endstream endobj 869 0 obj <>/Size 832/Type/XRef>>stream Sunner, J.; Magnera, T.F. The solution was centrifuged again. Applications of collision dynamics in quadrupole mass spectrometry. Breuker, K.; McLafferty, F.W. The mass spectrum of this antibody-peptide mixture showed in the high mass range three narrowly spaced multiply charged ion triplets at each charge state (, In solution, the bi-molecular association of the heme group to apo-myoglobin is thought to follow a bimodal process. A few microliters of complex-containing solution are loaded into a nano-electrospray capillary and all solubilized components including the protein-ligand complex are simultaneously transferred into the gas phase by electrospray. ; visualization: B.D.D. 0000005929 00000 n ; Opuni, K.F.M. (a) “neutral and resting” complex; (b) unitless number. Electrospray Mass Spectrometry of Biomacromolecular Complexes with Noncovalent Interactions—New Analytical Perspectives for Supramolecular Chemistry and Molecular Recognition Processes. Finally, 100 µL of the 0.5 µg/µL myoglobin solution were further diluted to 200 µL with 200 mM ammonium acetate buffer, pH 7, to obtain a final concentration of 0.25 µg/µL. Then 200 mM ammonium acetate buffer, pH 7, were added to reach a volume of 500 µL. 1 mg/mL was first prepared by dissolving the lyophilized powder (0.26 mg) of RNAse S (Lot # 52H7034, Sigma-Aldrich, Steinheim, Germany) in 0.26 mL of 200 mM ammonium acetate, pH 7.0. Protein concentration was determined to be 0.33 µg/µL using the Qubit, NanoESI capillaries for offline measurements were prepared in-house from borosilicate glass tubes of 1 mm outer and 0.5 mm inner diameters (BF 100-50-10, Sutter Instruments, Novato, CA, USA), using a P-1000 Flaming/BrownTM micropipette puller system (Sutter Instruments, Novato, CA, USA). Plotting the normalized intensities of the educts of the complex dissociation reaction in the gas phase as a function of collision cell voltage difference (, Since, in the gas phase of a Q-ToF mass spectrometer, the collision of multiply charged and accelerated complex ions takes place upon reaching elevated energies, the collision temperature (, According to the Eyring–Polanyi equation [, The entire procedure, which is termed “Intact Transition Epitope Mapping—Thermodynamic Weak-force Order (ITEM-TWO)”, starts with either generating the protein-ligand complex by mixing the two components in solution or by simply maintaining the natively obtained protein-ligand complex in an electrospray-compatible solution. Different theoretical models are useful to explain the fragmentation occurred in MS. All authors have read and agreed to the published version of the manuscript. We express our thanks to Michael Kreutzer for providing his expertise on bioinformatics and to Peter Lorenz and Hans-Juergen Thiesen for providing the FLAG peptide. x�b```b``�g`c`�db@ !V�(G���#���� �oz�8�5�_Y��@?�K{"Põ�g��=��N&`1@�G-�� "��\5��.�Z�M�S�N��mZ� 0000005422 00000 n Experimental and theoretical investigation of overall energy deposition in surface-induced unfolding of protein ions. ; Lyutvinskiy, Y.; Zubarev, R.A. Heme Binding in Gas-Phase Holo-Myoglobin Cations: Distal Becomes Proximal? 0000038816 00000 n Three-Dimensional Ion Mobility/TOFMS Analysis of Electrosprayed Biomolecules. Box LG53 Legon, Ghana. 0000032346 00000 n Thermodynamics and Mechanisms for Decomposition of Protonated Glycine and Its Protonated Dimer. The peptide was dissolved in 200 mM ammonium acetate buffer, pH 7, to obtain a peptide concentration of 1.31 µg/µL. It is an independent reimplementation of the SEQUEST algorithm, which identifies peptides by comparing the observed spectra to a catalog of theoretical spectra derived in silico from a database of known proteins. ; Valentine, S.J. and M.O.G. The concentration of the resulting solution was determined to be 0.5 µg/µL using a QubitTM 2.0 Fluorometer (Carlsbad, California, USA). Armentrout, P.B. 10, Nano-ESI-MS measurements were performed using a Q-TOF 2 instrument (Waters MS-Technologies, Manchester, UK). The ligand dissociation reaction encompasses in its most simple approximation a single transition state, as is