RESUMO
Myosin VI (Myo6) is the only minus-end directed nanomotor on actin, allowing it to uniquely contribute to numerous cellular functions. As for other nanomotors, the proper functioning of Myo6 relies on precise spatiotemporal control of motor activity via a poorly defined off-state and interactions with partners. Our structural, functional, and cellular studies reveal key features of myosin regulation and indicate that not all partners can activate Myo6. TOM1 and Dab2 cannot bind the off-state, while GIPC1 binds Myo6, releases its auto-inhibition and triggers proximal dimerization. Myo6 partners thus differentially recruit Myo6. We solved a crystal structure of the proximal dimerization domain, and show that its disruption compromises endocytosis in HeLa cells, emphasizing the importance of Myo6 dimerization. Finally, we show that the L926Q deafness mutation disrupts Myo6 auto-inhibition and indirectly impairs proximal dimerization. Our study thus demonstrates the importance of partners in the control of Myo6 auto-inhibition, localization, and activation.
Assuntos
Actinas , Cadeias Pesadas de Miosina , Humanos , Células HeLa , Dimerização , Actinas/metabolismo , Cadeias Pesadas de Miosina/metabolismoRESUMO
Charge-detection mass spectrometry (CDMS) enables direct measurement of the charge of an ion alongside its mass-to-charge ratio. CDMS offers unique capabilities for the analysis of samples where isotopic resolution or the separation of charge states cannot be achieved, i.e., heterogeneous macromolecules or highly complex mixtures. CDMS is usually performed using static nano-electrospray ionization-based direct infusion with acquisition times in the range of several tens of minutes to hours. Whether CDMS analysis is also attainable on shorter time scales, e.g., comparable to chromatographic peak widths, has not yet been extensively investigated. In this contribution, we probed the compatibility of CDMS with online liquid chromatography interfacing. Size exclusion chromatography was coupled to CDMS for separation and mass determination of a mixture of transferrin and ß-galactosidase. Molecular masses obtained were compared to results from mass spectrometry based on ion ensembles. A relationship between the number of CDMS spectra acquired and the achievable mass accuracy was established. Both proteins were found to be confidently identified using CDMS spectra obtained from a single chromatographic run when peak widths in the range of 1.4-2.5 min, translating to 140-180 spectra per protein were achieved. After demonstration of the proof of concept, the approach was tested for the characterization of the highly complex glycoprotein α-1-acid glycoprotein and the Fc-fusion protein etanercept. With chromatographic peak widths of approximately 3 min, translating to â¼200 spectra, both proteins were successfully identified, demonstrating applicability for samples of high inherent molecular complexity.
Assuntos
Espectrometria de Massas por Ionização por Electrospray , Transferrina , Espectrometria de Massas por Ionização por Electrospray/métodos , Cromatografia Líquida , Cromatografia em Gel , OrosomucoideRESUMO
Polycomb repressive complex 2 (PRC2) mediates epigenetic silencing of target genes in animals and plants. In Arabidopsis, PRC2 is required for the cold-induced epigenetic silencing of the FLC floral repressor locus to align flowering with spring. During this process, PRC2 relies on VEL accessory factors, including the constitutively expressed VRN5 and the cold-induced VIN3. The VEL proteins are physically associated with PRC2, but their individual functions remain unclear. Here, we show an intimate association between recombinant VRN5 and multiple components within a reconstituted PRC2, dependent on a compact conformation of VRN5 central domains. Key residues mediating this compact conformation are conserved among VRN5 orthologs across the plant kingdom. In contrast, VIN3 interacts with VAL1, a transcriptional repressor that binds directly to FLC These associations differentially affect their role in H3K27me deposition: Both proteins are required for H3K27me3, but only VRN5 is necessary for H3K27me2. Although originally defined as vernalization regulators, VIN3 and VRN5 coassociate with many targets in the Arabidopsis genome that are modified with H3K27me3. Our work therefore reveals the distinct accessory roles for VEL proteins in conferring cold-induced silencing on FLC, with broad relevance for PRC2 targets generally.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Histonas/genética , Histonas/metabolismo , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Domínio MADS/genética , Flores/genética , Flores/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismoRESUMO
