25 research outputs found
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Intra-molecular cross-linking of acidic residues for protein structure studies.
Intra-molecular cross-linking has been suggested as a method of obtaining distance constraints that would be useful in developing structural models of proteins. Recent work published on intra-molecular cross-linking for protein structural studies has employed commercially available primary amine selective reagents that can cross-link lysine residues to other lysine residues or the amino terminus. Previous work using these cross-linkers has shown that for several proteins of known structure, the number of cross-links that can be obtained experimentally may be small compared to what would be expected from the known structure, due to the relative reactivity, distribution, and solvent accessibility of the lysines in the protein sequence. To overcome these limitations we have investigated the use of cross-linking reagents that can react with other reactive sidechains in proteins. We used 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) to activate the carboxylic acid containing residues, aspartic acid (D), glutamic acid (E), and the carboxy terminus (O), for cross-linking reactions. Once activated, the DEO sidechains can react to form 'zero-length' cross-links with nearby primary amine containing resides, lysines (K) and the amino terminus (X), via the formation of a new amide bond. We also show that the EDC-activated DEO sidechains can be cross-linked to each other using dihydrazides, two hydrazide moieties connected by an alkyl cross-linker ann of variable length. Using these reagents, we have found three new 'zero-length' cross-links in ubiquitin consistent with its known structure (M1-E16, M1-E18, and K63-E64). Using the dihydrazide cross-linkers, we have identified 2 new cross-links (D21-D32 and E24-D32) unambiguously. Using a library of dihydrazide cross-linkers with varying arm length, we have shown that there is a minimum arm length required for the DEO-DEO cross-links of 5.8 angstroms. These results show that additional structural information can be obtained by exploiting new cross-linker chemistry, increasing the probability that the protein target of choice will yield sufficient distance constraints to develop a structural model
Oxonium Ion-Guided Optimization of Ion Mobility-Assisted Glycoproteomics on the timsTOF Pro
Spatial separation of ions in the gas phase, providing information about their size as collisional cross-sections, can readily be achieved through ion mobility. The timsTOF Pro (Bruker Daltonics) series combines a trapped ion mobility device with a quadrupole, collision cell, and a time-of-flight analyzer to enable the analysis of ions at great speed. Here, we show that the timsTOF Pro is capable of physically separating N-glycopeptides from nonmodified peptides and producing high-quality fragmentation spectra, both beneficial for glycoproteomics analyses of complex samples. The glycan moieties enlarge the size of glycopeptides compared with nonmodified peptides, yielding a clear cluster in the mobilogram that, next to increased dynamic range from the physical separation of glycopeptides and nonmodified peptides, can be used to make an effective selection filter for directing the mass spectrometer to analytes of interest. We designed an approach where we (1) focused on a region of interest in the ion mobilogram and (2) applied stepped collision energies to obtain informative glycopeptide tandem mass spectra on the timsTOF Pro:glyco-polygon–stepped collision energy-parallel accumulation serial fragmentation. This method was applied to selected glycoproteins, human plasma– and neutrophil-derived glycopeptides. We show that the achieved physical separation in the region of interest allows for improved extraction of information from the samples, even at shorter liquid chromatography gradients of 15 min. We validated our approach on human neutrophil and plasma samples of known makeup, in which we captured the anticipated glycan heterogeneity (paucimannose, phosphomannose, high mannose, hybrid and complex glycans) from plasma and neutrophil samples at the expected abundances. As the method is compatible with off-the-shelve data acquisition routines and data analysis software, it can readily be applied by any laboratory with a timsTOF Pro and is reproducible as demonstrated by a comparison between two laboratories
Novel canonical and non-canonical viral antigens extend current targets for immunotherapy of HPV-driven cervical cancer
Current immunotherapeutic approaches for human papillomavirus (HPV)-driven cervical cancer target the viral oncogenes E6 and E7. We report viral canonical and alternative reading frame (ARF)-derived sequences presented on cervical tumor cells, including antigens encoded by the conserved viral gene E1. We confirm immunogenicity of the identified viral peptides in HPV-positive women, and women with cervical intraepithelial neoplasia. We observe consistent transcription of the E1, E6, and E7 genes in 10 primary cervical tumor resections from the four most common high-risk HPV subtypes (HPV16, 18, 31, and 45), suggesting the suitability of E1 as therapeutic target. We finally confirm HLA presentation of canonical peptides derived from E6 and E7, and ARF-derived viral peptides from a reverse-strand transcript spanning the HPV E1 and E2 genes in primary human cervical tumor tissue. Our results extend currently known viral immunotherapeutic targets in cervical cancer and highlight E1 as an important cervical cancer antigen
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Chemical crosslinking and mass spectrometry studies of the structure and dynamics of membrane proteins and receptors.
