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Top-Down Tandem Mass Spectrometry on RNase A and B Using a Qh/FT-ICR Hybrid Mass Spectrometer

Bourgoin-Voillard, Sandrine; Leymarie, Nancy; Costello, Catherine E.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Protein characterization using top-down approaches emerged with advances in high-resolution mass spectrometers and increased diversity of available activation modes: collision induced dissociation (CID), infrared multiphoton dissociation (IRMPD) electron capture dissociation (ECD) and electron transfer dissociation (ETD). Nevertheless, top-down approaches are still rarely used for glycoproteins. Hence, this work summarized the capacity of top-down approaches to improve sequence coverage and glycosylation site assignment on the glycoprotein Ribonuclease B (RNase B). The glycan effect on the protein fragmentation pattern was also investigated by comparing the fragmentation patterns of RNase B and its non-glycosylated analog RNase A. The experiments were performed on a Bruker 12-T-Qh/FTICR SolariX mass spectrometer using vibrational (CID/IRMPD) and radical activation (ECD/ETD) with/without pre- or post-activation (IRMPD or CID, respectively).

Unusual fragmentation of β-linked peptides by ExD tandem Mass Spectrometry

Sargaeva, Nadezda P.; Lin, Cheng; O’Connor, Peter B.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Ion-electron reaction based fragmentation methods (ExD) in tandem mass spectrometry (MS), such as Electron Capture Dissociation (ECD) and Electron Transfer Dissociation (ETD) represent a powerful tool for biological analysis ExD methods have been used to differentiate the presence of the isoaspartate (isoAsp) from the aspartate (Asp) in peptides and proteins. IsoAsp is a β3-type amino acid that has an additional methylene group in the backbone, forming a Cα-Cβ bond within the polypeptide chain. Cleavage of this bond provides specific fragments that allow differentiation of the isomers. The presence of a Cα-Cβ bond within the backbone is unique to β-amino acids, suggesting a similar application of ExD toward the analysis of peptides containing other β-type amino acids. In the current study, ECD and ETD analysis of several β-amino acid containing peptides was performed. It was found that N-Cβ and Cα-Cβ bond cleavages were rare, providing few c and z• type fragments, which was attributed to the instability of the Cβ radical. Instead, the electron capture resulted primarily in the formation of a• and y fragments, representing an alternative fragmentation pathway, likely initiated by the electron capture at a backbone amide nitrogen protonation site within the beta amino acid residues.

Partial de novo sequencing and unusual CID fragmentation of a 7 kDa, disulfide-bridged toxin

Medzihradszky, Katalin F.; Bohlen, Christopher J.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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A 7 kDa toxin isolated from the venom of the Texas coral snake (Micrurus tener tener) was subjected to collision-induced dissociation (CID) and electron-transfer dissociation (ETD) analyses both before and after reduction at low pH. Manual and automated approaches to de novo sequencing are compared in detail. Manual de novo sequencing utilizing the combination of high accuracy CID and ETD data and an acid-related cleavage yielded the N-terminal half of the sequence from the reduced species. The intact polypeptide, containing 3 disulfide bridges produced a series of unusual fragments in ion trap CID experiments: abundant internal amino acid losses were detected, and also one of the disulfide-linkage positions could be determined from fragments formed by the cleavage of two bonds. In addition, internal and c-type fragments were also observed.

Insulin degludec in combination with bolus insulin aspart is safe and effective in children and adolescents with type 1 diabetes

Thalange, Nandu; Deeb, Larry; Iotova, Violeta; Kawamura, Tomoyuki; Klingensmith, Georgeanna; Philotheou, Areti; Silverstein, Janet; Tumini, Stefano; Ocampo Francisco, Ann-Marie; Kinduryte, Ona; Danne, Thomas
Fonte: John Wiley & Sons A/S Publicador: John Wiley & Sons A/S
Tipo: Artigo de Revista Científica
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Insulin degludec (IDeg) once-daily was compared with insulin detemir (IDet) once- or twice-daily, with prandial insulin aspart in a treat-to-target, randomized controlled trial in children 1–17 yr with type 1 diabetes, for 26 wk (n = 350), followed by a 26-wk extension (n = 280). Participants were randomized to receive either IDeg once daily at the same time each day or IDet given once or twice daily according to local labeling. Aspart was titrated according to a sliding scale or in accordance with an insulin:carbohydrate ratio and a plasma glucose correction factor. Randomization was age-stratified: 85 subjects 1–5 yr. (IDeg: 43), 138 6–11 yr (IDeg: 70) and 127 12–17 yr (IDeg: 61) were included. Baseline characteristics were generally similar between groups overall and within each stratification. Non-inferiority of IDeg vs. IDet was confirmed for HbA1c at 26 wk; estimated treatment difference (ETD) 0.15% [−0.03; 0.32]95%CI. At 52 wk, HbA1c was 7.9% (IDeg) vs. 7.8% (IDet), NS; change in mean FPG was −1.29 mmol/L (IDeg) vs. +1.10 mmol/L (IDet) (ETD −1.62 mmol/L [−2.84; −0.41]95%CI, p = 0.0090) and mean basal insulin dose was 0.38 U/kg (IDeg) vs. 0.55 U/kg (IDet). The majority of IDet treated patients (64%) required twice-daily administration to achieve glycemic targets. Hypoglycemia rates did not differ significantly between IDeg and IDet...

