Exposure of Solvent-Inaccessible Regions in the Amyloidogenic Protein Human SOD1 Determined by Hydroxyl Radical Footprinting J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-10-16 Yuewei Sheng, Joseph Capri, Alan Waring, Joan Selverstone Valentine, Julian Whitelegge
Solvent-accessibility change plays a critical role in protein misfolding and aggregation, the culprit for several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Mass spectrometry-based hydroxyl radical (·OH) protein footprinting has evolved as a powerful and fast tool in elucidating protein solvent accessibility. In this work, we used fast photochemical oxidation of protein (FPOP) hydroxyl radical (·OH) footprinting to investigate solvent accessibility in human copper-zinc superoxide dismutase (SOD1), misfolded or aggregated forms of which underlie a portion of ALS cases. ·OH-mediated modifications to 56 residues were detected with locations largely as predicted based on X-ray crystallography data, while the interior of SOD1 β-barrel is hydrophobic and solvent-inaccessible and thus protected from modification. There were, however, two notable exceptions—two closely located residues inside the β-barrel, predicted to have minimal or no solvent accessibility, that were found modified by FPOP (Phe20 and Ile112). Molecular dynamics (MD) simulations were consistent with differential access of peroxide versus quencher to SOD1’s interior complicating surface accessibility considerations. Modification of these two residues could potentially be explained either by local motions of the β-barrel that increased peroxide/solvent accessibility to the interior or by oxidative events within the interior that might include long-distance radical transfer to buried sites. Overall, comparison of modification patterns for the metal-free apoprotein versus zinc-bound forms demonstrated that binding of zinc protected the electrostatic loop and organized the copper-binding site. Our study highlights SOD1 hydrophobic groups that may contribute to early events in aggregation and discusses caveats to surface accessibility conclusions.
Liquid Extraction Surface Analysis (LESA) Electron-Induced Dissociation and Collision-Induced Dissociation Mass Spectrometry of Small Molecule Drug Compounds J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-08-27 Andrea F. Lopez-Clavijo, Rian L. Griffiths, Richard J. A. Goodwin, Helen J. Cooper
Here, we present liquid extraction surface analysis (LESA) coupled with electron-induced dissociation (EID) mass spectrometry in a Fourier-transform ion cyclotron resonance mass spectrometer for the analysis of small organic pharmaceutical compounds directly from dosed tissue. First, the direct infusion electrospray ionisation EID and collision-induced dissociation (CID) behaviour of erlotinib, moxifloxacin, clozapine and olanzapine standards were compared. EID mass spectra were also compared with experimental or reference electron impact ionisation mass spectra. The results show that (with the exception of erlotinib) EID and CID result in complementary fragment ions. Subsequently, we performed LESA EID MS/MS and LESA CID MS/MS on singly charged ions of moxifloxacin and erlotinib extracted from a thin tissue section of rat kidney from a cassette-dosed animal. Both techniques provided structural information, with the majority of peaks observed for the drug standards also observed for the tissue-extracted species. Overall, these results demonstrate the feasibility of LESA EID MS/MS of drug compounds from dosed tissue and extend the number of molecular structures for which EID behaviour has been determined.
Coupling of a High-Resolution Ambient Pressure Drift Tube Ion Mobility Spectrometer to a Commercial Time-of-flight Mass Spectrometer J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-08-13 Maria Allers, Laila Timoumi, Ansgar T. Kirk, Florian Schlottmann, Stefan Zimmermann
Ion mobility spectrometry provides information about molecular structures of ions. Hence, high resolving power allows separation of isomers which is of major interest in several applications. In this work, we couple our high-resolution ion mobility spectrometer (IMS) with a resolving power of Rp = 100 to a time-of-flight mass spectrometer (TOF-MS). Besides, the benefit of an increased resolving power such an IMS-MS also helps analyzing and understanding the ionization processes in IMS. Usually, the coupling between IMS and TOF-MS is realized by synchronizing data acquisition of the IMS and MS resulting in two-dimensional data containing ion mobility and mass spectra. However, due to peak widths of less than 100 μs in our high-resolution IMS, this technique is not practicable due to significant peak broadening during the ion transfer into the MS and an insufficient data acquisition rate of the MS. Thus, a novel but simple interface between the IMS and MS has been designed which minimizes ion losses, allows recording of ion mobility at full IMS resolving power, and enables a shuttered transmission of ions into the MS. The interface is realized by replacing the Faraday plate used in IMS by a Faraday grid that is shielded by two additional aperture grids. For demonstration, positive product ions of benzene, toluene, and m-xylene in air are investigated. The IMS is equipped with a radioactive 3H source. Besides the well-known product ions M+ and M·NO+, a dimer ion is also observed for benzene and toluene, consisting of two molecules and three further hydrogen atoms.
Action and Ion Mobility Spectroscopy of a Shortened Retinal Derivative J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-07-30 Lihi Musbat, Shirrel Assis, Jonathan M. Dilger, Tarick J. El-Baba, Daniel R. Fuller, Jeppe Langeland Knudsen, Hjalte V. Kiefer, Amiram Hirshfeld, Noga Friedman, Lars H. Andersen, Mordechai Sheves, David E. Clemmer, Yoni Toker
The development of tandem ion mobility spectroscopy (IMS) known as IMS-IMS has led to extensive research into isomerizations of isolated molecules. Many recent works have focused on the retinal chromophore which is the optical switch used in animal vision. Here, we study a shortened derivative of the chromophore, which exhibits a rich IM spectrum allowing for a detailed analysis of its isomerization pathways, and show that the longer the chromophore is, the lower the barrier energies for isomerization are.
Operation and Performance of a Mass-Selective Cryogenic Linear Ion Trap J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-07-30 Larry F. Tesler, Adam P. Cismesia, Matthew R. Bell, Laura S. Bailey, Nicolas C. Polfer
We report on the performance of a cryogenic 2D linear ion trap (cryoLIT) that is shown to be mass-selective in the temperature range of 17–295 K. As the cryoLIT is cooled, the ejection voltages during the mass instability scan decrease, which results in an effective mass shift to lower m/z relative to room temperature. This is attributed to a decrease in trap radius caused by thermal contraction. Additionally, the cryoLIT generates reproducible mass spectra from day-to-day, and is capable of performing stored waveform inverse Fourier transform (SWIFT) mass isolation of fragile N2-tagged ions for the purpose of background-free infrared dissociation spectroscopy.
Evaluation of Digital Image Recognition Methods for Mass Spectrometry Imaging Data Analysis J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-10-15 Måns Ekelöf, Kenneth P. Garrard, Rika Judd, Elias P. Rosen, De-Yu Xie, Angela D. M. Kashuba, David C. Muddiman
Analyzing mass spectrometry imaging data can be laborious and time consuming, and as the size and complexity of datasets grow, so does the need for robust automated processing methods. We here present a method for comprehensive, semi-targeted discovery of molecular distributions of interest from mass spectrometry imaging data, using widely available image similarity scoring algorithms to rank images by spatial correlation. A fast and powerful batch search method using a MATLAB implementation of structural similarity (SSIM) index scoring with a pre-selected reference distribution is demonstrated for two sample imaging datasets, a plant metabolite study using Artemisia annua leaf, and a drug distribution study using maraviroc-dosed macaque tissue.
Conditions for Analysis of Native Protein Structures Using Uniform Field Drift Tube Ion Mobility Mass Spectrometry and Characterization of Stable Calibrants for TWIM-MS J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-10-15 Julian A. Harrison, Celine Kelso, Tara L. Pukala, Jennifer L. Beck
Determination of collisional cross sections (CCS) by travelling wave ion mobility mass spectrometry (TWIM-MS) requires calibration against standards for which the CCS has been measured previously by drift tube ion mobility mass spectrometry (DTIM-MS). The different extents of collisional activation in TWIM-MS and DTIM-MS can give rise to discrepancies in the CCS of calibrants across the two platforms. Furthermore, the conditions required to ionize and transmit large, folded proteins and assemblies may variably affect the structure of the calibrants and analytes. Stable hetero-oligomeric phospholipase A2 (PDx) and its subunits were characterized as calibrants for TWIM-MS. Conditions for acquisition of native-like TWIM (Synapt G1 HDMS) and DTIM (Agilent 6560 IM-Q-TOF) mass spectra were optimized to ensure the spectra exhibited similar charge state distributions. CCS measurements (DTIM-MS) for ubiquitin, cytochrome c, holo-myoglobin, serum albumin and glutamate dehydrogenase were in good agreement with other recent results determined using this and other DTIM-MS instruments. PDx and its β and γ subunits were stable across a wide range of cone and trap voltages in TWIM-MS and were stable in the presence of organic solvents. The CCS of PDx and its subunits were determined by DTIM-MS and were used as calibrants in determination of CCS of native-like cytochrome c, holo-myoglobin, carbonic anhydrase, serum albumin and haemoglobin in TWIM-MS. The CCS values were in good agreement with those measured by DTIM-MS where available. These experiments demonstrate conditions for analysis of native-like proteins using a commercially available DTIM-MS instrument, characterize robust calibrants for TWIM-MS, and present CCS values determined by DTIM-MS and TWIM-MS for native proteins to add to the current literature database.
Initial Benchmarking of the Liquid Sampling-Atmospheric Pressure Glow Discharge-Orbitrap System Against Traditional Atomic Mass Spectrometry Techniques for Nuclear Applications J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-10-05 Edward D. Hoegg, Benjamin T. Manard, E. Miller Wylie, K. J. Mathew, Chelsea F. Ottenfeld, R. Kenneth Marcus
The integration of the liquid sampling-atmospheric pressure glow discharge (LS-APGD) ion source with Orbitrap mass spectrometers has resulted in new opportunities in the field of isotope ratio mass spectrometry. In a field that has been dominated by thermal ionization mass spectrometry (TIMS) and inductively coupled plasma mass spectrometry (ICP-MS) on quadrupole and scanning-mode sector field analyzer platforms for highly accurate and precise measurements, the LS-APGD-Orbitrap system offers a benchtop instrument capable of meeting the rigorous International Target Values for measurement uncertainty for uranium (U). In order to benchmark the LS-APGD-Orbitrap, a series of U certified reference materials with increasing 235U isotopic composition were analyzed. By using U samples ranging in enrichment from 1 to 80%, the ability of the system to measure isotope ratios over a wide range is demonstrated. This analysis represents the first time that the LS-APGD-Orbitrap system has been used to analyze highly enriched U samples, allowing for the measurement of each of the U isotopes, including 234U and 236U-related species, which had not been achieved previously. Ultimately, the LS-APGD-Orbitrap system was able to measure CRM U-800 (assayed as 235U / 238U = 4.265622) as 4.266922, with a combined uncertainty, (uc), of 0.040%. These results are compared to those obtained using traditional elemental mass spectrometers including TIMS and ICP-MS-based instruments. The effectiveness of the LS-APGD-Orbitrap MS system for measuring U isotopes shows excellent promise in nuclear forensics, safeguards, and other nuclear weapon-based applications.
