Hello, everyone. This week, I’d like to share an article published on anal Chem, native ambient mass spectroscopy enablers analysis of indirect endogenous protein assemblies up to 145 kDa directly from tissue 。 The corresponding author of the article is Professor Helen J. Cooper from the University of Birmingham, UK.
Non denaturing in situ mass spectrometry (NAMS) is a new top-down mass spectrometry method. Combining the advantages of non denaturing mass spectrometry and in situ mass spectrometry, it can be directly characterized in the physiological environment of proteins and their complexes. Nams can provide information on protein structure, space and transient interaction, and has great potential to analyze endogenous protein assemblies directly from tissues. However, at present, nams is only successfully applied to directly detect proteins with low molecular weight (& lt; 20kDa) or high abundance, such as hemoglobin tetramer or Rida homotrimer.
At present, liquid extraction surface analysis (LESA) and Nano Spray analytical electrospray ionization (nano DESI) are applied to the nams analysis of proteins in tissues 。 LESA requires automatic micro liquid node sampling of tissue substrates, and then Nano Spray Ionization (nanoESI) for MS analysis. Nano desi forms a mobile solvent bridge with the mobile phase through the probe, which is in direct contact with the sample, and then carries out mass spectrometry analysis and imaging.
In this study, the author used nams to comprehensively analyze the protein assemblies in rat brain, kidney and liver tissue sections. By adjusting the ion optics and gas pressure in the mass spectrometer to improve the m/z transport and nano spray performance, the analysis of large protein assemblies can be realized. Results eight protein assemblies were identified, which proved that the accessible molecular weight of nams was up to 145 kDa. The spatial distribution of protein assemblies up to 94 kDa was mapped in the brain and kidney by nano desi mass spectrometry imaging.
In brain tissue, the authors mapped the spatial distribution and corresponding mass spectra of complete protein complexes with molecular weight of 37.0 – 66.4kda in rat brain slices by nano desi nams (Fig. 1a, b). The experiment successfully identified and imaged three protein assemblies in rat brain tissue, including homotrimeric cytokine macrophage migration inhibitory factor (MIF, 37.0kda), homodimeric phosphoglycerate mutase 1 (pgam1, 57.6kda) and malate dehydrogenase 2 (mdh2, 66.4kda). It was found that MIF was evenly distributed throughout the brain tissue region (Fig. 1c). The author identified MIF by analyzing the spectra of monomer (5 +, 4 +) and dimer (5 +) subunits and sequence ions produced by nano-desi-hcd MS2 (Fig. 1D). The authors also successfully identified pgam1 using nano-desi-hcd MS2 and found that pgam1 showed high abundance in the cerebral cortex and low abundance in the midbrain and corpus callosum (Fig. 1E, f). In contrast, the spatial distribution of mdh2 in brain tissue is basically uniform (Fig. 1g, H). The detected results are consistent with the actual distribution of protein in rat brain, and the reliability is high.
Figure 1 Ion images and HCD MS2 spectra showed the dissociation of subunits of protein complexes in rat brain. (a) H& Optical images of continuous tissue sections stained with E. Label:CE, cerebellum; C. Cerebral cortex; CC, corpus callosum; F. Fornix; 5. Lateral ventricle; MB, midbrain; Me, medulla and pons; H. Hippocampus; Th, thalamus; HT, hypothalamus; BG, basal ganglia; Or, olfactory region. (b) Nano desi full scan mass spectrometry, representing the pixels marked”(b)” in the optical image. (C, d) macrophage inhibitory factor homotrimers showed uniform distribution. (E, f) pgam1 homodimer distribution. (g, H) mdh2 homodimer distribution.
In addition, the authors identified four homodimeric protein components (61.2-94.2kda) in rat kidney, including ω- Amidase (61.2kda), mdh2 (66.4kda), malate dehydrogenase 1 (mdh1, 72.8kda) and α- Enolase (94.2kda) and imaged it (Fig. 2). The observed α- Enolase is a metal binding form, and two Mg 2 + ions are bound to each subunit.
Figure 2 (a) H & amp of rat kidney; E-stained serial sections showed cortical (c) and medullary (m) tissues. (b) Example full scan mass spectrometry of a single nano desi pixel in rat renal cortical tissue obtained during MSI. (c, d) α- Enolase homodimer. (E, f) malate dehydrogenase 1. (g, H) mdh2 homodimer. (i, j) ω- Amidase.
Homotrimeric ornithine transcarbamylase (OTC, 108.8kda) and homotetrameric lactate dehydrogenase A (ldhA, 145.4kda) were also identified from rat liver tissue (Fig. 3). In the full scan mode, nano desi can detect the weak signal of 145.4kda ldhA. Through the further confirmation of nano-desi-ptcr MS2, the detected substance is indeed ldhA.
Figure 3 (a) Nano desi PTCR MS 2 of intact OTC homotrimer directly from rat liver tissue. (b) Nano desi HCD MS 2 of intact OTC homotrimer showed a subunit mass of 36.2kda. (c) Nanoesi-ptcr MS2 of intact ldhA homotetramer (145.4kda). (d) Nanoesi-hcd MS2 of intact ldhA homotetramer.
In this study, the authors successfully detected and identified endogenous protein assemblies from tissues by using nams mass spectrometry. The molecular range is 37.0-145.4kda, including dimer, trimer and tetramer. The detected upper limit (145.4kda) exceeds twice the upper limit of quality reported by LESA MS, which is 100kda higher than the upper limit of quality reported by nano desi. By adjusting ion optics and high m/Z gas pressure, or the development of subsequent instruments and methods, nams may further break through the upper limit of 145.4kda and detect protein assemblies with greater molecular weight.
Hale OJ, Hughes JW, Sisley EK, Cooper HJ. Native Ambient Mass Spectrometry Enables Analysis of Intact Endogenous Protein Assemblies up to 145 kDa Directly from Tissue. Anal Chem. 2022 Apr 12; 94(14):5608-5614.
Hale OJ, Cooper HJ. Native Mass Spectrometry Imaging and In Situ Top-Down Identification of Intact Proteins Directly from Tissue. J Am Soc Mass Spectrom. 2020 Dec 2; 31(12):2531-2537.