Decoding Split and Pool Combinatorial Libraries with Electron Transfer Dissociation Tandem Mass Spectrometry

Sarkar, Mohosin; Pascal, Bruce D.; Steckler, Caitlin; Aquino, Claudio; Micalizio, Glenn C.; Kodadek, Thomas; Chalmers, Michael J.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Screening of bead-based split and pool combinatorial chemistry libraries is a powerful approach to aid the discovery of new chemical compounds able to interact with, and modulate the activities of, protein targets of interest. Split and pool synthesis provides for large and well diversified chemical libraries, in this case comprised of oligomers generated from a well-defined starting set. At the end of the synthesis, each bead in the library displays many copies of a unique oligomer sequence. Because the sequence of the oligomer is not known at the time of screening, methods for decoding of the sequence of each screening “hit” are essential. Here we describe an electron transfer dissociation (ETD) based tandem mass spectrometry approach for the decoding of mass-encoded split and pool libraries. We demonstrate that the newly described “chiral oligomers of pentenoic amides (COPAs)” yield non-sequence-specific product ions upon collisional activated dissociation; however, complete sequence information can be obtained with ETD. To aid in the decoding of libraries from MS and MS/MS data, we have incorporated 79Br/81Br isotope “tags” to differentiate N- and C-terminal product ions. In addition, we have created “Hit-Find,” a software program that allows users to generate libraries in silico. The user can then search all possible members of the chemical library for those that fall within a user-defined mass error.

Desenvolvimento de dispositivos orgânicos eletrocrômicos de transmissão

Guedes, André Felipe da Silva
Fonte: Pontifícia Universidade Católica do Rio Grande do Sul; Porto Alegre Publicador: Pontifícia Universidade Católica do Rio Grande do Sul; Porto Alegre
Tipo: Tese de Doutorado
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A síntese e a aplicação de novos materiais orgânicos, nanoestruturados, para o desenvolvimento de tecnologia em dispositivos orgânicos tem despertado grande interesse na comunidade científica. Atualmente encontram-se no mercado os primeiros produtos eletrônicos poliméricos (materiais orgânicos semicondutores), entre esses há os dispositivos eletrocrômicos, os quais são chamados de janelas inteligentes. Os principais aspectos funcionais de dispositivos eletrocrômicos para aplicação na indústria arquitetônica e automotiva são o controle da passagem de luz e de temperatura para o conforto térmico e visual. Podem ser flexíveis e muito finos, além de não conter metais pesados. Os dispositivos orgânicos são formados por camadas de materiais orgânicos depositados em diversas arquiteturas. Neste trabalho, a partir dos materiais orgânicos eletrodepositados, no caso Polianilina, PANI, que apresenta estabilidade nos parâmetros ópticos e elétricos, desenvolveram-se protótipos de dispositivos orgânicos eletrocrômicos. As técnicas utilizadas na caracterização desses materiais foram: espectroscopia de absorção ultravioleta-visível (UV-Vis), microscopia óptica, microscopia eletrônica de varredura (MEV), medida de espessura e medidas elétricas. O objetivo deste trabalho foi o desenvolvimento de dispositivos orgânicos eletrocrômicos de transmissão (DETs) que apresentem o Poli(3...