Membrane proteins make up a diverse and important subset of proteins for which structural information is limited. In this study, chemical cross-linking and mass spectrometry were used to explore the structure of the G-protein-coupled photoreceptor bovine rhodopsin in the dark-state conformation. All experiments were performed in rod outer segment membranes using amino acid 'handles' in the native protein sequence and thus minimizing perturbations to the native protein structure. Cysteine and lysine residues were covalently cross-linked using commercially available reagents with a range of linker arm lengths. Following chemical digestion of cross-linked protein, cross-linked peptides were identified by accurate mass measurement using liquid chromatography-fourier transform mass spectrometry and an automated data analysis pipeline. Assignments were confirmed and, if necessary, resolved, by tandem MS. The relative reactivity of lysine residues participating in cross-links was evaluated by labeling with NHS-esters. A distinct pattern of cross-link formation within the C-terminal domain, and between loop I and the C-terminal domain, emerged. Theoretical distances based on cross-linking were compared to inter-atomic distances determined from the energy-minimized X-ray crystal structure and Monte Carlo conformational search procedures. In general, the observed cross-links can be explained by re-positioning participating side-chains without significantly altering backbone structure. One exception, between C3 16 and K325, requires backbone motion to bring the reactive atoms into sufficient proximity for cross-linking. Evidence from other studies suggests that residues around K325 for a region of high backbone mobility. These findings show that cross-linking studies can provide insight into the structural dynamics of membrane proteins in their native environment
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The interfacial bioscience grand challenge.
This report is broken down into the following 3 sections: (1) Chemical Cross-linking and Mass Spectrometry Applied to Determination of Protein Structure and Dynamics; (2) Computational Modeling of Membrane Protein Structure and Dynamics; and (3) Studies of Toxin-Membrane Interactions using Single Molecule Biophysical Methods
Large-scale genome-wide analysis identifies genetic variants associated with cardiac structure and function
BACKGROUND: Understanding the genetic architecture of cardiac structure and function may help to prevent and treat heart disease. This investigation sought to identify common genetic variations associated with inter-individual variability in cardiac structure and function. METHODS: A GWAS meta-analysis of echocardiographic traits was performed, including 46,533 individuals from 30 studies (EchoGen consortium). The analysis included 16 traits of left ventricular (LV) structure, and systolic and diastolic function. RESULTS: The discovery analysis included 21 cohorts for structural and systolic function traits (n = 32,212) and 17 cohorts for diastolic function traits (n = 21,852). Replication was performed in 5 cohorts (n = 14,321) and 6 cohorts (n = 16,308), respectively. Besides 5 previously reported loci, the combined meta-analysis identified 10 additional genome-wide significant SNPs: rs12541595 near MTSS1 and rs10774625 in ATXN2 for LV end-diastolic internal dimension; rs806322 near KCNRG, rs4765663 in CACNA1C, rs6702619 near PALMD, rs7127129 in TMEM16A, rs11207426 near FGGY, rs17608766 in GOSR2, and rs17696696 in CFDP1 for aortic root diameter; and rs12440869 in IQCH for Doppler transmitral A-wave peak velocity. Findings were in part validated in other cohorts and in GWAS of related disease traits. The genetic loci showed associations with putative signaling pathways, and