RESUMO
Native ambient mass spectrometry enables the in situ analysis of proteins and their complexes directly from tissue, providing both structural and spatial information. Until recently, the approach was applied exclusively to the analysis of soluble proteins; however, there is a drive for new techniques that enable analysis of membrane proteins. Here we demonstrate native ambient mass spectrometry of membrane proteins, including ß-barrel and α-helical (single and multipass) integral membrane proteins and membrane-associated proteins incorporating lipid anchors, by integration of a simple washing protocol to remove soluble proteins. Mass spectrometry imaging revealed that washing did not disrupt the spatial distributions of the membrane and membrane-associated proteins. Some delocalization of the remaining soluble proteins was observed.
Assuntos
Proteínas de Membrana , Proteínas de Membrana/química , Espectrometria de Massas/métodosRESUMO
Label-free spatial mapping of the noncovalent interactions of proteins in their tissue environment has the potential to revolutionize life sciences research by providing opportunities for the interrogation of disease progression, drug interactions, and structural and molecular biology more broadly. Here, we demonstrate mass spectrometry imaging of endogenous intact noncovalent protein-ligand complexes in rat brain. The spatial distributions of a range of ligand-bound and metal-bound proteins were mapped in thin tissue sections by use of nanospray-desorption electrospray ionization. Proteins were identified directly from the tissue by top-down mass spectrometry. Three GDP-binding proteins (ADP ribosylation factor ARF3, ARF1, and GTPase Ran) were detected, identified, and imaged in their ligand-bound form. The nature of the ligand was confirmed by multiple rounds of tandem mass spectrometry. In addition, the metal-binding proteins parvalbumin-α and carbonic anhydrase 2 were detected, identified, and imaged in their native form, i.e., parvalbumin-α + 2Ca2+ and carbonic anhydrase + Zn2+. GTPase Ran was detected with both GDP and Mg2+ bound. Several natively monomeric proteins displaying distinct spatial distributions were also identified by top-down mass spectrometry. Protein mass spectrometry imaging was achieved at a spatial resolution of 200 µm.
Assuntos
Encéfalo/metabolismo , Espectrometria de Massas/métodos , Metais/química , Proteínas/química , Proteínas/metabolismo , Animais , Ligantes , Masculino , Metais/metabolismo , Modelos Moleculares , Conformação Proteica , RatosRESUMO
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.
Assuntos
Scrapie , Espectrometria de Massas por Ionização por Electrospray , Animais , Encéfalo/metabolismo , Rim/metabolismo , Proteínas/metabolismo , Ovinos , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
Liquid extraction surface analysis (LESA) is an ambient surface sampling technique that allows the analysis of intact proteins directly from tissue samples via mass spectrometry. Integration of ion mobility separation to LESA mass spectrometry workflows has shown significant improvements in the signal-to-noise ratios of the resulting protein mass spectra and hence the number of proteins detected. Here, we report the use of a quadrupole-cyclic ion mobility-time-of-flight mass spectrometer (Q-cIM-ToF) for the analysis of proteins from mouse brain and rat kidney tissues sampled via LESA. Among other features, the instrument allows multiple pass cyclic ion mobility separation, with concomitant increase in resolving power. Single-pass experiments enabled the detection of 30 proteins from mouse brain tissue, rising to 44 when quadrupole isolation was employed. In the absence of ion mobility separation, 21 proteins were detected in rat kidney tissue including the abundant α- and ß-globin chains from hemoglobin. Single-pass cyclic ion mobility mass spectrometry enabled the detection of 60 additional proteins. Multipass experiments of a narrow m/z range (m/z 870-920) resulted in the detection of 24 proteins (one pass), 37 proteins (two passes) and 54 proteins (three passes), thus demonstrating the benefits of improved mobility resolving power.
Assuntos
Espectrometria de Mobilidade Iônica/métodos , Proteínas/análise , Animais , Encéfalo/metabolismo , Hemoglobinas/análise , Rim/metabolismo , Extração Líquido-Líquido , Camundongos , Ratos , Propriedades de SuperfícieRESUMO
Previously, we have shown that native ambient mass spectrometry imaging allows the spatial mapping of folded proteins and their complexes in thin tissue sections. Subsequent top-down native ambient mass spectrometry of adjacent tissue section enables protein identification. The challenges associated with protein identification by this approach are (i) the low abundance of proteins in tissue and associated long data acquisition timescales and (ii) irregular spatial distributions which hamper targeted sampling of the relevant tissue location. Here, we demonstrate that these challenges may be overcome through integration of laser capture microdissection in the workflow. We show identification of intact protein assemblies in rat liver tissue and apply the approach to identification of proteins in the granular layer of rat cerebellum.
RESUMO
Liquid extraction surface analysis (LESA) is an ambient surface sampling technique that can be coupled with mass spectrometry (MS) to analyze analytes directly from biological substrates such as tissue sections. LESA MS involves liquid microjunction sampling of a substrate by use of a discrete volume of solvent followed by nano-electrospray ionization. As the technique makes use of electrospray ionization, it lends itself to the analysis of intact proteins. Here, we describe the use of LESA MS to analyze and image the distribution of intact denatured proteins from thin fresh frozen tissue sections.
Assuntos
Proteínas , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas/métodos , Proteínas/química , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
Liquid extraction surface analysis (LESA) coupled to native mass spectrometry (MS) presents unique analytical opportunities due to its sensitivity, speed, and automation. Here, we examine whether this tool can be used to quantitatively probe protein-ligand interactions through calculation of equilibrium dissociation constants (Kd values). We performed native LESA MS analyses for a well-characterized system comprising bovine carbonic anhydrase II and the ligands chlorothiazide, dansylamide, and sulfanilamide, and compared the results with those obtained from direct infusion mass spectrometry and surface plasmon resonance measurements. Two LESA approaches were considered: In one approach, the protein and ligand were premixed in solution before being deposited and dried onto a solid substrate for LESA sampling, and in the second, the protein alone was dried onto the substrate and the ligand was included in the LESA sampling solvent. Good agreement was found between the Kd values derived from direct infusion MS and LESA MS when the protein and ligand were premixed; however, Kd values determined from LESA MS measurements where the ligand was in the sampling solvent were inconsistent. Our results suggest that LESA MS is a suitable tool for quantitative analysis of protein-ligand interactions when the dried sample comprises both protein and ligand.
Assuntos
Inibidores da Anidrase Carbônica , Extração Líquido-Líquido , Animais , Inibidores da Anidrase Carbônica/análise , Bovinos , Ligantes , Extração Líquido-Líquido/métodos , Espectrometria de Massas/métodos , Proteínas/química , SolventesRESUMO
We have previously demonstrated native liquid extraction surface analysis (LESA) mass spectrometry imaging of small intact proteins in thin tissue sections. We also showed calculation of collision cross sections for specific proteins extracted from discrete locations in tissue by LESA traveling wave ion mobility spectrometry (TWIMS). Here, we demonstrate an integrated native LESA TWIMS mass spectrometry imaging (MSI) workflow, in which ion mobility separation is central to the imaging experiment and which provides spatial, conformational, and mass information on endogenous proteins in a single experiment. The approach was applied to MSI of a thin tissue section of mouse kidney. The results show that the benefits of integration of TWIMS include improved specificity of the ion images and the capacity to calculate collision cross sections for any protein or protein complex detected in any pixel (without a priori knowledge of the presence of the protein).