UF Recommended Data and File Formats for Electronic Theses and Dissertations (2012 and 2007 ); University of Florida Recommended Data and File Formats for Electronic Theses and Dissertations (2012 and 2007 )

Varied
Fonte: George A. Smathers Libraries, University of Florida; George A. Smathers Libraries, University of Florida ( Gainesville, FL ) Publicador: George A. Smathers Libraries, University of Florida; George A. Smathers Libraries, University of Florida ( Gainesville, FL )
Tipo: mixed material Formato: Documentation
Publicado em //2012 Português
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Additional related materials are available from the ETD Committee website: http://www.library.ufl.edu/committees/etd/

Análise de proteoma no carcinoma de células escamosas de laringe; Proteome analysis in laryngeal squamous cell carcinoma

Marília Bueno Santiago
Fonte: Biblioteca Digital da Unicamp Publicador: Biblioteca Digital da Unicamp
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 30/07/2015 Português
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O câncer de laringe representa cerca de 25% dos tumores malignos que acometem essa área e 2% de todas as doenças malignas. Ocorre em sua grande maioria em homens e é um dos mais comuns entre os tumores que atingem a região da cabeça e pescoço. Pode ocorrer em uma das três porções em que se divide o órgão: supraglote, glote e subglote, sendo que aproximadamente 2/3 dos tumores surgem na corda vocal verdadeira, localizada na glote, e 1/3 acomete a supraglote que se localiza acima das cordas vocais. O tipo histológico mais comum e que atinge mais de 90% dos pacientes, é o carcinoma de células escamosas (CEC) e é classificado de acordo com o grau de diferenciação celular. Estudos de expressão gênica têm sido realizados em vários tumores a fim de possibilitar o envolvimento de algumas proteínas na evolução tumoral, bem como determinar marcadores de gravidade. O objetivo deste trabalho foi estudar o contexto proteômico em células tumorais, displásicas e normais de pacientes com de CEC de laringe. Para isso após a coleta do material, foi realizada a confirmação histológica através de lâminas coradas com Hematoxilina-Eosina. A partir daí as células foram selecionadas e capturadas no Microdissecador a Laser da Zeiss. Em seguida foi realizada a análise proteômica...

Hunt, D.F.
Fonte: Association of Biomolecular Resource Facilities Publicador: Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em /10/2011 Português
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Electron transfer dissociation mass spectrometry is a break-though technology for sequencing post-translationally modified peptides. In this technique, radical anions of polyaromatic hydrocarbons (fluoranthene and azulene) are employed to transfer electrons to the carbonyl group in the polyamide backbone of multiply charged peptides generated by electrospray ionization. Capture of an electron into the peptide backbone reduces the positive charge on the ion by one, and forms a carbonyl radical anion that then abstracts a proton from a nearby protonated amino group. The resulting carbonyl radical triggers cleavage of the adjacent nitrogen-carbon bond to produce fragments of type c' and z·. The purpose of the workshop is to provide instruction on how to manually interpret peptide ETD mass spectra. Following a lengthy tutorial about ion structures, fragmentation pathways, predictable changes in fragment ion isotope patterns, etc., we will outline a general approach for the manual interpretation of peptide ETD spectra, solve the sequence of several post-translationally modified peptides, assign homework spectra, and reconvene on the second day of the workshop to go over the homework problems. A packet of lecture notes and handouts will be provided. Attendees should bring an inexpensive calculator...

Cation permeation through connexin 43 hemichannels is cooperative, competitive and saturable with parameters depending on the permeant species

Orellana, Juan A.; Díaz, Emilio; Schalper, Kurt A.; Vargas, Aníbal A.; Bennett, Michael V. L.; Sáez, Juan C.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Kinetics of permeation through connexin 43-EGFP hemichannels (Cx43-EGFP HCs) were evaluated in divalent cation-free solutions, which enhance HC open probability and thus, allow measurements during initial velocity. Three cations that become fluorescent upon binding to intracellular nucleic acids [ethidium (Etd), propidium (Prd) and 4',6-diamidino-2-phenylindole (DAPI)] and Cx43-EGFP or Cx43 wild type HeLa cell transfectants (Cx43-EGFP- and Cx43-WT-HeLa cells, respectively) were used. Levels of Cx43-EGFP at the cell periphery and rate of dye uptake were directly related. The rate of uptake of each dye reached saturation consistent with a facilitated transport mechanism. Before saturation, the relation between rate of uptake and concentration of each dye was sigmoidal with Hill coefficients >1, indicating positive cooperativity of transport at low concentrations. The maximal rate of Etd uptake was not affected by the presence of DAPI and vice versa, but under each condition the apparent affinity constant of the main permeant molecule increased significantly consistent with competitive inhibition or competition for binding sites within the channel. Moreover, Cx43-EGFP and Cx43-WT HCs had similar permeability properties, indicating that EGFP bound to the C-terminal of Cx43 does not significantly alter the permeability of Cx43 HCs to positively charged molecules.