General approaches for designing sequence-specific peptide-binding proteins would have wide utility in proteomics and synthetic biology. However, designing peptide-binding proteins is challenging, as most peptides do not have defined structures in isolation, and hydrogen bonds must be made to the buried polar groups in the peptide backbone1-3. Here, inspired by natural and re-engineered protein-peptide systems4-11, we set out to design proteins made out of repeating units that bind peptides with repeating sequences, with a one-to-one correspondence between the repeat units of the protein and those of the peptide. We use geometric hashing to identify protein backbones and peptide-docking arrangements that are compatible with bidentate hydrogen bonds between the side chains of the protein and the peptide backbone12. The remainder of the protein sequence is then optimized for folding and peptide binding. We design repeat proteins to bind to six different tripeptide-repeat sequences in polyproline II conformations. The proteins are hyperstable and bind to four to six tandem repeats of their tripeptide targets with nanomolar to picomolar affinities in vitro and in living cells. Crystal structures reveal repeating interactions between protein and peptide interactions as designed, including ladders of hydrogen bonds from protein side chains to peptide backbones. By redesigning the binding interfaces of individual repeat units, specificity can be achieved for non-repeating peptide sequences and for disordered regions of native proteins.
Assuntos
Peptídeos , Engenharia de Proteínas , Proteínas , Sequência de Aminoácidos , Modelos Moleculares , Peptídeos/química , Peptídeos/metabolismo , Proteínas/química , Proteínas/metabolismo , Engenharia de Proteínas/métodos , Ligação de Hidrogênio , Ligação Proteica , Dobramento de Proteína , Conformação ProteicaRESUMO
Ultraviolet photodissociation is a fast, photon-mediated fragmentation method that yields high sequence coverage and informative cleavages of biomolecules. In this work, 193 nm UVPD was coupled with a 12 Tesla FT-ICR mass spectrometer and 10.6 µm infrared multi-photon dissociation to provide gentle slow-heating of UV-irradiated ions. No internal instrument hardware modifications were required. Adjusting the timing of laser pulses to the ion motion within the ICR cell provided consistent fragmentation yield shot-to-shot and may also be used to monitor ion positions within the ICR cell. Single-pulse UVPD of the native-like 5+ charge state of ubiquitin resulted in 86.6% cleavage coverage. Additionally, IR activation post UVPD doubled the overall fragmentation yield and boosted the intensity of UVPD-specific x-type fragments up to 4-fold. This increased yield effect was also observed for the 6+ charge state of ubiquitin, albeit less pronounced. This indicates that gentle slow-heating serves to sever tethered fragments originating from non-covalently linked compact structures and makes activation post UVPD an attractive option to boost fragmentation efficiency for top-down studies. Lastly, UVPD was implemented and optimized as a fragmentation method for 2DMS, a data-independent acquisition method. UVPD-2DMS was demonstrated to be a viable method using BSA digest peptides as a model system.
Assuntos
Espectrometria de Massas em Tandem , Raios Ultravioleta , Espectrometria de Massas em Tandem/métodos , Íons , Peptídeos , UbiquitinaRESUMO
Understanding modification of synthetic polymer structures is necessary for their accurate synthesis and potential applications. In this contribution, a series of partially hydrolyzed poly(2-oxazoline) species were produced forming poly[(2-polyoxazoline)-co-(ethylenimine)] (P(EtOx-co-EI)) copolymers; EI being the hydrolyzed product of Ox. Bulk mass spectrometry (MS) measurements accurately measured the EI content. Tandem mass spectrometry analysis of the EI content in the copolymer samples determined the distribution of each monomer within the copolymer and corresponded to a theoretically modelled random distribution. The EI distribution across the polymers was shown to be effected by the choice of terminus, with a permanent hydrolysis event observed at an OH terminus. A neighbouring group effect wasn't observed at the polymer length analysed (approximately 25-mer species), suggesting that previously observed neighbouring group effects require a larger polymer chain. Although clearly useful for random polymer distribution this approach may be applied to many systems containing non-specific modifications to determine if they are directed or random locations across peptides, proteins, polymers, and nucleic acids.
