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  • Deep Intact Proteoform Characterization in Human Cell Lysate Using High-pH and Low-pH Reversed-Phase Liquid Chromatography
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2019-11-21
    Dahang Yu, Zhe Wang, Kellye A. Cupp-Sutton, Xiaowen Liu, Si Wu

    Abstract Post-translational modifications (PTMs) play critical roles in biological processes and have significant effects on the structures and dynamics of proteins. Top-down proteomics methods were developed for and applied to the study of intact proteins and their PTMs in human samples. However, the large dynamic range and complexity of human samples makes the study of human proteins challenging. To address these challenges, we developed a 2D pH RP/RPLC-MS/MS technique that fuses high-resolution separation and intact protein characterization to study the human proteins in HeLa cell lysate. Our results provide a deep coverage of soluble proteins in human cancer cells. Compared to 225 proteoforms from 124 proteins identified when 1D separation was used, 2778 proteoforms from 628 proteins were detected and characterized using our 2D separation method. Many proteoforms with critically functional PTMs including phosphorylation were characterized. Additionally, we present the first detection of intact human GcvH proteoforms with rare modifications such as octanoylation and lipoylation. Overall, the increase in the number of proteoforms identified using 2DLC separation is largely due to the reduction in sample complexity through improved separation resolution, which enables the detection of low-abundance PTM-modified proteoforms. We demonstrate here that 2D pH RP/RPLC is an effective technique to analyze complex protein samples using top-down proteomics.

    更新日期:2020-01-17
  • Quantitation of Single and Combinatorial Histone Modifications by Integrated Chromatography of Bottom-up Peptides and Middle-down Polypeptide Tails
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2019-09-11
    Kevin A. Janssen, Mariel Coradin, Congcong Lu, Simone Sidoli, Benjamin A. Garcia

    The analysis of histone post-translational modifications (PTMs) by mass spectrometry (MS) has been critical to the advancement of the field of epigenetics. The most sensitive and accurate workflow is similar to the canonical proteomics analysis workflow (bottom-up MS), where histones are digested into short peptides (4-20 aa) and quantitated in extracted ion chromatograms. However, this limits the ability to detect even very common co-occurrences of modifications on histone proteins, preventing biological interpretation of PTM crosstalk. By digesting with GluC rather than trypsin, it is possible to produce long polypeptides corresponding to intact histone N-terminal tails (50-60 aa), where most modifications reside. This middle-down MS approach is used to study distant PTM co-existence. However, the most sensitive middle-down workflow uses weak cation exchange-hydrophilic interaction chromatography (WCX-HILIC), which is less robust than conventional reversed-phase chromatography. Additionally, since the buffer systems for middle-down and bottom-up proteomics differ substantially, it is cumbersome to toggle back and forth between both experimental setups on the same LC system. Here, we present a new workflow using porous graphitic carbon (PGC) as a stationary phase for histone analysis where bottom-up and middle-down sized histone peptides can be analyzed simultaneously using the same reversed-phase buffer setup. By using this protocol for middle-down sized peptides, we identified 406 uniquely modified intact histone tails and achieved a correlation of 0.85 between PGC and WCX-HILIC LC methods. Together, our method facilitates the analysis of single and combinatorial histone PTMs with much simpler applicability for conventional proteomics labs than the state-of-the-art middle-down MS.

    更新日期:2020-01-17
  • High-Throughput Quantitative Top-Down Proteomics: Histone H4
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2019-11-18
    Matthew V. Holt, Tao Wang, Nicolas L. Young