with gene expression in whole blood, monocytes, and myocardial tissue. CONCLUSION: The additional genetic loci identified in this large meta-analysis of cardiac structure and function provide insights into the underlying genetic architecture of cardiac structure and warrant follow-up in future functional studies. FUNDING: For detailed information per study, see Acknowledgments.This work was supported by a grant from the US National Heart, Lung, and Blood Institute (N01-HL-25195; R01HL 093328 to RSV), a MAIFOR grant from the University Medical Center Mainz, Germany (to PSW), the Center for Translational Vascular Biology (CTVB) of the Johannes Gutenberg-University of Mainz, and the Federal Ministry of Research and Education, Germany (BMBF 01EO1003 to PSW). This work was also supported by the research project Greifswald Approach to Individualized Medicine (GANI_MED). GANI_MED was funded by the Federal Ministry of Education and Research and the Ministry of Cultural Affairs of the Federal State of Mecklenburg, West Pomerania (contract 03IS2061A). We thank all study participants, and the colleagues and coworkers from all cohorts and sites who were involved in the generation of data or in the analysis. We especially thank Andrew Johnson (FHS) for generation of the gene annotation database used for analysis. We thank the German Center for Cardiovascular Research (DZHK e.V.) for supporting the analysis and publication of this project. RSV is a member of the Scientific Advisory Board of the DZHK. Data on CAD and MI were contributed by CARDIoGRAMplusC4D investigators. See Supplemental Acknowledgments for consortium details. PSW, JFF, AS, AT, TZ, RSV, and MD had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis
Photoelectron Spectroscopy of Reactive Intermediates
Photoelectron Spectroscopy (PES) has been used to investigate the structure and thermochemistry of a number of alkyl radicals and their corresponding carbonium ions. The radicals have been produced by flash vacuum pyrolysis of alkyl nitrites. The shape of the first band in the photoelectron spectrum of a free radical is related to the structural changes that take place in forming the carbonium ion from the radical. The ionization potentials obtained from the photoelectron spectra of the radicals are combined with gas phase ion thermochemistry data to obtain alkyl radical heats of formation. The thermochemical data thus obtained is used to discuss substituent and structural effects on the stability of radicals and carbonium ions. In many cases the thermolytic decomposition pathways of the alkyl radicals have been elucidated using PES. The application of the PES technique to the analysis of reactive intermediates present in heterogeneous thermolysis mixtures is also discussed.
Chapter 1 presents an introduction to the technique employed in these studies, and a review of the studies performed in this laboratory. Chapter 2 presents results on the thermochemistry and structure of the 1- and 2-adamantyl radicals. The tricyclic 1-adamantyl radical and carbonium ion are important as model bridgehead compounds, and the question of the amount of strain energy caused by the non-planarity of the radical and ion center has been of great interest. The first bands in the photoelectron spectra of the o-, m- and p-methylbenzyl radicals are presented in Chapter 3. The methyl substituent effects on the stabilities of the radicals and ions are discussed.
In Chapter 4 results on investigations of heterogeneous processes in Chemical Vapor Deposition (CVD) systems using the chlorosilanes as feed gases are discussed. SiCl₂ is found to be the major silicon containing reactive intermediate produced by surface reactions at 600 - 1100 °C in CVD systems using dichlorosilane and trichlorosilane as feed gases.