RESUMO
Collisionally activated dissociation (CAD), infrared multiphoton dissociation (IRMPD), ultraviolet photodissociation (UVPD), electron capture dissociation and electron detachment dissociation (EDD) experiments were conducted on a set of phosphopeptides, in a Fourier transform ion cyclotron resonance mass spectrometer. The fragmentation patterns were compared and varied according to the fragmentation mechanisms and the composition of the peptides. CAD and IRMPD produced similar fragmentation profiles of the phosphopeptides, while UVPD produced a large number of complementary fragments. Electron-based dissociation techniques displayed lower fragmentation efficiencies, despite retaining the labile phosphate group, and drastically different fragmentation profiles. EDD produced complex spectra whose interpretation proved challenging.
Assuntos
Fosfopeptídeos , Espectrometria de Massas em Tandem , Ciclotrons , Elétrons , Análise de Fourier , Fosfopeptídeos/química , Espectrometria de Massas em Tandem/métodosRESUMO
Adeno-associated virus (AAV)-based gene therapy is a rapidly developing field, requiring analytical methods for detailed product characterization. One important quality attribute of AAV products that requires monitoring is the amount of residual empty capsids following downstream processing. Traditionally, empty and full particles are quantified via analytical ultracentrifugation as well as anion exchange chromatography using ultraviolet or fluorescence detection. Here, we present a native mass spectrometry-based approach to assess the ratio of empty to full AAV-capsids without the need for excessive sample preparation. We report the rapid determination of the relative amount of empty capsids in AAV5 and AAV8 samples. The results correlate well with more conventional analysis strategies, demonstrating the potential of native mass spectrometry for the characterization of viral particles.
Assuntos
Capsídeo , Dependovirus , Dependovirus/genética , Terapia Genética , Vetores Genéticos/genética , Espectrometria de MassasRESUMO
Disulfide bond reduction within antibody mass spectrometry workflows is typically carried out using chemical reducing agents to produce antibody subunits for middle-down and middle-up analysis. In this contribution we offer an online electrochemical reduction method for the reduction of antibodies coupled with liquid chromatography (LC) and mass spectrometry (MS), reducing the disulfide bonds present in the antibody without the need for chemical reducing agents. An electrochemical cell placed before the analytical column and mass spectrometer facilitated complete reduction of NISTmAb inter- and intrachain disulfide bonds. Reduction and analysis were carried out under optimal solvent conditions using a trapping column and switching valve to facilitate solvent exchange during analysis. The level of reduction was shown to be affected by electrochemical potential, temperature and solvent organic content, but with optimization, complete disulfide bond cleavage was achieved. The use of an inline electrochemical cell offers a simple, rapid, workflow solution for liquid chromatography mass spectrometry analysis of antibody subunits.
Assuntos
Dissulfetos , Técnicas Eletroquímicas , Cromatografia Líquida , Espectrometria de Massas , Fluxo de TrabalhoRESUMO
Due to the natural dispersity that is present in synthetic polymers, an added complexity is always present in the analysis of polymeric species. Tandem mass spectrometry analysis requires the isolation of individual precursors before a fragmentation event to allow the unambiguous characterization of these species and is not viable at certain levels of complexity due to achievable isolation widths. Two-dimensional mass spectrometry (2DMS) fragments ions and correlates fragments with their corresponding precursors without the need for isolation. In this study, 2DMS electron capture dissociation (ECD) fragmentation of a polyoxazoline and polyacrylamide species was carried out, resulting in the analysis of byproducts and individual polymer species without the use of chromatographic techniques. This study shows that 2DMS ECD is a powerful tool for the analysis of polyacrylamide and polyoxazoline species and offers a new dimension in the characterization of polymers.
