Native Top-Down Mass Spectrometry with Collisionally Activated Dissociation Yields Higher-Order Structure Information for Protein Complexes.

Native mass spectrometry (MS) of proteins and protein assemblies reveals size and binding stoichiometry, but elucidating structures to understand their function is more challenging. Native top-down MS (nTDMS), i.e.

On the Mechanism of Theta Capillary Nanoelectrospray Ionization for the Formation of Highly Charged Protein Ions Directly from Native Solutions.

Theta capillary nanoelectrospray ionization (θ-nanoESI) can be used to "supercharge" protein ions directly from solution for detection by mass spectrometry (MS). In native top-down MS, the extent of protein charging is low. Given that ions with more charge fragment more readily, increasing charge can enhance the extent of sequence information obtained by top-down MS.

Internal Fragments Generated by Electron Ionization Dissociation Enhance Protein Top-Down Mass Spectrometry.

Top-down proteomics by mass spectrometry (MS) involves the mass measurement of an intact protein followed by subsequent activation of the protein to generate product ions. Electron-based fragmentation methods like electron capture dissociation and electron transfer dissociation are widely used for these types of analyses. Recently, electron ionization dissociation (EID), which utilizes higher energy electrons (>20 eV) has been suggested to be more efficient for top-down protein fragmentation compared to other electron-based dissociation methods.

PEPPI-MS: Polyacrylamide-Gel-Based Prefractionation for Analysis of Intact Proteoforms and Protein Complexes by Mass Spectrometry.

Prefractionation of complex mixtures of proteins derived from biological samples is indispensable for proteome analysis via top-down mass spectrometry (MS). Polyacrylamide gel electrophoresis (PAGE), which enables high-resolution protein separation based on molecular size, is a widely used technique in biochemical experiments and has the potential to be useful in sample fractionation for top-down MS analysis. However, the lack of a means to efficiently recover the separated proteins in-gel has always been a barrier to its use in sample prefractionation.

Biosample Concentration Using Microscale Forward Osmosis with Electrochemical Monitoring.

We report the design and operation of an integrated microfluidics system that uses cellulose ester dialysis membranes coupled with disposable carbon and copper electrodes for monitoring and concentration of microliter scale biofluid samples. Dialysis membranes are typically used for buffer exchange, but in this work, membranes with 100-500 Da MWCO were evaluated for feasibility in concentrating small volume samples. This is an alternative to the use of centrifugation, ultrafiltration, and evaporative methods, where quantitative inline monitoring of sample concentration is challenging.

Proteomics identification of radiation-induced changes of membrane proteins in the rat model of arteriovenous malformation in pursuit of targets for brain AVM molecular therapy.

Background: Rapid identification of novel targets and advancement of a vascular targeting strategy requires a comprehensive assessment of AVM endothelial membrane protein changes in response to irradiation. The aim of this study is to provide additional potential target protein molecules for evaluation in animal trials to promote intravascular thrombosis in AVM vessels post radiosurgery.

Methods: We employed in vivo biotinylation methodology that we developed, to label membrane proteins in the rat model of AVM post radiosurgery.

Structural Characterization of a Thrombin-Aptamer Complex by High Resolution Native Top-Down Mass Spectrometry.

Native mass spectrometry (MS) with electrospray ionization (ESI) has evolved as an invaluable tool for the characterization of intact native proteins and non-covalently bound protein complexes. Here we report the structural characterization by high resolution native top-down MS of human thrombin and its complex with the Bock thrombin binding aptamer (TBA), a 15-nucleotide DNA with high specificity and affinity for thrombin. Accurate mass measurements revealed that the predominant form of native human α-thrombin contains a glycosylation mass of 2205 Da, corresponding to a sialylated symmetric biantennary oligosaccharide structure without fucosylation.

Top-down/Bottom-up Mass Spectrometry Workflow Using Dissolvable Polyacrylamide Gels.