Sequence Ion Structures and Dissociation Chemistry of Deprotonated Sucrose Anions J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-10-03 Benjamin J. Bythell, Jordan M. Rabus, Ashley R. Wagoner, Maha T. Abutokaikah, Philippe Maître
We investigate the tandem mass spectrometry of regiospecifically labeled, deprotonated sucrose analytes. We utilize density functional theory calculations to model the pertinent gas-phase fragmentation chemistry of the prevalent glycosidic bond cleavages (B1-Y1 and C1-Z1 reactions) and compare these predictions to infrared spectroscopy experiments on the resulting B1 and C1 product anions. For the C1 anions, barriers to interconversion of the pyranose [α-glucose-H]−, C1 anions to entropically favorable ring-open aldehyde-terminated forms were modest (41 kJ mol−1) consistent with the observation of a band assigned to a carbonyl stretch at ~ 1680–1720 cm−1. For the B1 anions, our transition structure calculations predict the presence of both deprotonated 1,6-anhydroglucose and carbon 2-ketone ((4S,5S,6R)-4,5-dihydroxy-6-(hydroxymethyl)dihydro-2H-pyran-3(4H)-one) anion structures, with the latter predominating. This hypothesis is supported by our spectroscopic data which show diagnostic bands at 1600, 1674, and 1699 cm−1 (deprotonated carbon 2-ketone structures), and at ~ 1541 cm−1 (both types of structure) and RRKM rate calculations. The deprotonated carbon 2-ketone structures are also the lowest energy product B1 anions.
On-substrate Enzymatic Reaction to Determine Acetylcholinesterase Activity in Whole Blood by Paper Spray Mass Spectrometry J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-10-02 Daniel O. Carmany, Phillip M. Mach, Gabrielle M. Rizzo, Elizabeth S. Dhummakupt, Ethan M. McBride, Jennifer W. Sekowski, Bernard Benton, Paul S. Demond, Michael W. Busch, Trevor Glaros
Currently, all assays measuring acetylcholinesterase (AChE) activity following a suspected nerve agent exposure leverage methodologies that fail to identify the agent. This limits the overall effectiveness and ability to administer proper countermeasures. As such, there is an urgent need to identify novel, rapid, and more comprehensive approaches to establish AChE activity, including identification of the toxicant. Paper spray mass spectrometry was used to monitor the activity of acetylcholinesterase, both in-solution and on modified hydrophobic paper surface. Hydrophobic paper surfaces were prepared using vaporized trichloro(3,3,3-trifluoropropyl)silane. In both approaches, mixtures of diluted human whole blood with and without VX were mixed with a non-endogenous AChE specific substrate, 1,1-dimethyl-4-acetylthiomethylpiperidinium (MATP+). Formation of the cleaved MATP+ product was monitored over time and compared to MATP+ to determine relative AChE activity. This on-substrate assay was effective at determining AChE activity and identifying the toxicant; however, determination of AChE activity in-solution proceeded at a slower rate. The on-substrate assay serves as a pioneering example of an enzymatic reaction occurring on the surface of a paper spray ionization ticket. This work broadens the range of applications relating to paper spray ionization-based clinical diagnostic assays.
Quantitative Evaluation of Native Protein Folds and Assemblies by Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-10-02 Matthew J. Harris, Deepika Raghavan, Antoni J. Borysik
Hydrogen deuterium exchange mass spectrometry (HDX-MS) has significant potential for protein structure initiatives but its relationship with protein conformations is unclear. We report on the efficacy of HDX-MS to distinguish between native and non-native proteins using a popular approach to calculate HDX protection factors (PFs) from protein structures. The ability of HDX-MS to identify native protein conformations is quantified by binary structural classification such that merits of the approach for protein modelling can be quantified and better understood. We show that highly accurate PF calculations are not a prerequisite for HDX-MS simulations that are capable of effectively discriminating between native and non-native protein folds. The simulations can also be performed directly on unique structures facilitating high-throughput evaluation of many alternate conformations. The ability of HDX-MS to classify the conformations of homo-protein assemblies is also investigated. In contrast to protein monomers, we show a significant lack of correspondence between the simulated and experimental HDX-MS data for these systems with a subsequent decrease in the ability of HDX-MS to identify native states. However, we demonstrate surprisingly high diagnostic ability of the simulated data for assemblies in which a significant proportion of the individual chains occupy protein-protein interfaces. We relate this to the number of peptides that can sample alternate subunit orientations and discuss these observations within the larger context of applying HDX-MS to evaluate protein structures.
Resolution-Enhanced Kendrick Mass Defect Analysis of Polycyclic Aromatic Hydrocarbons and Fullerenes in the Diffusion Flame from a Butane Torch J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-08-06 Robert B. Cody, Thierry Fouquet
A modified Kendrick Mass Defect (KMD) analysis was applied to the analysis of polycyclic aromatic hydrocarbons (PAHs) and fullerenes in the diffusion flame from a handheld butane torch.
Large-Area Graphene Films as Target Surfaces for Highly Reproducible Matrix-Assisted Laser Desorption Ionization Suitable for Quantitative Mass Spectrometry J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-07-11 Yoon Kyung Choi, Joo Yeon Oh, Sang Yun Han
Due to the known sweet-spot issues that intrinsically arise from inhomogeneous formation of matrix-analyte crystals utilized as samples in matrix-assisted laser desorption ionization (MALDI) mass spectrometry, its reproducibility and thus its applications for quantification have been somewhat limited. In this paper, we report a simple strategy to improve the uniformity of matrix-analyte crystal spots, which we realized by adapting large-area graphene films, i.e., non-inert, interacting surfaces, as target surfaces. In this example, the graphitic surfaces of the graphene films interact with excess matrix molecules during the sample drying process, which induces spontaneous formation of optically uniform MALDI sample crystal layers on the film surfaces. Further, mass spectrometric imaging reveals that the visible uniformity is indeed accompanied by reproducible MALDI ionization over an entire sample spot, which greatly suppresses the appearance of sweet spots. The results of this study confirm that the proposed method achieves good linear responses of ion intensity to the analyte concentration (R2 > 0.99) with small relative standard deviations (σ < 10%), which is a range applicable for quantitative measurements using MALDI mass spectrometry.
Analysis of Fragmentation Pathways of New-Type Synthetic Cannabinoids Using Electrospray Ionization J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-06-27 Karolina Sekuła, Dariusz Zuba, Karolina Lorek
Recently, dozens of new psychoactive substances have appeared on the European drug market every year. The most abundant group of these compounds is synthetic cannabinoids. In the first few years of the “legal highs” phenomenon, JWH (John W. Huffman) compounds were especially popular among drug users. However, the group of synthetic cannabinoids is constantly expanding, as new compounds are created by replacing known structural elements with different chemical groups. The problem with the identification of novel substances in forensic laboratories results from the structural similarity of the compounds and the rapid introduction of newer designer drugs on the black market. In this study, the fragmentation patterns of 29 new-type synthetic cannabinoids using electrospray ionization were investigated. The analysis was performed using quadrupole time-of-flight mass spectrometry. Based on measurements carried out under various conditions, the way of fragmentation of the tested compounds that were divided into groups due to their chemical structure was established. The study showed that the bond between the carbon atom of the carbonyl group and the ring or NH group attached to the ring was mainly cleaved. This mechanism was adequate for the fragmentation of first-generation synthetic cannabinoids. This paper presents characteristic ions formed by synthetic cannabinoids (i.e., ions originating from an indole/indazole ring and an adamanyl/naphthalene/quinoline ring) using electrospray ionization. Knowledge of these specific fragments can be used in forensic laboratories to determine the structure of novel compounds from the group of synthetic cannabinoids.
Simulation of Unidirectional Ion Ejection in an Asymmetric Half-Round Rod Electrode Linear Ion Trap Mass Analyzer J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-07-11 HaiYan Wu, LiPeng Zhang, ZaiYue Zhang, Jie Qian, ShuGuang Zhang, YingJun Zhang, SaiJin Ge, XiaoXu Li
An asymmetric trapping field was generated from an asymmetric half-round rod electrode linear ion trap (A-HreLIT), and its performance of unidirectional ion ejection was studied. Two different asymmetric structures of A-HreLITs were constructed, one rotating y electrode pairs toward an x electrode with an angle θ, and the other stretching one x electrode with a distance α. The center of trapping field was displaced away from the geometrical center of the ion trap, defined to be the midpoint along the axis of y between x electrodes, which leads to unidirectional ion ejection through one x electrode. Computer simulations were used to investigate the relationship between asymmetric geometric parameter of θ (or α) and analytical performance. Both structures could result in similar asymmetric trapping fields, which mainly composed of dipole, quadrupole, and hexapole fields. The dipole and hexapole fields were approximately proportional to the asymmetric geometric parameter of rotation angle θ (or stretch distance α). In simulation, ion trajectories and ion kinetic energy were calculated. For ions with m/z 609 Th, the simulation results showed that mass resolution of over 2400 (FWHM) and ion unidirectional ejection efficiency of nearly 90% were achieved in an optimized A-HreLIT. Ion detection efficiency of A-HreLIT could be improved significantly with only one ion detector, while maintaining a considerable mass resolution. Furthermore, the A-HreLIT could be driven by a traditional balanced RF power supply. These advantages make A-HreLIT suitable for developing miniaturized mass spectrometer with high performance.