Wet and dry deposition of nitrogen c ompounds in the southeast Pacific coast: Montemar, central Chile

Fuenzalida, Humberto A.; Prado Fiedler, Roberto
Fonte: Universidade do Chile Publicador: Universidade do Chile
Tipo: Artículo de revista
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A tmosphericco ncentratioinn them arineb oundarlya yera ndd irectd epositiomn easurements of totaln itratea nda mmoniumfo r an easternS outhP acificc oastasl iten earV alparaisoC, hile,w ere carriedo utd uringt hep eriodJ anuary1 992t hroughM arch 1993.C oncentrationins a ir for bothn itrogen formse xhibita seasonapla tternw ith low valuesin summera nda pronounceidn creasein winter, havinga meana nnuacl oncentratioonf 32.6 nmolm ' 3f or totaln itratea ndo f 44.8 nmolm ' 3f ort otal ammoniumW. et depositioins characterizebdy annuapl recipitatiown eightedc oncentrationosf 6 .0 and 8.1 gmold m' • forn itratea nda mmoniumr,e spectivelTyh. e mainf actorr egulatingw etd epositiofno r bothn itratea nda mmoniumis rainfalla mounts, ot hatc hangeisn precipitatioanm ounat loneo riginated by climatologicfaalc torsc anp roducec hangeisn theirr elatived epositiolne velsE. xistingc limatological patternd etermineasn overaldl ominancoef dryo verw etd epositionw,i tht otalso f 12.9a nd7 .34m mol 2 m' pery ear,t her educedfo rmso f nitrogenb eingg enerallym orea bundant.

Cohomological aspects of non-K\"ahler manifolds

Angella, Daniele
Fonte: Universidade Cornell Publicador: Universidade Cornell
Tipo: Artigo de Revista Científica
Publicado em 03/02/2013 Português
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In this thesis, we study cohomological properties of non-K\"ahler manifolds. In particular, we are concerned in investigating the cohomology of compact (almost-)complex manifolds, and of manifolds endowed with special structures, e.g., symplectic structures, D-complex structures in the sense of F. R. Harvey and H. B. Lawson, exhaustion functions satisfying positivity conditions.; Comment: Ph.D. thesis, Universit\`a di Pisa, advisor prof. Adriano Tomassini; http://etd.adm.unipi.it/theses/available/etd-01072013-231626/

Mixed-Symmetry Shell-Model Calculations in Nuclear Physics

Gueorguiev, V. G.
Fonte: Universidade Cornell Publicador: Universidade Cornell
Tipo: Artigo de Revista Científica
Publicado em 16/02/2010 Português
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We consider a novel approach to the nuclear shell model. The one-dimensional harmonic oscillator in a box is used to introduce the concept of an oblique-basis shell-model theory. By implementing the Lanczos method for diagonalization of large matrices, and the Cholesky algorithm for solving generalized eigenvalue problems, the method is applied to nuclei. The mixed-symmetry basis combines traditional spherical shell-model states with SU(3) collective configurations. We test the validity of this mixed-symmetry scheme on 24Mg and 44Ti. Results for 24Mg, obtained using the Wilthental USD intersection in a space that spans less than 10% of the full-space, reproduce the binding energy within 2% as well as an accurate reproduction of the low-energy spectrum and the structure of the states - 90% overlap with the exact eigenstates. In contrast, for an m-scheme calculation, one needs about 60% of the full space to obtain compatible results. Calculations for 44Ti support the mixed-mode scheme although the pure SU(3) calculations with few irreps are not as good as the standard m-scheme calculations. The strong breaking of the SU(3) symmetry results in relatively small enhancements within the combined basis. However, an oblique-basis calculation in 50% of the full pf-shell space is as good as a usual m-scheme calculation in 80% of the space. Results for the lower pf-shell nuclei 44-48Ti and 48Cr...

