Native Ambient Mass Spectrometry Enables Analysis of Intact Endogenous Protein Assemblies up to 145 kDa Directly from Tissue

Abstract

Untargeted label-free interrogation of proteins in their functional form directly from their physiological environment promises to transform life sciences research by providing unprecedented insight into their transient interactions with other biomolecules and xenobiotics. Native ambient mass spectrometry (NAMS) shows great potential for the structural analysis of endogenous protein assemblies directly from tissues; however, to date, this has been limited to assemblies of low molecular weight (<20 kDa) or very high abundance (hemoglobin tetramer in blood vessels, RidA homotrimer in kidney cortex tissues). The present work constitutes a step change for NAMS of protein assemblies: we demonstrate the detection and identification of a range of intact endogenous protein assemblies with various stoichiometries (dimer, trimer, and tetramer) from a range of tissue types (brain, kidney, liver) by the use of multiple NAMS techniques. Crucially, we demonstrate a greater than twofold increase in accessible molecular weight (up to 145 kDa). In addition, spatial distributions of protein assemblies up to 94 kDa were mapped in brain and kidney by nanospray desorption electrospray ionization (nano-DESI) mass spectrometry imaging.

Mass Spectrometry Detection and Imaging of a Non‐Covalent Protein–Drug Complex in Tissue from Orally Dosed Rats

“…Nano‐DESI is less well‐established than the more mainstream imaging techniques of matrix assisted laser desorption ionisation (MALDI) [14] and desorption electrospray ionisation (DESI) [15] . Although MALDI and DESI have been applied to protein imaging under denaturing conditions, [16] to date only LESA and nano‐DESI have been demonstrated for native mass spectrometry imaging [1c,d, 17] . Nano‐DESI offers higher spatial resolution than LESA.…”

Native Ambient Mass Spectrometry of an Intact Membrane Protein Assembly and Soluble Protein Assemblies Directly from Lens Tissue

“…The combined benefits of native ambient MS for the analysis of protein assemblies and protein‐ligand complexes include measurement of accurate mass and stoichiometry, identification of both protein and non‐covalently bound ligands, together with information on spatial distribution within the sample. Recent efforts using liquid extraction surface analysis (LESA) [3, 4] and nanospray‐desorption electrospray ionization (nano‐DESI), have advanced native ambient MS for the analysis of fresh frozen tissue, allowing the spatial analysis of protein assemblies up to 145 kDa in molecular weight [5, 6] . Endogenous protein assemblies and their constituents (including small molecule ligands) can be identified by top‐down dissection of assemblies in the gas phase, potentially allowing the discovery of new protein‐ligand interactions [6–8] .…”

“…[5,6] Endogenous protein assemblies and their constituents (including small molecule ligands) can be identified by top-down dissection of assemblies in the gas phase, potentially allowing the discovery of new protein-ligand interactions. [6][7][8] In addition, Sharon and co-workers have demonstrated direct native mass spectrometry of crude cell lysates. [9] To date, all protein assemblies analysed directly from tissue by native ambient MS have been soluble in aqueous solution.…”

Evaluation of Quantitative Platforms for Single Target Mass Spectrometry Imaging

“…Mass spectrometry imaging (MSI) has pushed the boundary of pharmaceutical studies by allowing for ever more precise understanding of the distribution of drugs [ 1 , 2 ], metabolites [ 3 , 4 ], and endogenous products [ 5 , 6 ] within tissue sections. Advancements in MSI technology have improved both the sensitivity and resolution with sensitivity routinely approaching 1 microgram of analyte (for m / z ~300–800) per gram of tissue [ 7 , 8 ], and resolution reaching 5 μm on commercial platforms [ 9 , 10 ].…”

“…There are 3 broad categories of MSI ionization sources that see the most use: secondary ion mass spectrometry (SIMS) [ 16 , 19 ], matrix-assisted laser desorption ionization (MALDI) [ 9 , 21 , 27 , 28 , 29 , 30 ], and desorption electrospray ionization (DESI) and its emergent developments [ 1 , 6 , 10 , 14 , 31 ]. SIMS is the highest spatial resolution technique available, achieving nanometer-scale resolution, with such resolution accompanied by high rates of fragmentation [ 32 , 33 ].…”