Using Digital Waveforms to Mitigate Solvent Clustering During Mass Filter Analysis of Proteins J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-07-09 Bojana Opačić, Nathan M. Hoffman, Zachary P. Gotlib, Brian H. Clowers, Peter T. A. Reilly
With advances in the precision of digital electronics, waveform generation technology has progressed to a state that enables the creation of m/z filters that are purely digitally driven. These advances present new methods of performing mass analyses that provide information from a chemical system that are inherently difficult to achieve by other means. One notable characteristic of digitally driven mass filters is the capacity to transmit ions at m/z ratios that vastly exceed the capabilities of traditional resonant systems. However, the capacity to probe ion m/z ratios that span multiple orders of magnitudes across multiple orders of magnitude presents a new set of issues requiring a solution. In the present work, when probing multiply charged protein species beyond m/z 2000 using a gentle atmospheric pressure interface, the presence of solvent adducts and poorly resolved multimers can severely degrade spectral fidelity. Increasing energy imparted into a target ion population is one approach minimizing these clusters; however, the use of digital waveform technology provides an alternative that maximizes ion transport efficiency and simultaneously minimizes solvent clustering. In addition to the frequency of the applied waveform, digital manipulation also provides control over the duty cycle of the target waveform. This work examines the conditions and approach leading to optimal digital waveform operation to minimize solvent clustering.
Improvement in the Mass Resolution of Single Particle Mass Spectrometry Using Delayed Ion Extraction J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-08-13 Lei Li, Liu Liu, Li Xu, Mei Li, Xue Li, Wei Gao, Zhengxu Huang, Ping Cheng
A specific delayed ion extraction (DIE) technique, which combines a standard rectangular extraction pulse with an exponential pulse, was introduced for a single particle mass spectrometry (SPMS) instrument, and it can focus ions in a wide mass range and results in a mass resolution improvement for the mass range of the studied ions. The experimental results indicate that the average mass resolution for positive ions is about 1000 when the mass-to-charge ratio (m/z) is greater than 70, and for negative ions, when the m/z is greater than 70, the average resolution can reach 2000. The highest mass resolutions achieved so far are 1260 for positive ions and 2400 for negative ions for SPMS, which are very beneficial for mass peak interpretation and chemical compound identification. The primary applications for atmospheric particle measurements show that the high mass resolution of SPMS with the DIE technique is very beneficial for the analysis of carbon and metallic element containing particles, and 39K+ with C3H3+ and 41K+ and C3H5+ in organic particles were successfully differentiated using SPMS. The results indicate that SPMS with DIE technique can significantly ease mass peak interpretation and improve the mass assignment ability during analysis. Furthermore, existing SPMS instruments can be improved by a facile retrofitting process to implement the DIE technique.
Exploring the Potential of Dendritic Oligoglycerol Detergents for Protein Mass Spectrometry J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-10-01 Leonhard H. Urner, Yasmine B. Maier, Rainer Haag, Kevin Pagel
The ability to design detergents that are suitable for protein analysis by mass spectrometry (MS) represents an on-going challenge in the field of native MS. Desirable detergent characteristics include charge-reducing properties and low gas-phase stabilities of complexes formed with proteins. In this work, the gas-phase properties of oligoglycerol detergents (OGDs) are optimized by fine tuning of their molecular structure. Furthermore, a tandem mass spectrometry (MS/MS) approach is presented that estimates the gas-phase properties of detergents simply by studying the dissociation behaviour of protein-detergent complexes (PDCs) formed with the soluble protein β-lactoglobulin (BLG).
Method for Quantifying Oxidized Methionines and Application to HIV-1 Env J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-07-09 Joshua T. Shipman, Eden P. Go, Heather Desaire
Recombinantly expressed proteins are susceptible to oxidation during expression, purification, storage, and analysis; the residue most susceptible to oxidation is methionine. Methionine oxidation can be overestimated using current quantitative analysis methods because oxidation can occur during sample preparation, and researchers often do not use methods that account for this possibility. An experimental strategy had been developed previously to solve this problem through the use of an 18O-labeled hydrogen peroxide reagent. However, the method did not address the analysis of peptides that contained multiple methionine residues. Herein, we develop and validate a new analysis method that uses theoretical isotope distributions and experimental spectra to quantify methionine oxidation that is present prior to sample preparation. The newly described approach is more rapid than the previously described method, and it needs only half the amount of protein for analysis. This method was validated using model proteins; then, it was applied to the analysis of recombinant HIV-1 Env, the key protein in HIV vaccine candidates. While Met oxidation of this protein could not be analyzed using previous methods, the approach described herein was useful for determining the oxidation state of HIV-Env.
Disulfide Connectivity Analysis of Peptides Bearing Two Intramolecular Disulfide Bonds Using MALDI In-Source Decay J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-07-09 Philippe Massonnet, Jean R. N. Haler, Gregory Upert, Nicolas Smargiasso, Gilles Mourier, Nicolas Gilles, Loïc Quinton, Edwin De Pauw
Disulfide connectivity in peptides bearing at least two intramolecular disulfide bonds is highly important for the structure and the biological activity of the peptides. In that context, analytical strategies allowing a characterization of the cysteine pairing are of prime interest for chemists, biochemists, and biologists. For that purpose, this study evaluates the potential of MALDI in-source decay (ISD) for characterizing cysteine pairs through the systematic analysis of identical peptides bearing two disulfide bonds, but not the same cysteine connectivity. Three different matrices have been tested in positive and/or in negative mode (1,5-DAN, 2-AB and 2-AA). As MALDI-ISD is known to partially reduce disulfide bonds, the data analysis of this study rests firstly on the deconvolution of the isotope pattern of the parent ions. Moreover, data analysis is also based on the formed fragment ions and their signal intensities. Results from MS/MS-experiments (MALDI-ISD-MS/MS) constitute the last reference for data interpretation. Owing to the combined use of different ISD-promoting matrices, cysteine connectivity identification could be performed on the considered peptides.
Coaxial Electrospray Ionization for the Study of Rapid In-source Chemistry J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-06-13 Brynn N. Sundberg, Anthony F. Lagalante
Coaxial electrospray has been used effectively for several dual-emitter applications, but has not been utilized for the study of rapid in-source chemistry. In this paper, we report the fabrication of a coaxial, micro-volume dual-emitter through the modification of a manufacturer’s standard electrospray probe. This modification creates rapid mixing inside the Taylor cone and the ability to manipulate fast reactions using a variety of solvents and analytes. We demonstrate its potential as a low-cost, dual-emitter assembly for diverse applications through three examples: relative ionization in a biphasic electrospray, hydrogen-deuterium exchange, and protein supercharging.
Conjugation of para -benzoquinone of Cigarette Smoke with Human Hemoglobin Leads to Unstable Tetramer and Reduced Cooperative Oxygen Binding J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-07-02 Amrita Mitra, Amit Kumar Mandal
Besides multiple life-threatening diseases like lung cancer and cardiovascular disease, cigarette smoking is known to produce hypoxia, a state of inadequate oxygen supply to tissues. Hypoxia plays a pivotal role in the development of chronic obstructive pulmonary disease. Smoking during pregnancy imposes risk for the unborn child. In addition to carbon monoxide, conjugation of para-benzoquinone (pBQ), derived from cigarette smoke, with human hemoglobin (HbA) was also reported to contribute in hypoxia. In fact, conjugation of pBQ is more alarming than carbon monoxide as it is an irreversible covalent modification. In the present study, the functional assay of Hb-pBQ, performed through oxygen equilibrium curve, showed a significant decrease in both P50 and cooperativity. However, the structural changes associated with the observed functional perturbation of the hemoglobin conjugate (Hb-pBQ) are unknown to date. Enhanced sensitivity and high resolution of nano-ESI mass spectrometry platform have enabled to investigate the native structure of oligomers of hemoglobin in a single scan. The structural integrity of Hb-pBQ measured through the dissociation equilibrium constants (Kd) indicated that compared to HbA, Kd of tetramer-dimer and dimer-monomer equilibria were increased by 4.98- and 64.3-folds, respectively. Using isotope exchange mass spectrometry, we observed perturbations in the inter-subunit interactions of deoxy and oxy states of Hb-pBQ. However, the three-dimensional architecture of Hb-pBQ, monitored through collision cross-sectional area, did not show any change. We propose that the significant destabilization of the functionally active structure of hemoglobin upon conjugation with pBQ results in tighter oxygen binding that leads to hypoxia.
Charging and Charge Switching of Unsaturated Lipids and Apolar Compounds Using Paternò-Büchi Reactions J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-07-16 Patrick Esch, Sven Heiles
The ability to control the charge state and ionization efficiency of lipids and hydrocarbons by means of in-source Paternò-Büchi functionalization in nano-electrospray ionization mass spectrometry experiments is investigated. Ultraviolet light irradiation of acetylpyridine filled nano-electrospray emitter tips, containing unsaturated analytes, generates protonated lipid and hydrocarbon ions. Comparison of reaction yields and fragment ion abundances of functionalized unsaturated fatty acids indicate that acetylpyridine Paternò-Büchi functionalization allows to readily detect fatty acids and determine double bond positions, but fragmentation efficiency and reactivity depend on double bond position and varies between different acetylpyridine isomers. Results for methyl oleate and olefins suggest that fragment ion abundances of unsaturated compounds depend on interactions between acetylpyridine and nearby functional groups. Paternò-Büchi functionalization with acetylpyridine was used to detect and assign double bond positions of mono- and polyunsaturated fatty acid, cholesterol ester, triglyceride, and hydrocarbon standards with ion abundances that are up to 631 times higher than abundances of the same compounds prior Paternò-Büchi reaction. To demonstrate the scope and analytical robustness of the newly developed method, free fatty acids in mouse brain as well as male Schistosoma mansoni extracts and hydrocarbons in an olefin mixture are investigated. For this complex set of analytes, charging and charge switching using acetylpyridine Paternò-Büchi functionalization enable double bond position assignment and relative quantification in positive ion mode.