Common Powering Mechanism of Intermediate Luminosity Optical Transients and Luminous Blue Variables

Kashi, Amit; Soker, Noam
Fonte: Universidade Cornell Publicador: Universidade Cornell
Tipo: Artigo de Revista Científica
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We study recent Intermediate Luminosity Optical Transients (ILOTs) and major eruptions of Luminous Blue Variables (LBVs), and strengthen claims for a similar mechanism powering both. This process is a short duration release of gravitational energy in a binary system. In some ILOTs a merger occurs and one of the stars does not survive the transient event, e.g., V838 Mon and V1309 Sco. In some transient events a rapid and short mass transfer process takes place and the two stars survive the transient event, e.g., the Great Eruption of Eta Carinae. We study new ILOTs and reanalyze known ones in light of new observations and models. We reach our conclusion by analyzing these ILOTs using the Energy-Time Diagram (ETD) where we plot the total energy of the eruption against its eruption timescale. ILOTs and major LBV eruptions occupy the Optical Transient Stripe (OTS) in the ETD. The upper boundary of the stripe is explained by our proposed model where a main sequence (or a slightly off-main sequence) star accretes at a very high rate (<~ 1 M_\odot yr^{-1}) from a companion. We identify one LBV, NGC 3432 OT, with two eruptions; one with weak total energy and the other with large total energy. It bridges the regions of the ILOTs and LBVs in the OTS. We further study the ILOT M85 OT2006 and show that it cannot be a nova...

Nonlinear Processes in Coronal Heating and Slow Solar Wind Acceleration

Rappazzo, A. F.
Fonte: Universidade Cornell Publicador: Universidade Cornell
Tipo: Artigo de Revista Científica
Publicado em 11/01/2010 Português
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This work consists of two parts: the first devoted to the study of the heating of the magnetically confined Solar Corona, and the second to the acceleration of the Slow Solar Wind. Direct 3D reduced MHD simulations are presented. They model the heating of coronal loops in the solar atmosphere via the tangling of coronal field lines by photospheric footpoints motions within the framework of the "Parker scenario". We have derived scalings of physical quantities with loop length, and the ratio of photospheric to coronal Alfven velocities. The development of a turbulent dynamics makes the dissipation rate independent of the Reynolds number. The dynamics in physical space are desribed by weak turbulence, which develops when an MHD system is embedded in a strong axial magnetic field. The slow wind originates in and around the coronal streamer belt. The LASCO instrument onboard the SOHO spacecraft has observed plasma density enhancements forming beyond the cusp of a helmet streamer. Previous theoretical models for the formation and initial motion of these density enhancements are improved. The average expansion suffered by a parcel of plasma propagating outward, and the diamagnetic force due to the overall magnetic field radial gradients are now included. It is found that the magnetized wake configuration is resistively unstable...

Extended topological defects as sources and outlets of dislocations in spherical hexagonal crystals

Roshal, D. S.; Petrov, K. Yu.; Myasnikova, A. E.; Rochal, S. B.
Fonte: Universidade Cornell Publicador: Universidade Cornell
Tipo: Artigo de Revista Científica
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Extended topological defects (ETDs) arising in spherical hexagonal crystals due to their curvature are considered. These prevalent defects carry a unit total topological charge and are surrounded by scalene pentagonal boundaries. Topological peculiarities of reactions between ETDs and dislocations are considered. Similarly to boundaries of the usual planar crystalline order the ETDs emit and absorb the dislocations without preservation of their dislocational charge. Dislocations located inside the ETD area lose it and the enforced ETD decay can proceed in different ways without conservation of the total Burgers vector of the dislocations emitted.; Comment: 9 pages, 5 figures. arXiv admin note: substantial text overlap with arXiv:1309.7906

The long-term spectroscopic misadventures of AG Dra with a nod toward V407 Cyg: Degenerates behaving badly

Shore, S. N.; Genovali, K.; Wahlgren, G. M.
Fonte: Universidade Cornell Publicador: Universidade Cornell
Tipo: Artigo de Revista Científica
Publicado em 25/09/2011 Português
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We present some results of an ongoing study of the long-term spectroscopic variations of AG Dra, a prototypical eruptive symbiotic system. We discuss the effects of the environment and orbital modulation in this system and some of the physical processes revealed by a comparison with the nova outburst of the symbiotic-like recurrent nova V407 Cyg 2010.; Comment: 11 pages, invited review to appear in the proceedings of the 2011 Asiago Workshop on Symbiotic Stars, A. Siviero and U. Munari eds., Baltic Astronomy special issue. See also http://etd.adm.unipi.it/theses/available/etd-05052010-002805/