RESUMO
Ultraviolet photodissociation (UVPD) has been shown to produce extensive structurally informative data for a variety of chemically diverse compounds. Herein, we demonstrate the performance of the 193 nm UVPD fragmentation technique on structural/moiety characterization of 14 singly charged agrochemicals. Two-dimensional mass spectrometry (2DMS) using infrared multiphoton dissociation (IRMPD) and electron-induced dissociation (EID) have previously been applied to a select range of singly charged pesticides. The ≥80% moiety coverage achieved for the majority of the species by the UVPD and 2D-UVPD methods was on par with and, in some cases, superior to the data obtained by other fragmentation techniques in previous studies, demonstrating that UVPD is viable for these types of species. A three-dimensional (3D) peak picking method was implemented to extract the data from the 2DMS spectrum, overcoming the limitations of the line extraction method used in previous studies, successfully separating precursor specific fragments with milli-Dalton accuracy. Whole spectrum internal calibration combined with 3D peak picking obtained sub-part-per-million (ppm) to part-per-billion (ppb) mass accuracies across the entire 2DMS spectrum.
Assuntos
Agroquímicos , Elétrons , Espectrometria de Massas , Raios UltravioletaRESUMO
Two-dimensional mass spectrometry (2DMS) is a new, and theoretically ideal, data-independent analysis tool, which allows the characterization of a complex mixture and was used in the bottom-up analysis of IgG1 for the identification of post-translational modifications. The new peak picking algorithm allows the distinction between chimeric peaks in proteomics. In this application, the processing of 2DMS data correlates fragments to their corresponding precursors, with fragments from precursors which are <0.1 m/z at m/z 840 easily resolved, without the need for quadrupole or chromatographic separation.
Assuntos
Imunoglobulina G/análise , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Humanos , Imunoglobulina G/química , Processamento de Proteína Pós-TraducionalRESUMO
The structure and sequence elucidation of complex homo- and copolymers is key for further understanding polymers, polymer synthesis, and polymer interactions in biological processes. In this contribution, poly(dimethylacrylamide) homo- and dimethylacrylamide/4-acryloylmorpholine block copolymers were synthesized and analyzed by electron capture dissociation (ECD) and Fourier transform ion cyclotron resonance (FT-ICR) tandem mass spectrometry. Double-resonance experiments were carried out, providing a better understanding of the fragmentation process. A novel radical dissociation process is presented, and electron capture caused a specific cleavage at the terminal butyl-trithiocarbonate group, which initiated a free radical dissociation process.
RESUMO
Analysis of agrochemicals in an environmental matrix is challenging because these samples contain multiple agrochemicals, their metabolites, degradation products, and endogenous compounds. The analysis of such complex samples is achieved using chromatographic separation techniques coupled to mass spectrometry. Herein, we demonstrate a two-dimensional mass spectrometry (2DMS) technique on a 12 T Fourier transform ion cyclotron resonance mass spectrometer that can analyze a mixture of agrochemicals without using chromatography or quadrupole isolation in a single experiment. The resulting 2DMS contour plot contains abundant tandem MS information for each component in the sample and correlates product ions to their corresponding precursor ions. Two different fragmentation methods are employed, infrared multiphoton dissociation (IRMPD) and electron-induced dissociation (EID), with 2DMS to analyze the mixture of singly charged agrochemicals. The product ions of one of the agrochemicals, pirimiphos-methyl, present in the sample was used to internally calibrate the entire 2DMS spectrum, achieving sub part per million (ppm) to part per billion (ppb) mass accuracies for all species analyzed. The work described in this study will show the advantages of the 2DMS approach, by grouping species with common fragments/core structure and mutual functional groups, using precursor lines and neutral loss lines. In addition, the rich spectral information obtained from IRMPD and EID 2DMS contour plots can accurately identify and characterize agrochemicals.
Assuntos
Agroquímicos/química , Espectrofotometria Infravermelho/métodos , Espectrometria de Massas em Tandem/métodos , Elétrons , HumanosRESUMO
Detection and characterization of phosphopeptides by infrared multiphoton dissociation two-dimensional mass spectrometry (IRMPD 2DMS) is shown to be particularly effective. A mixture of phosphopeptides was analyzed by 2DMS without any prior separation. 2DMS enables the data independent analysis of the mixture and the correlation of the fragments to their precursor ions. The extraction of neutral loss lines corresponding to the loss of phosphate under IRMPD fragmentation allows the selective identification of phosphopeptides. Resonance of the 10.6 µm infrared radiation with the vibrational modes of the phosphate functional group produced efficient absorption and high cleavage coverage of the phosphopeptides at much lower irradiation fluence than for nonphosphorylated peptides improving discrimination. Additionally, the localization of the phosphate group was determined.