Optimized Electrostatic Linear Ion Trap for Charge Detection Mass Spectrometry J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-07-09 Joanna A. Hogan, Martin F. Jarrold
In charge detection mass spectrometry (CDMS), ions are passed through a detection tube and the m/z ratio and charge are determined for each ion. The uncertainty in the charge and m/z determinations can be dramatically reduced by embedding the detection tube in an electrostatic linear ion trap (ELIT) so that ions oscillate back and forth through the detection tube. The resulting time domain signal can be analyzed by fast Fourier transforms (FFTs). The ion’s m/z is proportional to the square of the oscillation frequency, and its charge is derived from the FFT magnitude. The ion oscillation frequency is dependent on the physical dimensions of the trap as well as the ion energy. A new ELIT has been designed for CDMS using the central particle method. In the new design, the kinetic energy dependence of the ion oscillation frequency is reduced by an order of magnitude. An order of magnitude reduction in energy dependence should have led to an order of magnitude reduction in the uncertainty of the m/z determination. In practice, a factor of four improvements was achieved. This discrepancy is probably mainly due to the trajectory dependence of the ion oscillation frequency. The new ELIT design uses a duty cycle of 50%. We show that a 50% duty cycle produces the lowest uncertainty in the charge determination. This is due to the absence of even-numbered harmonics in the FFT, which in turn leads to an increase in the magnitude of the peak at the fundamental frequency.
Extracting Charge and Mass Information from Highly Congested Mass Spectra Using Fourier-Domain Harmonics J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-07-12 Sean P. Cleary, Huilin Li, Dhanashri Bagal, Joseph A. Loo, Iain D. G. Campuzano, James S. Prell
Native mass spectra of large, polydisperse biomolecules with repeated subunits, such as lipoprotein Nanodiscs, can often be challenging to analyze by conventional methods. The presence of tens of closely spaced, overlapping peaks in these mass spectra can make charge state, total mass, or subunit mass determinations difficult to measure by traditional methods. Recently, we introduced a Fourier Transform-based algorithm that can be used to deconvolve highly congested mass spectra for polydisperse ion populations with repeated subunits and facilitate identification of the charge states, subunit mass, charge-state-specific, and total mass distributions present in the ion population. Here, we extend this method by investigating the advantages of using overtone peaks in the Fourier spectrum, particularly for mass spectra with low signal-to-noise and poor resolution. This method is illustrated for lipoprotein Nanodisc mass spectra acquired on three common platforms, including the first reported native mass spectrum of empty “large” Nanodiscs assembled with MSP1E3D1 and over 300 noncovalently associated lipids. It is shown that overtone peaks contain nearly identical stoichiometry and charge state information to fundamental peaks but can be significantly better resolved, resulting in more reliable reconstruction of charge-state-specific mass spectra and peak width characterization. We further demonstrate how these parameters can be used to improve results from Bayesian spectral fitting algorithms, such as UniDec.
Characterization of biomass and biochar by LDI-FTICRMS – Effect of the laser wavelength and biomass material J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-07-30 Frédéric Aubriet, Thierry Ghislain, Jasmine Hertzog, Alexander Sonnette, Anthony Dufour, Guillain Mauviel, Vincent Carré
The pyrolysis of the lignocellulosic biomass is a promising process to produce biofuels or green chemicals. Specific analytical methods have to be developed in order to better understand the composition of biomass and of its pyrolysis products and therefore to optimize the design of pyrolysis processes. For this purpose, different biomasses (Douglas and Miscanthus) and one biochar were analyzed by laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (LDI FT-ICR MS). This method allowed the biomass and biochar to be analyzed without any sample preparation and with a spatial resolution of about 100 μm. The influence of LDI conditions (laser wavelength and laser irradiance) and the nature of the biomass and biochar on the obtained mass spectrum were investigated. The nature and origin of the observed ions highly depended on LDI conditions. In the softest laser–biomass interaction conditions (low laser irradiance), the detected ions were related to the nature of the investigated biomass. Indeed, the main part of the detected species came from the different biomass subunits and was produced by photolysis of covalent bonds. When more severe laser irradiation conditions were used, the obtained mass spectra gathered the ions relative to (i) the chemical components of the investigated samples, (ii) the recombination products of these species in the gas phase after their ejection from the sample surface, and (iii) the compounds produced by laser pyrolysis of the sample. This was expected to be useful to mimic thermal pyrolysis.
Resolving the Discrepancies Between Empirical and Rayleigh Charge Limiting Models for Globular Proteins J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-07-24 Karen C. B. De Freitas
Starting with the Rayleigh charge limiting model, a slightly different approach is used to account for the well-known discrepancy that exists between the said model and experimental ESI MS data for globular proteins. It is shown using published datasets that for globular proteins, the mass density ρ exhibits a weak second-order dependence on its mass M, according to ρ(M)∝ M-α, α ~ 0.14. A direct equivalence established between ESI MS and x-ray techniques suggests a minimum but critical surface tension of 15.6 ± 5.2 mN/m for the droplet at the liquid-to-gas phase transition point. The packing density factor η for globular proteins is believed to lie between 1 (very tightly packed) and 4.6 (less tight, natively packed). While the Rayleigh charge limiting model has been linked historically to the CRM (J. Chem. Phys. 49:2240–2249, 1968; Anal. Chim. Acta 406:93–104, 2000), this paper does not expressly seek to justify the CRM, but rather uses empirical data and existing knowledge across subfields to help build a consistent picture of ESI MS phenomena that might be difficult to explain otherwise. These results would be useful in molecular dynamics (MD) simulations, understanding liquid-to-gas phase transitions and in opening up new routes for cross-calibration between ESI MS, IM MS, NMR and x-ray crystallography studies.
Liquid Native MALDI Mass Spectrometry for the Detection of Protein-Protein Complexes J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-07-31 Martine Beaufour, David Ginguené, Rémy Le Meur, Bertrand Castaing, Martine Cadene
Native mass spectrometry (MS) encompasses methods to keep noncovalent interactions of biomolecular complexes intact in the gas phase throughout the instrument and to measure the mass-to-charge ratios of supramolecular complexes directly in the mass spectrometer. Electrospray ionization (ESI) in nondenaturing conditions is now an established method to characterize noncovalent systems. Matrix-assisted laser desorption/ionization (MALDI), on the other hand, consumes low quantities of samples and largely tolerates contaminants, making it a priori attractive for native MS. However, so-called native MALDI approaches have so far been based on solid deposits, where the rapid transition of the sample through a solid state can engender the loss of native conformations. Here we present a new method for native MS based on liquid deposits and MALDI ionization, unambiguously detecting intact noncovalent protein complexes by direct desorption from a liquid spot for the first time. To control for aggregation, we worked with HUαβ, a heterodimer that does not spontaneously rearrange into homodimers in solution. Screening through numerous matrix solutions to observe first the monomeric protein, then the dimer complex, we settled on a nondenaturing binary matrix solution composed of acidic and basic organic matrices in glycerol, which is stable in vacuo. The role of temporal and spatial laser irradiation patterns was found to be critical. Both a protein-protein and a protein-ligand complex could be observed free of aggregation. To minimize gas-phase dissociation, source parameters were optimized to achieve a conservation of complexes above 50% for both systems.
Comparing Hydrogen Deuterium Exchange and Fast Photochemical Oxidation of Proteins: a Structural Characterisation of Wild-Type and ΔN6 β 2 -Microglobulin J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-09-28 Owen Cornwell, Sheena E. Radford, Alison E. Ashcroft, James R. Ault
Hydrogen deuterium exchange (HDX) coupled to mass spectrometry (MS) is a well-established technique employed in the field of structural MS to probe the solvent accessibility, dynamics and hydrogen bonding of backbone amides in proteins. By contrast, fast photochemical oxidation of proteins (FPOP) uses hydroxyl radicals, liberated from the photolysis of hydrogen peroxide, to covalently label solvent accessible amino acid side chains on the microsecond-millisecond timescale. Here, we use these two techniques to study the structural and dynamical differences between the protein β2-microglobulin (β2m) and its amyloidogenic truncation variant, ΔN6. We show that HDX and FPOP highlight structural/dynamical differences in regions of the proteins, localised to the region surrounding the N-terminal truncation. Further, we demonstrate that, with carefully optimised LC-MS conditions, FPOP data can probe solvent accessibility at the sub-amino acid level, and that these data can be interpreted meaningfully to gain more detailed understanding of the local environment and orientation of the side chains in protein structures.
Single-Run Mass Spectrometry Analysis Provides Deep Insight into E. coli Proteome J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-09-26 Bhaswati Chatterjee, Suman S. Thakur
Single-run mass spectrometry has enabled the detection and quantifications of E. coli proteins. A total of 2068 proteins quantified by intensity-based absolute quantification (iBAQ) Schwanhäusser et al.: (Nature. 473, 337–342, 2011) procedure were obtained with single enzyme-trypsin, without pre-fractionation, by quadruplicate long liquid chromatography runs coupled with high-resolution linear trap quadrupole (LTQ)-Orbitrap Velos mass spectrometry. The single-run of 12 h has ability to cover almost 98% of the quadruplicate LC-MS/MS runs of E. coli proteome and is therefore almost equivalent to quadruplicate LC-MS/MS runs. These quantified proteins are about 52% of the total proteins present in E. coli genome according to Uniprot database. The quantified proteins covered almost all of the proteins in folate biosynthesis. Remarkably greater part of Gene Ontology (GO) Barrell et al.: (Nucleic Acids Res. 37, D396–D403, 2009), Ashburner et al.: (Nat. Genet. 25, 25–29, 2000) annotations, signaling pathways along with protein-protein interactions were covered. Some of the important biological processes-cell cycle, DNA repair, ion transport, ubiquinone biosynthetic process, pseudouridine synthesis, peptidoglycan biosynthetic process, RNA processing, and translation-revealed protein-protein interaction network generated by Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) Jensen, et al.:(Nucleic Acids Res 37, D412-D126, 2009) database. Therefore, to achieve the saturation point of detection of maximum number of proteins in single LC-MS/MS run, 12-h liquid chromatography gradient is appropriate.
