressure MALDI source (Spectroglyph LLC, Kennewick, WA) coupled to a Thermo Scientific Q Exactive HF Orbitrap MS upgraded with ultra-high mass range (UHMR) boards (29). Spectra were acquired over the m/z range of 3,500 to 20,000 in positive polarity mode with a resolving power of 240k at m/z 200 (512 ms transient) and 250 laser shots per pixel.…”

193 nm ultraviolet photodissociation for the characterization of singly charged proteoforms generated by MALDI

“…An elevated pressure MALDI source from Spectroglyph (Kennewick, WA) 15 was mounted on a Thermo Scientific Q Exactive HF Orbitrap MS upgraded with UHMR boards and operated under custom privilege licenses, as described in great depth elsewhere. 2 Detailed notes for instrumental parameters and sample preparation are presented within Supplementary Information. Briefly, the spectrometer was operated under default parameters with several specifically optimized for MALDI-UVPD.…”

“…Fourier transform ion cyclotron resonance (FTICR) are commonly used mass analyzers that have both been applied to analyze intact proteins. 1 High mass resolution measurements by FTICR, and recently ultra-high mass range (UHMR) Orbitrap, 2,3 offer advantages over TOF for more precise identification of proteoforms, distinct forms of proteins potentially harboring multiple post-translational modifications (PTMs). 4,5 However, assigning proteoforms solely based on accurate mass can be challenging regardless of the analyzer employed, and often requires supplemental analyses.…”

Spatially resolved top-down proteomics of tissue sections based on a microfluidic nanodroplet sample preparation platform

“…The flow rate of the matrix was 150 µL/min with a nozzle temperature of 30.0 o C, with a velocity set to 1300 mm/min with 10 PSI of nitrogen gas. The matrix was then recrystallized with 5% acetic acid solution in water at 38.5 o C and dried for 3.5 minutes and then immediately analyzed using an elevated pressure MALDI source (Spectroglyph LLC, Kennewick, WA) coupled to a Thermo Scientific Q Exactive HF Orbitrap MS upgraded with ultra-high mass range (UHMR) boards (29). Spectra were acquired over the m/z range of 3,500 to 20,000 in positive polarity mode with a resolving power of 240k at m/z 200 (512 ms transient) and 250 laser shots per pixel.…”