Direct Imaging and Identification of Proteoforms up to 70 kDa from Human Tissue

Abstract

Imaging of proteoforms in human tissues is hindered by low molecular specificity and limited proteome coverage. Here, we introduce proteoform imaging mass spectrometry (PiMS), which increases the size limit for proteoform detection and identification by 4-fold compared to reported methods, and reveals tissue localization of proteoforms at <80 μm spatial resolution. PiMS advances proteoform imaging by combining liquid sampling (nanospray desorption electrospray ionization, nano-DESI) with ion detection using individual ion mass spectrometry (I2MS). We demonstrate the first proteoform imaging of human kidney, identifying 169 of 400 proteoforms <70 kDa using top-down mass spectrometry and database lookup from the human proteoform atlas, including dozens of key enzymes in primary metabolism. Moreover, PiMS images visualize kidney anatomical structures and cellular neighborhoods in the vasculature versus the medulla or the cortex. The benefits of PiMS are poised to increase proteome coverage for label-free protein imaging of intact tissues.

Native Mass Spectrometry at the Convergence of Structural Biology and Compositional Proteomics

“…When combined with the enhanced sensitivity of I 2 MS in the form of "proteoform imaging MS," proteoforms up to 70 kDa can be simultaneously mapped across the tissue sample regardless of heterogeneity. 59 While proteoform imaging MS has only been demonstrated using denaturing solutions, nano-DESI has been utilized in native mode, 60 which asserts that proteoform imaging MS will also be able to image and characterize large protein complexes in a state closer to how they appear in biology. As visual proteomics reaches its full potential, it may be possible to noninvasively map proteoforms in living patients.…”

“…One form of tissue imaging is nanospray desorption electrospray ionization (nano-DESI) MS. Nano-DESI MS uses a traveling probe to continuously extract proteins for MS analysis. When combined with the enhanced sensitivity of I 2 MS in the form of “proteoform imaging MS,” proteoforms up to 70 kDa can be simultaneously mapped across the tissue sample regardless of heterogeneity . While proteoform imaging MS has only been demonstrated using denaturing solutions, nano-DESI has been utilized in native mode, which asserts that proteoform imaging MS will also be able to image and characterize large protein complexes in a state closer to how they appear in biology.…”

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

“…This lends credence to multiplexed LCM based proteomics and alternative TD-MS strategies such as implementation of MALDI in-source decay (ISD) or ultraviolet photodissociation (UVPD) both of which have been demonstrated within lower mass ranges on MALDI enabled Orbitrap MS. 59 Routine TD fragmentation of proteoforms has not yet been demonstrated directly from tissue for MALDI analyses, but dissociation methods are more commonly for ambient ionization techniques which create multiply charged protein ions. 21,23,60 Nonetheless, the variance noted within the proteoforms detected by MALDI-MSI highlights the stability and promise of this platform for the mapping of distinct (histone) proteoform signatures, as exemplified above within regions of tubular atrophy. Case Study: MALDI-MSI on a Section of Kidney from a Renal Cell Carcinoma Nephrectomy: Hereditary disorders, as well as external risk factors such as smoking and exposure to toxins, play roles in the pathogenesis of cancer, suggesting a strong link to the epigenetics and histone profiles.…”

“…18 Several ambient imaging approaches have recently gained more traction for protein applications, allowing for the detection and TD dissociation of multiply charged proteoforms and even native protein complexes. [19][20][21][22][23] Despite these advances, matrix-assisted laser desorption/ ionization (MALDI) remains the most commonly used tool for MSI based proteomic applications. 17,24 MALDI has been capable of cellular resolution MSI for intact protein analyses for nearly a decade, 25,26 with several reports documenting distributions of proteins with masses over 100 kDa to recent upper bounds of 200 kDa.…”

“…Despite the widespread proteomic applications within MSI, the path forward is a challenging multidisciplinary effort reliant on instrument and method development . Several ambient imaging approaches have recently gained more traction for protein applications, allowing for the detection and TD dissociation of multiply charged proteoforms and even native protein complexes. − Despite these advances, matrix-assisted laser desorption/ ionization (MALDI) remains the most commonly used tool for MSI-based proteomic applications. , MALDI has been capable of cellular resolution MSI for intact protein analyses for nearly a decade, , with several reports documenting distributions of proteins with masses over 100 kDa to recent upper bounds of 200 kDa. , However, several distinct challenges arise, both from the sample preparation requirements and limited proteome coverage afforded by MSI …”