    Proteins physiologically exist as “proteoforms” that arise from one gene and acquire additional function by post-translational modifications (PTM). When multiple PTMs coexist on single protein molecules, top-down proteomics becomes the only feasible method of characterization; however, most top-down methods have limited quantitative capacity and insufficient throughput to truly address proteoform biology. Here we demonstrate that top-down proteomics can be quantitative, reproducible, sensitive, and high throughput. The proteoforms of histone H4 are well studied both as a challenging proteoform identification problem and due to their essential role in the regulation of all eukaryotic DNA-templated processes. Much of histone H4’s function is obfuscated from prevailing methods due to combinatorial mechanisms. Starting from cells or tissues, after an optimized protein purification process, the H4 proteoforms are physically separated by on-line C3 chromatography, narrowly isolated in MS1 and sequenced with ETD fragmentation. We achieve more than 30 replicates from a single 35-mm tissue culture dish by loading 55 ng of H4 on column. Parallelization and automation yield a sustained throughput of 12 replicates per day. We achieve reproducible quantitation (average biological Pearson correlations of 0.89) of hundreds of proteoforms (about 200–300) over almost six orders of magnitude and an estimated LLoQ of 0.001% abundance. We demonstrate the capacity of the method to precisely measure well-established changes with sodium butyrate treatment of SUM159 cells. We show that the data produced by a quantitative top-down method can be amenable to parametric statistical comparisons and is capable of delineating relevant biological changes at the full proteoform level.

    更新日期:2020-01-17
  • Comprehensive Characterization of the Recombinant Catalytic Subunit of cAMP-Dependent Protein Kinase by Top-Down Mass Spectrometry
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2019-12-02
    Zhijie Wu, Yutong Jin, Bifan Chen, Morgan K. Gugger, Chance L. Wilkinson-Johnson, Timothy N. Tiambeng, Song Jin, Ying Ge

    Reversible phosphorylation plays critical roles in cell growth, division, and signal transduction. Kinases which catalyze the transfer of γ-phosphate groups of nucleotide triphosphates to their substrates are central to the regulation of protein phosphorylation and are therefore important therapeutic targets. Top-down mass spectrometry (MS) presents unique opportunities to study protein kinases owing to its capabilities in comprehensive characterization of proteoforms that arise from alternative splicing, sequence variations, and post-translational modifications. Here, for the first time, we developed a top-down MS method to characterize the catalytic subunit (C-subunit) of an important kinase, cAMP-dependent protein kinase (PKA). The recombinant PKA C-subunit was expressed in Escherichia coli and successfully purified via his-tag affinity purification. By intact mass analysis with high resolution and high accuracy, four different proteoforms of the affinity-purified PKA C-subunit were detected, and the most abundant proteoform was found containing seven phosphorylations with the removal of N-terminal methionine. Subsequently, the seven phosphorylation sites of the most abundant PKA C-subunit proteoform were characterized simultaneously using tandem MS methods. Four sites were unambiguously identified as Ser10, Ser11, Ser18, and Ser30, and the remaining phosphorylation sites were localized to Ser2/Ser3, Ser358/Thr368, and Thr[215-224]Tyr in the PKA C-subunit sequence with a 20mer 6xHis-tag added at the N-terminus. Interestingly, four of these seven phosphorylation sites were located at the 6xHis-tag. Furthermore, we have performed dephosphorylation reaction by Lambda protein phosphatase and showed that all phosphorylations of the recombinant PKA C-subunit phosphoproteoforms were removed by this phosphatase.

    更新日期:2020-01-17
  • Discovery of Missing Methylation Sites on Endogenous Peptides of Human Cell Lines
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2019-08-19
    Xin Yan, Lingjun Li, Chenxi Jia

    Abstract Methylation of proteins has considerable impacts on physiological processes including signal transduction, DNA damage repair, transcriptional regulation, gene activation, and inhibition of gene expression. However, the traditional proteomics-based approach suffers from limited identification rates of these critical methylation sites on endogenous peptides. In this work, a peptidomics-based workflow was established to discover and characterize the global methylome of endogenous peptides in human cells. The reliability of our strategy was validated by methyl-SILAC labeling, resulting in 83% true-positive identifications in the HeLa cell line. We applied this approach to seven human cell lines, and 700 methylated forms on 646 putative methylation sites were identified in total, with over 61% of the methylation sites being newly identified. This study provides a complementary strategy for a traditional proteomics-based approach that enables identification of missing methylation sites and creates a first methylome draft of endogenous peptides of human cell lines, offering a valuable resource for in-depth studies of biological functions of methylated endogenous peptides.