Biologists' preeminent toolbox for separating, analyzing, and visualizing proteins is SDS-PAGE, yet recovering the proteins embedded in these polyacrylamide media as intact species is a long-standing challenge for mass spectrometry. In conventional workflows, protein mixtures from crude biological samples are electrophoretically separated at high-resolution within N,N'-methylene-bis-acrylamide cross-linked polyacrylamide gels to reduce sample complexity and facilitate sensitive characterization. However, low protein recoveries, especially for high molecular weight proteins, often hinder characterization by mass spectrometry.

Identification of protein targets in cerebral endothelial cells for brain arteriovenous malformation (AVMs) molecular therapies.

Background: To develop a new molecular targeted treatment for brain (AVMs), identification of membrane proteins that are localised on the AVM endothelium is crucial. Current treatment methods are surgery and radiosurgery. However, complete occlusion post radiosurgery are achieved within 3 years, while patient remain at risk of haemorrhage.

Improving Proteome Coverage and Sample Recovery with Enhanced FASP (eFASP) for Quantitative Proteomic Experiments.

Enhanced Filter Aided Sample Preparation (eFASP) incorporates plastics passivation and digestion-enhancing surfactants into the traditional FASP workflow to reduce sample loss and increase hydrophobic protein representation in qualitative and quantitative proteomics experiments. Resulting protein digests are free of contaminants and can be analyzed directly by LC-MS.

CryoEM structure of the Methanospirillum hungatei archaellum reveals structural features distinct from the bacterial flagellum and type IV pilus.

Archaea use flagella known as archaella-distinct both in protein composition and structure from bacterial flagella-to drive cell motility, but the structural basis of this function is unknown. Here, we report an atomic model of the archaella, based on the cryo electron microscopy (cryoEM) structure of the Methanospirillum hungatei archaellum at 3.4 Å resolution.

Salt Bridge Rearrangement (SaBRe) Explains the Dissociation Behavior of Noncovalent Complexes.

Native electrospray ionization-mass spectrometry, with gas-phase activation and solution compositions that partially release subcomplexes, can elucidate topologies of macromolecular assemblies. That so much complexity can be preserved in gas-phase assemblies is remarkable, although a long-standing conundrum has been the differences between their gas- and solution-phase decompositions. Collision-induced dissociation of multimeric noncovalent complexes typically distributes products asymmetrically (i.

Enhancing Protein Disulfide Bond Cleavage by UV Excitation and Electron Capture Dissociation for Top-Down Mass Spectrometry.

The application of ion pre-activation with 266 nm ultraviolet (UV) laser irradiation combined with electron capture dissociation (ECD) is demonstrated to enhance top-down mass spectrometry sequence coverage of disulfide bond containing proteins. UV-based activation can homolytically cleave a disulfide bond to yield two separated thiol radicals. Activated ECD experiments of insulin and ribonuclease A containing three and four disulfide bonds, respectively, were performed.

Addressing the needs of traumatic brain injury with clinical proteomics.

Background: Neurotrauma or injuries to the central nervous system (CNS) are a serious public health problem worldwide. Approximately 75% of all traumatic brain injuries (TBIs) are concussions or other mild TBI (mTBI) forms. Evaluation of concussion injury today is limited to an assessment of behavioral symptoms, often with delay and subject to motivation.

Enhanced FASP (eFASP) to increase proteome coverage and sample recovery for quantitative proteomic experiments.

The integrity of quantitative proteomic experiments depends on the reliability and the robustness of the protein extraction, solubilization, and digestion methods utilized. Combinations of detergents, chaotropes, and mechanical disruption can yield successful protein preparations; however, the methods subsequently required to eliminate these added contaminants, in addition to the salts, nucleic acids, and lipids already in the sample, can result in significant sample losses and incomplete contaminant removal. A recently introduced method for proteomic sample preparation, filter-aided sample preparation (FASP), cleverly circumvents many of the challenges associated with traditional protein purification methods but is associated with significant sample loss.

Thermal-stable proteins of fruit of long-living Sacred Lotus Gaertn var. China Antique.

Single-seeded fruit of the sacred lotus Gaertn var. China Antique from NE China have viability as long as ~1300 years determined by direct radiocarbon-dating, having a germination rate of 84%. The pericarp, a fruit tissue that encloses the single seeds of , is considered one of the major factors that contribute to fruit longevity.