Electrospray Generated from the Tip-Sealed Fine Glass Capillary Inserted with an Acupuncture Needle Electrode J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-09-20 Dilshadbek T. Usmanov, Satoshi Ninomiya, Kenzo Hiraoka, Hiroshi Wada, Hiroshi Nakano, Masaya Matsumura, Sachiyo Sanada-Morimura, Hiroshi Nonami
In electrospray, excess charges are supplied to a sample solution by the occurrence of electrochemical reactions. Recently, different versions of electrospray, e.g., dielectric barrier electrospray ionization, inductive desorption electrospray ionization, and electrostatic-ionization driven by dielectric polarization, have been reported in which the sample solution was not in direct contact with the metal electrode but separated by dielectric materials. The objective of the current work is to elucidate the mechanism of dielectric barrier electrospray. A sealed borosilicate glass capillary inserted with a fine acupuncture needle was used as a probe. A sample solution (~ 400 nL) was captured on the glass capillary tip and a positive high voltage (HV) pulse (+ 4.5 kV) was applied to the internal metal electrode. Mass spectra were measured as a function of the HV pulse width from μs to 10 s. Ions started to be detected with the pulse width of ~ 5 ms. The ion intensities increased slowly with time and reached a plateau in a few seconds. The charge distribution of cytochrome c [M + nH]n+ shifted to higher n values from a few ms to seconds. In addition to cone-jet mode normal electrospray that lasted until all the liquid sample was depleted from the glass tip, the polarization-induced electrospray ionization was observed at the early stage of the HV application.
Comparing the Effects of Additives on Protein Analysis Between Desorption Electrospray (DESI) and Electrospray Ionization (ESI) J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-09-19 Elahe Honarvar, Andre R. Venter
It is frequently said that DESI-MS follows a similar ionization mechanism as ESI because of similarities usually observed in their respective mass spectra. However, practical use of DESI-MS for protein analysis is limited to proteins with lower molecular weights (< 25 kDa) due to a mass-dependent loss in signal intensity. Here we investigated commonly used volatile acids and their ammonium salt buffers for DESI-MS analysis of protein. We noticed that, surprisingly, some additives influence the analysis differently in DESI compared to ESI. Improved signal intensities with both DESI and ESI were obtained when acetic and formic acid were added into aqueous methanol spray solvents with both DESI and ESI. On the other hand, while with ESI the addition of ammonium salts into spray solutions strongly reduced both signal and S/N, with DESI signal intensities and S/N were improved dramatically. Ammonium bicarbonate when used with DESI reduced the total amount of adduction and delivered excellent signal-to-noise ratios with high intensity; however, it also denatures protein. When native state protein mass spectra are preferred, ammonium acetate would also deliver reasonable adduct removal and improved S/N. The amount of total adduction of individual adducting species and of all species could not be correlated with differences in either solutions pH values or with proton affinities of the anions. An obvious difference between DESI and ESI mass spectrometry is the effects of protein solubility during droplet pickup (desorption), but differences in the sizes, velocities, and composition of ionizing droplets were also discussed as important factors.
LILBID and nESI: Different Native Mass Spectrometry Techniques as Tools in Structural Biology J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-09-17 Oliver Peetz, Nils Hellwig, Erik Henrich, Julija Mezhyrova, Volker Dötsch, Frank Bernhard, Nina Morgner
Native mass spectrometry is applied for the investigation of proteins and protein complexes worldwide. The challenge in native mass spectrometry is maintaining the features of the proteins of interest, such as oligomeric state, bound ligands, or the conformation of the protein complex, during transfer from solution to gas phase. This is an essential prerequisite to allow conclusions about the solution state protein complex, based on the gas phase measurements. Therefore, soft ionization techniques are required. Widely used for the analysis of protein complexes are nanoelectro spray ionization (nESI) mass spectrometers. A newer ionization method is laser induced liquid bead ion desorption (LILBID), which is based on the release of protein complexes from solution phase via infrared (IR) laser desorption. We use both methods in our lab, depending on the requirements of the biological system we are interested in. Here we benchmark the performance of our LILBID mass spectrometer in comparison to a nESI instrument, regarding sample conditions, buffer and additive tolerances, dissociation mechanism and applicability towards soluble and membrane protein complexes.
Proton Transfer Accounting for Anomalous Collision-Induced Dissociation of Proton-Bound Hoogsteen Base Pair of Cytosine and Guanine J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-09-13 Jeong Ju Park, Choong Sik Lee, Sang Yun Han
To understand the anomalous collision-induced dissociation (CID) behavior of the proton-bound Hoogsteen base pair of cytosine (C) and guanine (G), C:H+∙∙∙G, we investigated CID of a homologue series of proton-bound heterodimers of C, 1-methylcytosine, and 5-methylcytosine with G as a common base partner. The CID experiments were performed in an energy-resolved way (ER-CID) under both multiple and near-single collision conditions. The relative stabilities of the protonated complexes examined by ER-CID suggested that the proton-bound complexes produced by electrospray ionization in this study are proton-bound Hoogsteen base pairs. On the other hand, in contrast to the other base pairs, CID of C:H+∙∙∙G exhibited more abundant productions of C:H+, the fragment protonated on the moiety with a smaller proton affinity, than that of G:H+. This appeared to contradict general prediction based on the kinetic method. However, further theoretical exploration of potential energy surfaces found that there can be facile proton transfers in the proton-bound Hoogsteen base pairs during the CID process, which makes the process accessible to an additional product state of O-protonated C for C:H+ fragments. The presence of an additional dissociation channel, which in other words corresponds to twofold degeneracy in the transition state leading to C:H+ fragments, effectively doubles the apparent reaction rate for production of C:H+. In this way, the process gives rise to the anomaly, the observed pronounced formation of C:H+ in the CID of the proton-bound Hoogsteen base pair, C:H+∙∙∙G.
Production of Gas-Phase Uranium Fluoroanions Via Solubilization of Uranium Oxides in the [1-Ethyl-3-Methylimidazolium] + [F(HF) 2.3 ] − Ionic Liquid J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-06-28 Christopher A. Zarzana, Gary S. Groenewold, Michael T. Benson, James E. Delmore, Tetsuya Tsuda, Rika Hagiwara
A new methodology for gas-phase uranium ion formation is described in which UO2 is dissolved in neat N-ethyl,N′-methylimidazolium fluorohydrogenate ionic liquid [EMIm+][F(HF)2.3−], yielding a blue-green solution. The solution was diluted with acetonitrile and then analyzed by electrospray ionization mass spectrometry. UF6− (a U(V) species) was observed at m/z = 352, and other than cluster ions derived from the ionic liquid, nothing else was observed. When the sample was analyzed using infusion desorption chemical ionization, UF6− was the base peak, and it was accompanied by a less intense UF5− that most likely was formed by elimination of a fluorine radical from UF6−. Formation of UF6− required dissolution of UO2 followed by or concurrent with oxidation of uranium from the + 4 to the + 5 state and finally formation of the fluorouranate. Dissolution of UO3 produced a bright yellow solution indicative of a U(VI) species; however, electrospray ionization did not produce abundant U-containing ions. The abundant UF6− provides a vehicle for accurate measurement of uranium isotopic abundances free from interference from minor isotopes of other elements and a convenient ion synthesis route that is needed gas-phase structure and reactivity studies like infrared multiphoton dissociation and ion-molecule dissociation and condensation reactions. The reactive fluorohydrogenate ionic liquid may also enable conversion of uranium in oxidic matrices into uranium fluorides that slowly oxidize to uranyl fluoride under ambient conditions, liberating the metal for facile measurement of isotope ratios without extensive chemical separations.
Detection of Neutral CO Lost During Ionic Dissociation Using Atmospheric Pressure Thermal Dissociation Mass Spectrometry (APTD-MS) J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-09-11 Pengyi Zhao, Travis White, R. Graham Cooks, Qinghao Chen, Yong Liu, Hao Chen
Elucidation of ion dissociation patterns is particularly important to structural analysis by mass spectrometry (MS). However, typically, only the charged fragments from an ion dissociation event are detected in tandem MS experiments; neutrals are not identified. In recent years, we have developed an atmospheric pressure thermal dissociation (APTD) technique that can be applied to dissociate ions at atmosphere pressure and thus provide one way to characterize neutral fragments. In this paper, we focus on the detection of neutral CO resulting from amino acid and peptide ion dissociation. In the first set of experiments, several protonated amino acids (e.g., + 1 ion of phenylalanine) were found to undergo loss of a neutral (s) of total mass 46 Da, a process leading to iminium ion formation. We successfully detected the neutral species CO by using a CO sensor, UV-Vis and MS analysis following selective CO trapping with a rhodium complex. The capture of CO from dissociation of protonated amino acids supports the assignment of the loss of 46 Da to neutral losses of CO and H2O, rather than loss of formaldehyde or dihydroxycarbene, other possible fragmentation pathways that have been subject of debate for a long time. In a second experiment, we used the APTD method in combination with the CO detection technique, to demonstrate the formation of CO in the conversion of b ions to a ions during peptide ion dissociations. These results showed the potential of APTD in the elucidation of ion dissociation mechanisms, using simple home-built apparatus.
Optimized Workflow for Selecting Peptides for HDX-MS Data Analyses J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-08-31 Lars Sørensen, Rune Salbo
Hydrogen deuterium exchange measured by mass spectrometry (HDX-MS) is a commonly used technique for studying the structural dynamics of proteins in solution. The first part of any bottom-up HDX-MS experiment is to identify the peptides generated from a digestion step. This requires manual inspection of the identified peptides to determine their use for HDX-MS analysis, which is a time-consuming task. Throughout the literature, there have been different approaches for removing peptides that do not yield quantifiable HDX information. This includes using validity scores from the software used in the generation of the peptide map and that the peptide should be found in two out of three technical replicate experiments. Here, we analyze the previously available methods for filtering the identified peptides in regard to their ability to predict whether a peptide will provide quantifiable HDX-MS data or not. We also present a new score-based system relying on a combination of MS/MS parameters that offers an improved method for separating quantifiable peptides from the nonquantifiable. Using this score-based method reduces the number of peptide spectra that needs to be manually inspected and thereby the time spent curating HDX-MS data.