Assuntos
Fosfopeptídeos/análise , Espectrometria de Massas , FosforilaçãoRESUMO
Investigating the structure of active ingredients, such as agrochemicals and their associated metabolites, is a crucial requisite in the discovery and development of these molecules. In this study, structural characterization by electron-induced dissociation (EID) was compared to collisionally activated dissociation (CAD) on a series of agrochemicals. EID fragmentation produced a greater variety of fragment ions and complementary ion pairs leading to more complete functional group characterization compared to CAD. The results obtained displayed many more cross-ring fragmentation of the pyrimidine ring compared to the pyridine ring. Compounds that consisted of one aromatic heterocyclic moiety (azoxystrobin, fluazifop acid, fluazifop-p-butyl, and pirimiphos-methyl) displayed cross-ring fragmentation while compounds with only aromatic hydrocarbon rings (fenpropidin and S-metolachlor) displayed no cross-ring fragmentation. The advantages of high-resolution accurate mass spectrometry (HRAM MS) are shown with the majority of assignments at ppb range error values and the ability to differentiate ions with the same nominal mass but different elemental composition. This highlights the potential for HRAM MS and EID to be used as a tool for structural characterization of small molecules with a wide variety of functional groups and structural motifs.
RESUMO
Two-dimensional mass spectrometry (2D MS) is a data-independent tandem mass spectrometry technique in which precursor and fragment ion species can be correlated without the need for prior ion isolation. The behavior of phase in 2D Fourier transform mass spectrometry is investigated with respect to the calculation of phase-corrected absorption-mode 2D mass spectra. 2D MS datasets have a phase that is defined differently in each dimension. In both dimensions, the phase behavior of precursor and fragment ions is found to be different. The dependence of the phase for both precursor and fragment ion signals on various parameters (e.g., modulation frequency, shape of the fragmentation zone) is discussed. Experimental data confirms the theoretical calculations of the phase in each dimension. Understanding the phase relationships in a 2D mass spectrum is beneficial to the development of possible algorithms for phase correction, which may improve both the signal-to-noise ratio and the resolving power of peaks in 2D mass spectra.
RESUMO
With increasing focus on the structural elucidation of polymers, advanced tandem mass spectrometry techniques will play a crucial role in the characterization of these compounds. In this contribution, synthesis and analysis of methyl-initiated and xanthate-terminated poly(2-ethyl-2-oxazoline) using Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) was achieved. Electron capture dissociation (ECD) produced full end group characterization as well as backbone fragmentation including complete sequence coverage of the polymer. A method of fragment ion characterization is also presented with the use of the high-resolution-modified Kendrick mass defect plots as a means of grouping fragments from the same fragmentation pathways together. This type of data processing is applicable to all tandem mass spectrometry techniques for polymer analysis but is made more effective with high mass accuracy methods. ECD FT-ICR MS demonstrates its promising role as a structural characterization technique for polyoxazoline species.
RESUMO
Two-dimensional mass spectrometry (2D MS) correlates precursor and fragment ions without ion isolation in a Fourier transform ion cyclotron resonance mass spectrometer (FTICR MS) for tandem mass spectrometry. Infrared activated electron capture dissociation (IR-ECD), using a hollow cathode configuration, generally yields more information for peptide sequencing in tandem mass spectrometry than ECD (electron capture dissociation) alone. The effects of the fragmentation zone on the 2D mass spectrum are investigated as well as the structural information that can be derived from it. The enhanced structural information gathered from the 2D mass spectrum is discussed in terms of how de novo peptide sequencing can be performed with increased confidence. 2D IR-ECD MS is shown to sequence peptides, to distinguish between leucine and isoleucine residues through the production of w ions as well as between C-terminal ( b/ c) and N-terminal ( y/ z) fragments through the use of higher harmonics, and to assign and locate peptide modifications.