Star formation rate in the solar neighborhood

Cignoni, Michele
Fonte: Universidade Cornell Publicador: Universidade Cornell
Tipo: Artigo de Revista Científica
Publicado em 07/08/2006 Português
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This thesis develops a method for obtaining the star formation histories of a mixed, resolved population through the use of color-magnitude diagrams (CMDs). The method is applied to the derivation of the local star formation rate, modeling observations of the Hipparcos satellite wigth synthetic CMDs computed for different star formation histories with an updated stellar evolution library. Parallax and photometric uncertainties are included explicitly and corrected using the Bayesian Richardson-Lucy algorithm. We find that the solar neighborhood star formation rate has a characteristic timescale for variation of about 6 Gyr, with a maximum activity close to 3 Gyr ago. This suggests a global, rather than local, star forming event. The summary and conclusions are included here, the full thesis is available at the URL listed above.; Comment: PhD thesis/Physics 2006, University of Pisa, 167 pages, complete pdf version is available at: http://etd.adm.unipi.it/theses/available/etd-04032006-122227/

Designer Reagents for Mass Spectrometry-Based Proteomics: Clickable Cross-Linkers for Elucidation of Protein Structures and Interactions

Sohn, Chang Ho; Agnew, Heather D.; Lee, J. Eugene; Sweredoski, Michael J.; Graham, Robert L. J.; Smith, Geoffrey T.; Hess, Sonja; Czerwieniec, Gregg; Loo, Joseph A.; Heath, James R.; Deshaies, Raymond J.; Beauchamp, J. L.
Fonte: American Chemical Society Publicador: American Chemical Society
Tipo: Article; PeerReviewed Formato: application/pdf; application/pdf
Publicado em 20/03/2012 Português
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We present novel homobifunctional amine-reactive clickable cross-linkers (CXLs) for investigation of three-dimensional protein structures and protein–protein interactions (PPIs). CXLs afford consolidated advantages not previously available in a simple cross-linker, including (1) their small size and cationic nature at physiological pH, resulting in good water solubility and cell-permeability, (2) an alkyne group for bio-orthogonal conjugation to affinity tags via the click reaction for enrichment of cross-linked peptides, (3) a nucleophilic displacement reaction involving the 1,2,3-triazole ring formed in the click reaction, yielding a lock-mass reporter ion for only clicked peptides, and (4) higher charge states of cross-linked peptides in the gas-phase for augmented electron transfer dissociation (ETD) yields. Ubiquitin, a lysine-abundant protein, is used as a model system to demonstrate structural studies using CXLs. To validate the sensitivity of our approach, biotin-azide labeling and subsequent enrichment of cross-linked peptides are performed for cross-linked ubiquitin digests mixed with yeast cell lysates. Cross-linked peptides are detected and identified by collision induced dissociation (CID) and ETD with linear quadrupole ion trap (LTQ)-Fourier transform ion cyclotron resonance (FTICR) and LTQ-Orbitrap mass spectrometers. The application of CXLs to more complex systems (e.g....

High resolution data-independent acquisition with electron transfer dissociation mass spectrometry: Multiplexed analysis of post-translationally modified proteins

Sweredoski, Michael J.; Second, Tonya Pekar; Broeker, Jenny; Moradian, Annie; Hess, Sonja
Fonte: Elsevier Publicador: Elsevier
Tipo: Article; PeerReviewed Formato: application/pdf
Publicado em 20/07/2015 Português
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Data-dependent acquisition (DDA) mode is the most commonly used method in bottom-up proteomics. Recently, data-independent acquisition (DIA) modes have become popular alternatives because of their unbiased analysis, leading in general to more comprehensive, global qualitative profiling of proteome systems and also higher quantitative reproducibility in such profiling. Most of the previously established DIA methods are based on collision-induced dissociation (CID). However, when it comes to the analysis of labile post-translational modifications (PTMs), electron capture/transfer dissociation (ECD/ETD) may be better suited. In addition to the bottom-up approach, the middle-down approach, which analyzes peptides in the range of 3,000–10,000 Da has emerged as an attractive alternative, including the analysis of highly modified and highly variable protein variants that exist in key system functions, such as histone signaling cascades. Here, we establish that a data-independent (DIA) middle-down ETD approach is a superior strategy in the differential characterization of PTM changes in histone H2B. We suggest that this strategy can further be used for other approaches where dynamic PTM characterization or changes due to different conditions are fundamental to accurate understanding of biological systems and function.