indicated by a “one stage” chemical reaction where. Thus, the energy diagram of the complex dissociation reaction requires the introduction of, ESI-MS of large biomolecules and macromolecular non-covalent complexes in positive ion mode records series of multiply protonated ions which represent a Gaussian ion intensity distribution of individual charge states for a given molecular/supra-molecular species (complex) [, Mean charge states of each ion species, e.g., holo-protein ions (educts) and of apo-protein as well as of ligand ions (products) can be separately determined from the mass spectrum. ; Prell, J.S. 0000001699 00000 n 0000038602 00000 n 0000011267 00000 n The ESI mass spectrum of myoglobin (, Mean charge states of holo-myoglobin ions (m+), apo-myoglobin (n+) and heme (p+) as well as apex heights (holo-myoglobin ions (h, educts), heme ions (i, product), and apo-myoglobin ions (j, product) were extracted (, After recording mass spectra under increasing collision cell voltage difference settings (, The gas phase dissociation reaction of RNAse S was investigated in the same manner. Opuni, K.F.M. Rapid Determination of Activation Energies for Gas-Phase Protein Unfolding and Dissociation in a Q-IM-ToF Mass Spectrometer. and M.O.G. Please let us know what you think of our products and services. Although the QTAIM analysis gives a good correlation in order to explain the formation of p-quinoid resonance forms; however, the best correlation has been obtained using the NBO approximation as well as from the Wiberg indexes. ; McLuckey, S.A. Collision-Induced Dissociation (CID) of Peptides and Proteins. 0000012390 00000 n xref 0000008519 00000 n ; Heaton, A.L. ; Mroz, A.M.; Prell, J.S. Generation of the immune complex in solution and subsequent electrospraying of the entire mixture started data acquisition with the respective instrument settings as mentioned (for details see Methods section). and M.O.G. This solution was centrifuged for 30 min at 13,000 rpm and 23 °C. Wells, J.M. startxref Danquah, B.D. The WATERS Synapt G2S mass spectrometer has been bought through an EU grant [EFRE-UHROM 9] made available to MOG. 0000001897 00000 n With an energy input above a critical threshold, immune complex dissociation proceeds irreversibly but comparatively slowly under CID conditions. Tandem mass spectrometry (MS/MS) acts as a key technique for peptide identification. Yefremova, Y.; Melder, F.T.I. The pressure in the source region of the mass spectrometer was manually adjusted to 2.24 mbar using the speedy valve [, Nano-ESI-MS measurements were performed using a Synapt G2S instrument (Waters MS-Technologies, Manchester, UK). 0000013458 00000 n ; Cairo, C.W. Fractions of educts and products were derived from heights of ion signals of complex (educt) and its dissociated constituents (products) at all applied collision cell voltage differences (, The mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE [, ITEM-TWO is able (i) to determine epitopes and (ii) to investigate the epitopes´ binding strengths in the gas phase. We obtained a quantitative explanation about the methyl loss from methoxy coumarins. High-Pressure Mass Spectrometric Investigations of the Potential Energy Surfaces of Gas-Phase SN2 Reactions. ; Mauk, A.G.; Douglas, D.J. Some recent advances in concepts and technique. The PRIDE database and related tools and resources in 2019: Improving support for quantification data. Price, W.D. Quantitative Proteomics Analysis by Sequential Window Acquisition of All Theoretical Mass Spectra–Mass Spectrometry Reveals a Cross-Protection Mechanism for Monascus To Tolerate High-Concentration Ammonium Chloride. Normalization of ion intensities is achieved by summation of all apex values and setting the sum to 100% (equations and calculations are shown in the, A series of mass spectra in which the collision cell voltage difference is increased stepwise records ion signals with varying intensities of all ion species, i.e., educts and products, as they emerge from the collision induced complex dissociation reaction. The apparent dissociation constant, Electrospray mass spectrometric methods have gained broad acceptance for investigation of the constituents of supramolecular complexes and determination of binding surfaces, e.g., for identifying the locations of partial surfaces on antigens which are recognized by an antibody of interest [, Recently, and still uncommonly, the combination of fast and robust gas-phase epitope mapping methods [, In a typical Q-ToF instrument, dissociation of protein-ligand complex ions in the gas phase is induced by collisions of multiply charged complex ions with noble gas atoms in a collision chamber at a given collision energy. 