Optimised Desorption Electrospray Ionisation Mass Spectrometry Imaging (DESI-MSI) for the Analysis of Proteins/Peptides Directly from Tissue Sections on a Travelling Wave Ion Mobility Q-ToF J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-08-30 Mark W. Towers, Tamas Karancsi, Emrys A. Jones, Steven D. Pringle, Emmanuelle Claude
Desorption electrospray ionisation mass spectrometry imaging (DESI-MSI) is typically known for the ionisation of small molecules such as lipids and metabolites, in singly charged form. Here we present a method that allows the direct detection of proteins and peptides in multiply charged forms directly from tissue sections by DESI. Utilising a heated mass spectrometer inlet capillary, combined with ion mobility separation (IMS), the conditions with regard to solvent composition, nebulising gas flow, and solvent flow rate have been explored and optimised. Without the use of ion mobility separation prior to mass spectrometry analysis, only the most abundant charge series were observed. In addition to the dominant haemoglobin subunit(s) related trend line in the m/z vs drift time (DT) 2D plot, trend lines were found relating to background solvent peaks, residual lipids and, more importantly, small proteins/large peptides of lower abundance. These small proteins/peptides were observed with charge states from 1+ to 12+, the majority of which could only be resolved from the background when using IMS. By extracting charge series from the 2D m/z vs DT plot, a number of proteins could be tentatively assigned by accurate mass. Tissue images were acquired with a pixel size of 150 μm showing a marked improvement in protein image resolution compared to other liquid-based ambient imaging techniques such as liquid extraction surface analysis (LESA) and continuous-flow liquid microjunction surface sampling probe (LMJ-SSP) imaging.
Dual-Channel Enzymatic Inhibition Measurement (DEIM) Coupling Isotope Substrate via Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-08-29 Min Tao, Li Zhang, Yinlong Guo
A novel dual-channel enzymatic inhibition measurement (DEIM) method was developed to improve the repeatability with light/heavy isotope substrates, producing reliable relative standard deviations (< 3%) by employing acetylcholinesterase (AChE) as the model enzyme. The matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) was adapted for enzyme-inhibited method due to its good salt-tolerance and high throughput; meanwhile, dual-channel enzymatic reactions were performed to improve the repeatability of each well. The acetylcholinesterase inhibition measurement was conducted by mixing the quenched enzyme reaction solution of blank group (with heavy isotope as substrate) and experimental group (with light isotope as substrate), of which the inhibition rate might be affected by isotope effects. Hence, inverse study and Km measurement were implemented to validate the method. The inverse study shows similar inhibition rate (68.9 and 70.3%) and the Km of isotope substrates are analogous (0.139 and 0.135 mM), which demonstrated that the novel method is feasible to AChE inhibition measurement. Finally, the method was applied to herb extracts, half of which exhibit inhibition to AChE. The precise dual-channel enzymatic inhibition measurement (DEIM) method could be regarded as a promising approach to potential enzyme inhibitor screening.
Correction to: Characterization of Long-Chain Fatty Acid as N-(4-Aminomethylphenyl) Pyridinium Derivative by MALDI LIFT-TOF/TOF Mass Spectrometry J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-08-27 Cheryl Frankfater, Xuntian Jiang, Fong-Fu Hsu
In the preceding article “Characterization of Long-Chain Fatty Acid as N-(4-Aminomethylphenyl) Pyridinium Derivative by MALDI LIFT-TOF/TOF Mass Spectrometry” by Frankfater et al., errors in Figs. 2 and 3 have occurred. The legend (Fig. 2)
Formation and Characterization of Zr 4+ Stabilized by Neutral Tridentate Ligands in the Gas Phase J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-08-23 Xiuting Chen, Qingnuan Li, Yu Gong
Ligated tetrapositive metal ions are rare gas-phase species which tend to form complexes with lower charges due to the high 4th ionization energies of metals. We report the observation of tetrapositive Zr(TMPDA)34+ and Zr(TMOGA)34+ complexes in the gas phase by electrospray ionization of Zr(ClO4)4/TMPDA and Zr(ClO4)4/TMOGA mixtures. The Zr4+ center in both complexes is coordinated by nine atoms from three neutral diamide ligands forming nine-coordinate twisted tricapped trigonal prismatic geometry on the basis of DFT calculations. Collision-induced dissociation of both complexes resulted in the loss of protonated ligands to form tripositive Zr(TMPDA)(TMPDA-H)3+ and Zr(TMOGA)(TMOGA-H)3+ products which retain the IV oxidation state of zirconium at the cost of charge reduction from 4+ to 3+ of the whole complexes. The very high 4th ionization energy of zirconium (34.34 eV) makes tetrapositive zirconium complex the most challenging tetracation to be stabilized against charge reduction in the gas phase to date.
Collision-Induced Unfolding Reveals Unique Fingerprints for Remote Protein Interaction Sites in the KIX Regulation Domain J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-08-22 Jessica N. Rabuck-Gibbons, Jean M. Lodge, Anna K. Mapp, Brandon T. Ruotolo
The kinase-inducible domain (KIX) of the transcriptional coactivator CBP binds multiple transcriptional regulators through two allosterically connected sites. Establishing a method for observing activator-specific KIX conformations would facilitate the discovery of drug-like molecules that capture specific conformations and further elucidate how distinct activator-KIX complexes produce differential transcriptional effects. However, the transient and low to moderate affinity interactions between activators and KIX are difficult to capture using traditional biophysical assays. Here, we describe a collision-induced unfolding-based approach that produces unique fingerprints for peptides bound to each of the two available sites within KIX, as well as a third fingerprint for ternary KIX complexes. Furthermore, we evaluate the analytical utility of unfolding fingerprints for KIX complexes using CIUSuite, and conclude by speculating as to the structural origins of the conformational families created from KIX:peptide complexes following collisional activation.
Modified Quadrupole Ion Trap Mass Spectrometer for Infrared Ion Spectroscopy: Application to Protonated Thiated Uridines J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-08-22 L. A. Hamlow, Y. Zhu, Zachary J. Devereaux, N. A. Cunningham, G. Berden, J. Oomens, M. T. Rodgers
Modifications to a Paul-type quadrupole ion trap mass spectrometer providing optical access to the trapped ion cloud as well as hardware and software for coupling to a table-top IR optical parametric oscillator laser (OPO) are detailed. Critical experimental parameters for infrared multiple photon dissociation (IRMPD) on this instrument are characterized. IRMPD action spectra, collected in the hydrogen-stretching region with this instrument, complemented by spectra in the IR fingerprint region acquired at the FELIX facility, are employed to characterize the structures of the protonated forms of 2-thiouridine, [s2Urd+H]+, and 4-thiouridine, [s4Urd+H]+. The measured spectra are compared with predicted linear IR spectra calculated at the B3LYP/6-311+G(d,p) level of theory to determine the conformers populated in the experiments. This comparison indicates that thiation at the 2- or 4-positions shifts the protonation preference between the 2,4-H tautomer and 4-protonation in opposite directions versus canonical uridine, which displays a roughly equal preference for the 2,4-H tautomer and O4 protonation. As found for canonical uridine, protonation leads to a mixture of conformers exhibiting C2′-endo and C3′-endo sugar puckering with an anti nucleobase orientation being populated for both 2- and 4-thiated uridine.
Analysis of the Thermal Stability of Very Thin Surface Layers of Corrosion Inhibitors by Time-of-Flight Secondary Ion Mass Spectrometry J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-08-17 Janez Kovač, Matjaž Finšgar
The powerful nature of the secondary ion mass spectrometry (SIMS) technique was explored in order to analyse very thin surface layers that were self-assembled on steel material from acidic solution. These surface layers are adsorbed corrosion inhibitors. The SIMS technique proved useful to characterise the molecular structure and homogeneity of thin surface layers in the nanometre range of specific analytes on the metallic substrate. Using SIMS, the thermal stability of these layers was further investigated and the desorption energy at a certain temperature was determined, where special attention was devoted to the method’s static limit. In order to compare, and for certain cases emphasise, the benefits gained by using SIMS in such surface analysis compared with the X-ray photoelectron spectroscopy (XPS) method, the same samples were also analysed by means of the latter. XPS is usually considered to be the most powerful analytical tool in surface analysis studies, but, as shown herein, it has certain limitations compared to SIMS. Finally, the surface topography was investigated by employing atomic force microscopy (AFM) in order to carry out a comprehensive surface analysis.
Performance Enhancements in Differential Ion Mobility Spectrometry-Mass Spectrometry (DMS-MS) by Using a Modified CaptiveSpray Source J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-08-16 Ri Wu, Wei-Jing Wu, Ze Wang, Y.-L. Elaine Wong, Y.-L. Winnie Hung, H. T. Wong, Xiangfeng Chen, T.-W. Dominic Chan
Differential ion mobility spectrometry (DMS) spatially separates ions in the gas phase using the mobility differences of the ions under applied low and high electric fields. The use of DMS as an ion filter (or ion selector) prior to mass spectrometry analysis has been compromised by the limited ion transmission efficiency. This paper reports enhancement of the DMS-MS sensitivity and signal stability using a modified CaptiveSpray™ source. In terms of the ion sampling and transmission efficiency, the modified CaptiveSpray source swept ~ 89% of the ions generated by the tapered capillary through the DMS device (compared to ~ 10% with a conventional microspray source). The signal fluctuation improved from 11.7% (relative standard deviation, RSD) with microspray DMS-MS to 3.6% using CaptiveSpray-DMS-MS. Coupling of LC to DMS-MS via the modified CaptiveSpray source was simple and robust. Using DMS as a noise-filtering device, LC-DMS-MS performed better than conventional LC-MS for analyzing a BSA digest standard. Although LC-DMS-MS had a lower sequence coverage (55%), a higher Mascot score (283) was obtained compared to those of LC-MS (sequence coverage 65%; Mascot score 192) under the same elution conditions. The improvement in the confidence of the search result was attributed to the preferential elimination of noise ions.