Protein complexes: breaking up is hard to do well.

Mass spectrometry of protein assemblies reveals size and stoichiometry. In this issue of Structure, Hall and colleagues demonstrate that gas-phase dissociations can recapitulate solution structure for complexes with few intersubunit salt bridges, high charge density, inflexible subunits, or small intersubunit interfaces.

Cofilin-induced changes in F-actin detected via cross-linking with benzophenone-4-maleimide.

Cofilin is a member of the actin depolymerizing factor (ADF)/cofilin family of proteins. It plays a key role in actin dynamics by promoting disassembly and assembly of actin filaments. Upon its binding, cofilin has been shown to bridge two adjacent protomers in filamentous actin (F-actin) and promote the displacement and disordering of subdomain 2 of actin.

Concomitant inhibition of HSP90, its mitochondrial localized homologue TRAP1 and HSP27 by green tea in pancreatic cancer HPAF-II cells.

Pancreatic cancer is a deadly disease characterized by poor prognosis and patient survival. Green tea polyphenols have been shown to exhibit multiple antitumor activities in various cancers, but studies on the pancreatic cancer are very limited. To identify the cellular targets of green tea action, we exposed a green tea extract (GTE) to human pancreatic ductal adenocarcinoma HPAF-II cells and performed two-dimensional gel electrophoresis of the cell lysates.

Synthesis of Maleimide-End Functionalized Star Polymers and Multimeric Protein-Polymer Conjugates.

Protein-polymer conjugates exhibit superior properties to unmodified proteins, generating a high demand for these materials in the fields of medicine, biotechnology, and nanotechnology. Multimeric conjugates are predicted to surpass the activity of monomeric conjugates. Herein, we report a straightforward method to synthesize multimeric polymer-conjugates.

New reagents for increasing ESI multiple charging of proteins and protein complexes.

The addition of m-nitrobenzyl alcohol (m-NBA) was shown previously (Lomeli et al., J. Am.

The heat shock protein inhibitor Quercetin attenuates hepatitis C virus production.

Unlabelled: The hepatitis C viral (HCV) genome is translated through an internal ribosome entry site (IRES) as a single polyprotein precursor that is subsequently cleaved into individual mature viral proteins. Nonstructural protein 5A (NS5A) is one of these proteins that has been implicated in regulation of viral genome replication, translation from the viral IRES and viral packaging. We sought to identify cellular proteins that interact with NS5A and determine whether these interactions may play a role in viral production.

Reconstitution of the mia40-erv1 oxidative folding pathway for the small tim proteins.

Mia40 and Erv1 execute a disulfide relay to import the small Tim proteins into the mitochondrial intermembrane space. Here, we have reconstituted the oxidative folding pathway in vitro with Tim13 as a substrate and determined the midpoint potentials of Mia40 and Tim13. Specifically, Mia40 served as a direct oxidant of Tim13, and Erv1 was required to reoxidize Mia40.

S-layer, surface-accessible, and concanavalin A binding proteins of Methanosarcina acetivorans and Methanosarcina mazei.

The outermost cell envelope structure of many archaea and bacteria contains a proteinaceous lattice termed the surface layer or S-layer. It is typically composed of only one or two abundant, often posttranslationally modified proteins that self-assemble to form the highly organized arrays. Surprisingly, over 100 proteins were annotated to be S-layer components in the archaeal species Methanosarcina acetivorans C2A and Methanosarcina mazei Gö1, reflecting limitations of current predictions.

Connecting actin monomers by iso-peptide bond is a toxicity mechanism of the Vibrio cholerae MARTX toxin.

The Gram-negative bacterium Vibrio cholerae is the causative agent of a severe diarrheal disease that afflicts three to five million persons annually, causing up to 200,000 deaths. Nearly all V. cholerae strains produce a large multifunctional-autoprocessing RTX toxin (MARTX(Vc)), which contributes significantly to the pathogenesis of cholera in model systems.