0000006215 00000 n ; Al-Majdoub, M.; Yefremova, Y.; El-Kased, R.F. 0000009428 00000 n 0000002805 00000 n All mass spectra were acquired in positive-ion mode applying a mass window of, The Savitzky–Golay method was used for smoothing in five cycles with a window of 10 for the high mass range and in three cycles with a window of 5 for the low mass range, respectively. By continuing you agree to the use of cookies. Native mass spectrometry: A bridge between interactomics and structural biology. The mixture of antiFLAG antibody with seven peptides was investigated. Mixing of antigen or epitope peptide and antibody solutions is the only required in-solution handling step when the complex components are dissolved in electrospray-compatible solutions. Capillaries were gold-coated using a sputter coater BalTec SCD 0045 (Bal-Tech, Balzers, Liechtenstein) with the following conditions: current was set to 20 mA for 150 s, table distance was 5 cm, while vacuum was ca. 0000014313 00000 n 870 0 obj <>stream ; Rower, C.; Opuni, K.F.M. A complementary explanation about the fragmentation way of the methyl loss from … Role of Heme in the Unfolding and Assembly of Myoglobin. Stability of complementary and mismatched DNA duplexes: Comparison and contrast in gas versus solution phases. A resulting supernatant of approximately 800 µL was collected and the protein concentration was determined to be 0.78 µg/µL using a Qubit, A volume of 20 µL of 1 µg/µL of mouse monoclonal antiFLAG M2 antibody (product code F 1804, Sigma-Aldrich, Steinheim, Germany) was first re-buffered into 200 mM ammonium acetate buffer, pH 7, using a centrifugal filter (Amicon Ultra cutoff 50 K; Merck Millipore Ltd., Tullagreen, Carrigtwohill Co Cork, Ireland), as described [, The human cardiac Troponin I epitope peptide (ENREVGDWRKNIDAL; peptides&elephants, Hennigsdorf, Germany) was obtained as lyophilized powder. The authors thank the German Academic Exchange Service (DAAD) for providing a postdoctoral fellowship (Re-invitation Programme for Former Scholarship Holders) for KO (91548123) and a doctoral scholarship for BD (No. Schmidt, A.; Karas, M. The influence of electrostatic interactions on the detection of heme-globin complexes in ESI-MS. Ito, W.; Kurosawa, Y. A stock solution of ca. %PDF-1.4 %���� Ion mobility–mass spectrometry for structural proteomics. 0000006260 00000 n ; Kebarle, P. Ion–molecule equilibria measurements by high pressure mass spectrometry. On the basis of the wide spectrum eddy contact concept and the isotropic turbulence theory, a multi‐scale theoretical model for the prediction of liquid‐side mass transfer coefficient in gas–liquid system was developed. As a consequence, from all above considerations it appears well possible to semi-quantitatively compare apparent gas phase binding strengths between complexes and to relate these to in-solution dissociation constants of antibody-antigen complexes, after correcting the energy terms, i.e., by subtracting external energy contributions. Effects of substitutions of closely related amino acids at the contact surface in an antigen-antibody complex on thermodynamic parameters. 0000004139 00000 n Seo, J.; Hoffmann, W.; Warnke, S.; Bowers, M.T. We obtained the best correlation using the NBO approximation and the Wiberg indexes. Then, 100 µL were transferred onto a Microcon centrifuge filter with a 3 kDa cutoff (Millipore Corp., Bedford, MA, USA) together with further 200 µL of 200 mM ammonium acetate solution. Find support for a specific problem on the support section of our website.


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