The FUNPET—a New Hybrid Ion Funnel-Ion Carpet Atmospheric Pressure Interface for the Simultaneous Transmission of a Broad Mass Range J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-08-15 Benjamin E. Draper, Staci N. Anthony, Martin F. Jarrold
An atmospheric pressure interface transports ions from ambient pressure to the low-pressure environment of a mass spectrometer. A capillary coupled to an ion funnel is widely used. However, conventional ion funnels do little to negate the large amount of energy picked up by high-mass ions from the gas flow through the capillary. There has been little work done on the effects of gas flow on ion transmission, and the previous studies have all been limited to low-mass, low-charge ions. In this work, we account for the effects of gas flow, diffusion, and electric fields (static and oscillating) on ion trajectories and use simulations to design a new hybrid ion funnel-ion carpet (FUNPET) interface that transmits a broad mass range with a single set of instrument conditions. The design incorporates a virtual jet disruptor where pressure buildup and counter flow dissipate the supersonic jet that results from gas flow into the interface. This, and the small exit aperture that can be used with the FUNPET, reduces the gas flow into the next stage of differential pumping. The virtual jet disruptor thermalizes ions with a broad range of masses (1 kDa to 1 GDa), and once thermalized, they are transmitted into next region of the mass spectrometer with low excess kinetic energy. The FUNPET interface is easy to fabricate from flexible printed circuit board and a support frame made by 3D printing. The performance of the interface was evaluated using charge detection mass spectrometry.
Cell-Free Identification of S. cerevisiae Strains by Analysis of Supernatant Using LC-MS J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-08-13 Cathy Muste, Kevin G. Owens
Current literature shows a gap for methods which can identify yeast sub-species (strains or serovars) in samples where there are no viable cells remaining. Presented here is a technique for the analysis of yeast supernatant, including solid phase extraction, data-dependent acquisition liquid chromatography/mass spectrometry (LC-MS), and two chemometric methods to identify and classify yeast strains. Five strains of Saccharomyces cerevisiae were successfully identified in various stages of growth. In addition, peptide/protein identification was performed, without the need for additional data acquisition.
Variation in FPOP Measurements Is Primarily Caused by Poor Peptide Signal Intensity J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-06-25 Niloofar Abolhasani Khaje, Charles K. Mobley, Sandeep K. Misra, Lindsey Miller, Zixuan Li, Evgeny Nudler, Joshua S. Sharp
Fast photochemical oxidation of proteins (FPOP) may be used to characterize changes in protein structure by measuring differences in the apparent rate of peptide oxidation by hydroxyl radicals. The variability between replicates is high for some peptides and limits the statistical power of the technique, even using modern methods controlling variability in radical dose and quenching. Currently, the root cause of this variability has not been systematically explored, and it is unknown if the major source(s) of variability are structural heterogeneity in samples, remaining irreproducibility in FPOP oxidation, or errors in LC-MS quantification of oxidation. In this work, we demonstrate that coefficient of variation of FPOP measurements varies widely at low peptide signal intensity, but stabilizes to ≈ 0.13 at higher peptide signal intensity. We dramatically reduced FPOP variability by increasing the total sample loaded onto the LC column, indicating that the major source of variability in FPOP measurements is the difficulties in quantifying oxidation at low peptide signal intensities. This simple method greatly increases the sensitivity of FPOP structural comparisons, an important step in applying the technique to study subtle conformational changes and protein-ligand interactions.
Directed-Backbone Dissociation Following Bond-Specific Carbon-Sulfur UVPD at 213 nm J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-04-05 Lance E. Talbert, Ryan R. Julian
Ultraviolet photodissociation or UVPD is an increasingly popular option for tandem-mass spectrometry experiments. UVPD can be carried out at many wavelengths, and it is important to understand how the results will be impacted by this choice. Here, we explore the utility of 213 nm photons for initiating bond-selective fragmentation. It is found that bonds previously determined to be labile at 266 nm, including carbon-iodine and sulfur-sulfur bonds, can also be cleaved with high selectivity at 213 nm. In addition, many carbon-sulfur bonds that are not subject to direct dissociation at 266 nm can be selectively fragmented at 213 nm. This capability can be used to site-specifically create alaninyl radicals that direct backbone dissociation at the radical site, creating diagnostic d-ions. Furthermore, the additional carbon-sulfur bond fragmentation capability leads to signature triplets for fragmentation of disulfide bonds. Absorption of amide bonds can enhance dissociation of nearby labile carbon-sulfur bonds and can be used for stochastic backbone fragmentation typical of UVPD experiments at shorter wavelengths. Several potential applications of the bond-selective fragmentation chemistry observed at 213 nm are discussed.
Carbon Nanoparticles and Graphene Nanosheets as MALDI Matrices in Glycomics: a New Approach to Improve Glycan Profiling in Biological Samples J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-06-18 Alireza Banazadeh, Wenjing Peng, Lucas Veillon, Yehia Mechref
Glycomics continues to be a highly dynamic and interesting research area due to the need to comprehensively understand the biological attributes of glycosylation in many important biological functions such as the immune response, cell development, cell differentiation/adhesion, and host-pathogen interactions. Although matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) has proven to be suitable for glycomic profiling studies, there is a need for improved sensitivity in the detection of native glycans, which ionize inefficiently. In this study, we investigated the efficiencies of graphene nanosheets (GNs) and carbon nanoparticles (CNPs) as MALDI matrices and co-matrices in glycan profiling. Our results indicated an enhancement of signal intensity by several orders of magnitude upon using GNs and CNPs in MALDI analysis of N-glycans derived from a variety of biological samples. Interestingly, increasing the amounts of CNPs and GNs improved not only the signal intensities but also prompted in-source decay (ISD) fragmentations, which produced extensive glycosidic and cross-ring cleavages. Our results indicated that the extent of ISD fragmentation could be modulated by CNP and GN concentrations, to obtain MS2 and pseudo-MS3 spectra. The results for glycan profiling in high salt solutions confirmed high salt-tolerance capacities for both CNPs and GNs. Finally, the results showed that by using CNPs and GNs as co-matrices, DHB crystal formation was more homogeneous which improved shot-to-shot reproducibility and sensitivity.
Native Top-Down Mass Spectrometry and Ion Mobility MS for Characterizing the Cobalt and Manganese Metal Binding of α-Synuclein Protein J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-06-27 Piriya Wongkongkathep, Jong Yoon Han, Tae Su Choi, Sheng Yin, Hugh I. Kim, Joseph A. Loo
Structural characterization of intrinsically disordered proteins (IDPs) has been a major challenge in the field of protein science due to limited capabilities to obtain full-length high-resolution structures. Native ESI-MS with top-down MS was utilized to obtain structural features of protein-ligand binding for the Parkinson’s disease-related protein, α-synuclein (αSyn), which is natively unstructured. Binding of heavy metals has been implicated in the accelerated formation of αSyn aggregation. Using high-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry, native top-down MS with various fragmentation methods, including electron capture dissociation (ECD), collisional activated dissociation (CAD), and multistage tandem MS (MS3), deduced the binding sites of cobalt and manganese to the C-terminal region of the protein. Ion mobility MS (IM-MS) revealed a collapse toward compacted states of αSyn upon metal binding. The combination of native top-down MS and IM-MS provides structural information of protein-ligand interactions for intrinsically disordered proteins.
Screening and Identification of the Metabolites of Water Extracts of Raw and Honey-Processed Astragalus in Rat Urine Based on UHPLC/ESI-Q-TOF-MS and Multivariate Statistical Analysis J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-06-21 Jing Huang, Hongyuan Chen, Chanyi Li, Wuping Liu, Wenjie Ma, Wen Rui
Radix Astragali is a famous traditional Chinese medicine and honey-processed Astragalus is a product of Radix Astragali acquired by honey-processing. These two products are widely utilized to treat various diseases. In this study, we screened bioactive components and metabolites of raw and honey-processed Astragalus in rat urine by ultra-performance liquid chromatography equipped with electrospray ionization/quadrupole time-of-flight mass spectrometry (UHPLC/ESI-Q-TOF-MS) combined with multivariate statistical analysis. In total, 62 compounds, including 7 parent compounds and 55 metabolites, were detected and 11 metabolites were characterized for the first time. The identified metabolites indicated that the metabolic reactions of Astragalus in rats included hydroxylation, glucuronidation, deglucosidation, monomethylation, demethylation, sulfation, hydrogenation, and dehydroxylation. The metabolic pathways of raw and honey-processed Astragalus in rat urine also were clarified. Through multivariate statistical analysis of the data of the raw and honey-processed Astragalus groups, we found that 20 compounds were differential components and that 1 metabolite only existed in the honey-processed Astragalus group. The differences in these ingredients between these two groups might provide the basis for interpreting the biologic activity differences in traditional Chinese medicine treatments.
Epitope Ligand Binding Sites of Blood Group Oligosaccharides in Lectins Revealed by Pressure-Assisted Proteolytic Excision Affinity Mass Spectrometry J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-06-25 Yannick Baschung, Loredana Lupu, Adrian Moise, Michael Glocker, Stephan Rawer, Alexander Lazarev, Michael Przybylski
Affinity mass spectrometry using selective proteolytic excision and extraction combined with MALDI and ESI mass spectrometry has been applied to the identification of epitope binding sites of lactose, GalNac, and blood group oligosaccharides in two blood group-specific lectins, human galectin-3 and glycine max lectin. The epitope peptides identified comprise all essential amino acids involved in carbohydrate recognition, in complete agreement with available X-ray structures. Tryptic and chymotryptic digestion of lectins for proteolytic extraction/excision-MS was substantially improved by pressure-enhanced digestion using an automated Barocycler procedure (40 kpsi). Both previously established immobilization on affinity microcolumns using divinyl sulfone and coupling of a specific peptide glycoprobe to the gold surface of a biosensor chip were successfully employed for proteolytic excision and extraction of carbohydrate epitopes and affinity measurements. The identified epitope peptides could be differentiated according to the carbohydrate employed, thus demonstrating the specificity of the mass spectrometric approach. The specificities of the epitope ligands for individual carbohydrates were further ascertained by affinity studies using synthetic peptide ligands with immobilized carbohydrates. Binding affinities of the synthetic ligand peptides to lactose, in comparison to the intact full-length lectins, were determined by surface acoustic wave (SAW) biosensor analysis and provided micromolar KD values for the intact lectins, in agreement with results of previous ITC and SPR studies. Binding affinities of the epitope peptides were approximately two orders of magnitude lower, consistent with their smaller size and assembled arrangement in the carbohydrate recognition domains.