Chapter 5 presents the spectra of the 1- and 2-methylnaphthyl radicals. The relative stabilities of the radicals and carbonium ions are discussed based on proton affinities determined by Fourier transform mass spectrometric equilibrium studies, combined with ionization potentials obtained from photoelectron spectra of the radicals.</p
Energetics and structure of the 1- and 2-adamantyl radicals and their corresponding carbonium ions by photoelectron spectroscopy
The first photoelectron bands of the 1- and 2-adamantyl radicals, formed by flash vacuum photolysis of 1- and 2-adamantylmethyl nitrite, have been obtained. The adiabatic (IP_a) and vertical (IP_v) ionization potentials of the 1-adamantyl radical are 6.21 ± 0.03 and 6.36 ± 0.05 eV, respectively. IP_a and IP_v for the 2-adamantyl radical are 6.73 ± 0.03 and 6.99 ± 0.05 eV, respectively. The difference in hydride affinities between the 1-adamantyl and tert-butyl cations (Sharma, R. B.; Sen Sharma, D. K.; Hiraoka, K.; Kebarle, P. J. Am. Chem. Soc. 1985, 107, 3747) combined with the difference in IP_a between the tert-butyl and 1-adamantyl radicals (0.49 ± 0.06 eV) yield a value of 99 kcal/mol for the tertiary C-H bond energy in adamantane, 3.7 ± 1.2 kcal/mol greater than the tertiary C-H bond energy in isobutane (assumed to be 95 kcal/mol). The effects of the geometrical constraints imposed by the adamantyl cage on the homolytic and heterolytic C-H bond cleavage energies are discussed for the 1- and 2-adamantyl cases. The width of the Franck-Condon envelope obtained is related to the geometry changes that occur upon ionization. The surprisingly broad envelope observed for the planar 2-adamantyl radical indicates that the Franck-Condon envelope for the 1-admantyl radical should not be interpreted as exclusively due to changes at the bridgehead position. Thermal decomposition products of the 1- and 2-adamantyl radicals are observed, and the pathways for thermal decompositions of the radicals are discussed. To confirm expected trends in ionization potentials and band shapes of tertiary radicals, the first photoelectron band of the 2-methyl-2-butyl radical has been obtained. The IP_a of the 2-metyl-2-butyl radical is 6.65 ± 0.04 eV with IPV = 6.91 ± 0.05 eV
Energetics and structure of the 1- and 2-adamantyl radicals and their corresponding carbonium ions by photoelectron spectroscopy
The first photoelectron bands of the 1- and 2-adamantyl radicals, formed by flash vacuum photolysis of 1- and 2-adamantylmethyl nitrite, have been obtained. The adiabatic (IP_a) and vertical (IP_v) ionization potentials of the 1-adamantyl radical are 6.21 ± 0.03 and 6.36 ± 0.05 eV, respectively. IP_a and IP_v for the 2-adamantyl radical are 6.73 ± 0.03 and 6.99 ± 0.05 eV, respectively. The difference in hydride affinities between the 1-adamantyl and tert-butyl cations (Sharma, R. B.; Sen Sharma, D. K.; Hiraoka, K.; Kebarle, P. J. Am. Chem. Soc. 1985, 107, 3747) combined with the difference in IP_a between the tert-butyl and 1-adamantyl radicals (0.49 ± 0.06 eV) yield a value of 99 kcal/mol for the tertiary C-H bond energy in adamantane, 3.7 ± 1.2 kcal/mol greater than the tertiary C-H bond energy in isobutane (assumed to be 95 kcal/mol). The effects of the geometrical constraints imposed by the adamantyl cage on the homolytic and heterolytic C-H bond cleavage energies are discussed for the 1- and 2-adamantyl cases. The width of the Franck-Condon envelope obtained is related to the geometry changes that occur upon ionization. The surprisingly broad envelope observed for the planar 2-adamantyl radical indicates that the Franck-Condon envelope for the 1-admantyl radical should not be interpreted as exclusively due to changes at the bridgehead position. Thermal decomposition products of the 1- and 2-adamantyl radicals are observed, and the pathways for thermal decompositions of the radicals are discussed. To confirm expected trends in ionization potentials and band shapes of tertiary radicals, the first photoelectron band of the 2-methyl-2-butyl radical has been obtained. The IP_a of the 2-metyl-2-butyl radical is 6.65 ± 0.04 eV with IPV = 6.91 ± 0.05 eV