    更新日期:2020-01-17
  • One-Pot Quantitative Top- and Middle-Down Analysis of GluC-Digested Histone H4
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2019-05-30
    Matthew V. Holt, Tao Wang, Nicolas L. Young

    Histone post-translational modifications (PTMs) have been intensively investigated due to their essential function in eukaryotic genome regulation. Histone modifications have been effectively studied using modified bottom-up proteomics approaches; however, the methods often do not capture single-molecule combinations of PTMs (proteoforms) that mediate known and expected biochemical mechanisms. Both middle-down mass spectrometry (MS) and top-down MS quantitation of H4 proteoforms present viable access to this important information. Histone H4 middle-down has previously avoided GluC digestion due to complex digestion products and interferences; however, the common AspN digestion cleaves at amino acid 23, disconnecting K31ac from other PTMs. Here, we demonstrate the effective use of GluC-based middle-down quantitation and compare it to top-down-based quantitation of proteoforms. Despite potential interferences in the m/z space, the proteoforms arising from all three GluC products (E52, E53, and E63) and intact H4 are chromatographically resolved and successfully analyzed in a single LC–MS analysis. Quantitative results and associated analytical metrics are compared between the different analytes of a single sample digested to different extents to reveal general concordance as well as the relative biases and complementarity of each approach. There is moderate proteoform discordance between digestion products (e.g., E52 and E53); however, each digestion product exhibits high concordance, regardless of digestion time. Under the conditions used, the GluC products are better chromatographically resolved yet show greater variance than the top-down quantitation that are more extensively sampled for MS2. GluC-based middle-down of H4 is thus viable. Both top-down and middle-down approaches have comparable quantitation capacity and are complementary.

    更新日期:2020-01-17
  • Finding the Sweet Spot in ERLIC Mobile Phase for Simultaneous Enrichment of N-Glyco and Phosphopeptides
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2019-07-08
    Yusi Cui, Ka Yang, Dylan Nicholas Tabang, Junfeng Huang, Weiping Tang, Lingjun Li

    Simultaneous enrichment of glyco- and phosphopeptides will benefit the studies of biological processes regulated by these posttranslational modifications (PTMs). It will also reveal potential crosstalk between these two ubiquitous PTMs. Unlike custom-designed multifunctional solid phase extraction (SPE) materials, operating strong anion exchange (SAX) resin in electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) mode provides a readily available strategy to analytical labs for enrichment of these PTMs for subsequent mass spectrometry (MS)-based characterization. However, the choice of mobile phase has largely relied on empirical rules from hydrophilic interaction chromatography (HILIC) or ion-exchange chromatography (IEX) without further optimization and adjustments. In this study, ten mobile phase compositions of ERLIC were systematically compared; the impact of multiple factors including organic phase proportion, ion pairing reagent, pH, and salt on the retention of glycosylated and phosphorylated peptides was evaluated. This study demonstrated good enrichment of glyco- and phosphopeptides from the nonmodified peptides in a complex tryptic digest. Moreover, the enriched glyco- and phosphopeptides elute in different fractions by orthogonal retention mechanisms of hydrophilic interaction and electrostatic interaction in ERLIC, maximizing the LC-MS identification of each PTM. The optimized mobile phase can be adapted to the ERLIC HPLC system, where the high resolution in separating multiple PTMs will benefit large-scale MS-based PTM profiling and in-depth characterization.