Spectroscopic Identification of the Carbyne Hydride Structure of the Dehydrogenation Product of Methane Activation by Osmium Cations J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-04-09 P. B. Armentrout, Stach E. J. Kuijpers, Olga V. Lushchikova, Randy L. Hightower, Georgia C. Boles, Joost M. Bakker
The present work explores the structures of species formed by dehydrogenation of methane (CH4) and perdeuterated methane (CD4) by the 5d transition metal cation osmium (Os+). Using infrared multiple photon dissociation (IRMPD) action spectroscopy and density functional theory (DFT), the structures of the [Os,C,2H]+ and [Os,C,2D]+ products are explored. This study complements previous work on the related species formed by dehydrogenation of methane by four other 5d transition metal cations (M+ = Ta+, W+, Ir+, and Pt+). Osmium cations are formed in a laser ablation source, react with methane pulsed into a reaction channel downstream, and the resulting products spectroscopically characterized through photofragmentation using the Free-Electron Laser for IntraCavity Experiments (FELICE) in the 300–1800 cm−1 range. Photofragmentation was monitored by the loss of H2/D2. Comparison of the experimental spectra and DFT calculated spectra leads to identification of the ground state carbyne hydride, HOsCH+ (2A′) as the species formed, as previously postulated theoretically. Further, a full description of the systematic spectroscopic shifts observed for deuterium labeling of these complexes, some of the smallest systems to be studied using IRMPD action spectroscopy, is achieved. A full rotational contour analysis explains the observed linewidths as well as the observation of doublet structures in several bands, consistent with previous observations for HIrCH+ (2A′).
Spontaneous Isomerization of Peptide Cation Radicals Following Electron Transfer Dissociation Revealed by UV-Vis Photodissociation Action Spectroscopy J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-01-16 Naruaki Imaoka, Camille Houferak, Megan P. Murphy, Huong T. H. Nguyen, Andy Dang, František Tureček
Peptide cation radicals of the z-type were produced by electron transfer dissociation (ETD) of peptide dications and studied by UV-Vis photodissociation (UVPD) action spectroscopy. Cation radicals containing the Asp (D), Asn (N), Glu (E), and Gln (Q) residues were found to spontaneously isomerize by hydrogen atom migrations upon ETD. Canonical N-terminal [z4 + H]+● fragment ion-radicals of the R-C●H-CONH- type, initially formed by N−Cα bond cleavage, were found to be minor components of the stable ion fraction. Vibronically broadened UV-Vis absorption spectra were calculated by time-dependent density functional theory for several [●DAAR + H]+ isomers and used to assign structures to the action spectra. The potential energy surface of [●DAAR + H]+ isomers was mapped by ab initio and density functional theory calculations that revealed multiple isomerization pathways by hydrogen atom migrations. The transition-state energies for the isomerizations were found to be lower than the dissociation thresholds, accounting for the isomerization in non-dissociating ions. The facile isomerization in [●XAAR + H]+ ions (X = D, N, E, and Q) was attributed to low-energy intermediates having the radical defect in the side chain that can promote hydrogen migration along backbone Cα positions. A similar side-chain mediated mechanism is suggested for the facile intermolecular hydrogen migration between the c- and [z + H]●-ETD fragments containing Asp, Asn, Glu, and Gln residues.
Disambiguation of Isomeric Procyanidins with Cyclic B-Type and Non-cyclic A-Type Structures from Wine and Peanut Skin with HPLC-HDX-HRMS/MS J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-08-10 Edoardo Longo, Fabrizio Rossetti, Vakare Merkyte, Emanuele Boselli
Hydrogen/deuterium exchange coupled with high-resolution mass spectrometry was successfully applied for the identification of A-type tetrameric, pentameric, and hexameric procyanidins in peanut skin. This extended a previous study on isomeric cyclic B-type unconventional tetramer, pentamer, and hexamer procyanidins found in wine and cranberries. Not only had the method successfully identified the procyanidins with a single A-linkage (e.g., tetrameric m/z 1153.2608) by means of distinguishing them from their isomeric cyclic B-type analogues, but this method also worked for procyanidins with two or more A-linkages (such as the tetrameric m/z 1151.2452). As a further consequence, B-type cyclic pentamers and hexamers in wine have been elucidated with hydrogen/deuterium exchange (HDX) for the first time.
Introducing a Cell-Free Approach for the Identification of Brewing Yeast ( Saccharomyces cerevisiae ) Strains Using MALDI-TOF MS J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-08-07 Elsa Gorre, Cathy Muste, Kevin G. Owens
Matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry (TOF MS) is now accepted as a quick, easy-to-use, cost-effective, and accurate technique for the identification of microorganisms. However, the successful identification of microorganisms is dependent upon careful attention to factors such as growth conditions, extraction methods, mass spectral data collection, and data analysis procedures. Currently, most microorganism identification has been limited to the species level, and only a limited number of publications have been successful in achieving strain-level identification. In this work, a “cell-free” approach is introduced where peptide analytes secreted by several Saccharomyces cerevisiae strains during their growth period are analyzed. The analysis of the cell supernatant generates mass spectral patterns that are specific to each strain. The patterns generated in combination with a robust data analysis workflow using the open-source programs MALDIquant and Mass-Up allows for strain-level identification of S. cerevisiae. The cell-free approach using the yeast supernatant to accurately identify yeast strains is presented here as a proof of concept.
Solvent Mediation of Peptide Conformations: Polyproline Structures in Water, Methanol, Ethanol, and 1-Propanol as Determined by Ion Mobility Spectrometry-Mass Spectrometry J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-08-01 Tarick J. El-Baba, Daniel R. Fuller, David A. Hales, David H. Russell, David E. Clemmer
Ion mobility spectrometry and circular dichroism spectroscopy are used to examine the populations of the small model peptide, polyproline-13 in water, methanol, ethanol, and 1-propanol over a range of solution temperatures (from 288 to 318 K). At low temperatures, the less-polar solvents (1-propanol and ethanol) favor the all-cis polyproline I helix (PPI); as the temperature is increased, the trans-configured polyproline II helix (PPII) is formed. In polar solvents (methanol and water), PPII is favored at all temperatures. From the experimental data, we determine the relative stabilities of the eight structures in methanol, ethanol, and 1-propanol, as well as four in water, all with respect to PPII. Although these conformers show relatively small differences in free energies, substantial variability is observed in the enthalpies and entropies across the structures and solvents. This requires that enthalpies and entropies be highly correlated: in 1-propanol, cis-configured PPI conformations are energetically favorable but entropically disfavored. In more polar solvents, PPI is enthalpically less favorable and entropy favors trans-configured forms. While either ΔH0 or ΔS0 can favor different structures, no conformation in any solvent is simultaneously energetically and entropically stabilized. These data present a rare opportunity to examine the origin of conformational stability.
Multi-electrode Harmonized Kingdon Traps J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-08-01 Evgeny Nikolaev, Mikhail Sudakov, Gleb Vladimirov, Luis Fernando Velásquez-García, Petr Borisovets, Anastasia Fursova
Based on the analysis of the results of the study of various designs of multi-electrode harmonized Kingdon traps, we propose a new type of trap with two merged internal electrodes that has the ability to capture and accumulate ions formed inside. We also investigated the influence of inaccuracies in the manufacture of the electrodes on the field inside such trap. The four-electrode trap, which actually degenerates into a two-electrode device with traces of two other electrodes present at the ends of the internal electrodes (their splitting) has been found as the less sensitive to inaccuracies caused by manufacturing and cutting the ends of trap electrodes. We show that a mass spectrometer with a relatively high resolving power can be created on the basis of such a trap. The creation of the traps requires the manufacture of complex electrodes with demanded accuracy of their surfaces. This becomes possible with the advent of 3D printers.
3-Hydroxy-2-Nitrobenzoic Acid as a MALDI Matrix for In-Source Decay and Evaluation of the Isomers J. Am. Soc. Mass Spectrom. (IF 2.869) Pub Date : 2018-07-30 Yuko Fukuyama, Shunsuke Izumi, Koichi Tanaka
In in-source decay (ISD) in matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS), 1,5-diaminonaphthalene (1,5-DAN) is a most frequently used matrix probably due to the highly sensitive detection of fragment ions. 1,5-DAN is a reducing matrix generating c- and z-series ions by N–Cα bond cleavage. However, it is difficult for reducing matrices to distinguish leucine and isoleucine, and generate c(n-1)-series ions owing to proline (Pro) at residues n. Oxidizing matrices providing a- and x-series ions accompanied by d-series ions by Cα–C bond cleavage solve the problem, but their sensitivity of the ISD fragment ions has been lower than reducing matrices such as 1,5-DAN. Recently, 3-hydroxy-4-nitrobenzoic acid (3H4NBA) had been reported as an oxidizing matrix generating a-series ions with higher intensity compared with conventional oxidizing matrices such as 5-nitrosalicylic acid, but a little lower intensity compared with 1,5-DAN (Anal Chem 88, 8058–8063, 2016). In this study, 3H4NBA isomers (2H3NBA, 2H4NBA, 2H5NBA, 2H6NBA, 3H2NBA, 3H5NBA, 4H2NBA, 4H3NBA, 5H2NBA, and 3H4NBA) were evaluated. All the isomers generated a-series ions accompanied by d-series ions, wherein 3H2NBA, 3H5NBA, 4H2NBA, 4H3NBA, and 5H2NBA were first confirmed as oxidizing matrices for ISD. Among the isomers, 3H2NBA and 4H3NBA generated a-series ions with higher peak intensity compared with 3H4NBA for several peptides. Especially, 3H2NBA generated a-series ions with almost the same or higher intensity, and clearly higher peak resolution compared with c-series ions using 1,5-DAN in several cases. 3H2NBA was expected to contribute to ISD analyses in MALDI-MS as one of the most effective oxidizing matrices.
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