    更新日期:2020-01-17
  • Top-down Mass Spectrometry of Sarcomeric Protein Post-translational Modifications from Non-human Primate Skeletal Muscle
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2019-03-04
    Yutong Jin, Gary M. Diffee, Ricki J. Colman, Rozalyn M. Anderson, Ying Ge

    Abstract Sarcomeric proteins, including myofilament and Z-disk proteins, play critical roles in regulating muscle contractile properties. A variety of isoforms and post-translational modifications (PTMs) of sarcomeric proteins have been shown to be associated with modulation of muscle functions and the occurrence of muscle diseases. Non-human primates (NHPs) are excellent research models for sarcopenia, a disease associated with alterations in sarcomeric proteins, due to their marked similarities to humans. However, the sarcomeric proteins in NHP skeletal muscle have not been well characterized. To gain a deeper understanding of sarcomeric proteins in NHP skeletal muscle, we employed top-down mass spectrometry (MS) to conduct a comprehensive analysis on isoforms and PTMs of sarcomeric proteins in rhesus macaque skeletal muscle. We identified 23 protein isoforms with 46 proteoforms of sarcomeric proteins, including 6 isoforms with 18 proteoforms from fast skeletal troponin T. Particularly, for the first time, a novel PDZ/LIM domain protein isoform, PDLIM7, was characterized with a newly identified protein sequence. Moreover, we also identified multiple PTMs on these proteins, including deamidation, methylation, acetylation, tri-methylation, phosphorylation, and S-glutathionylation. Most PTM sites were localized, including Asn13 deamidation on MLC-2S; His73 methylation on αactin; N-terminal acetylation on most identified proteins; N-terminal tri-methylation on MLC-1S, MLC-1F, MLC-2S, and MLC-2F; Ser14 phosphorylation on MLC-2S; and Ser15 and Ser16 phosphorylation on MLC-2F. In summary, a comprehensive characterization of sarcomeric proteins including multiple isoforms and PTMs in NHP skeletal muscle was achieved by analyzing intact proteins in the top-down MS approach.

    更新日期:2020-01-17
  • The Addition of Polar Organic Solvent Vapors During the Analysis of Proteins by DESI-MS
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2019-11-22
    Roshan Javanshad, Tara L. Maser, Elahe Honarvar, Andre R. Venter

    Exposure of electrospray droplets to organic vapors was shown to dramatically reduce alkali-metal adduction on protein ions and shift protein charge states. Since DESI-MS is affected by similar adduct species as ESI-MS and shares similar ionization mechanisms, polar organic vapor additives should likewise also improve the DESI-MS analysis of proteins. Here the DESI spray was exposed to a variety of polar organic vapor additives. Head space vapors of polar organic solvents were entrained in nitrogen gas and delivered to the atmosphere inside a semi-enclosed plastic enclosure surrounding the spray plume. The vapors of acetone, acetonitrile, ethyl acetate, methanol, and water were investigated. Vapor dependent effects were observed with respect to changes in protein charge state distributions and signal intensities. With ethyl acetate vapor addition, the signal intensities of all proteins investigated were significantly increased, including proteins larger than 25 kDa such as carbonic anhydrase II and bovine serum albumin.

    更新日期:2019-11-26
  • Backbone Cleavages of Protonated Peptoids upon Collision-Induced Dissociation: Competitive and Consecutive B-Y and A 1 -Y X Reactions
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2019-11-21
    Emilie Halin, Sébastien Hoyas, Vincent Lemaur, Julien De Winter, Sophie Laurent, Michael D. Connolly, Ronald N. Zuckermann, Jérôme Cornil, Pascal Gerbaux

    Mass spectrometric techniques and more particularly collision-induced dissociation (CID) experiments represent a powerful method for the determination of the primary sequence of (bio)molecules. However, the knowledge of the ion fragmentation patterns say the dissociation reaction mechanisms is a prerequisite to reconstitute the sequence based on fragment ions. Previous papers proposed that protonated peptoids dissociate following an oxazolone-ring mechanism starting from the O-protonation species and leading to high mass Y sequence ions. Here we revisit this backbone cleavage mechanism by performing CID and ion mobility experiments, together with computational chemistry, on tailor-made peptoids. We demonstrated that the B/Y cleavages of collisionally activated O-protonated peptoids must involve the amide nitrogen protonated structures as the dissociating species, mimicking the CID behavior of protonated peptides. Upon the nucleophilic attack of the oxygen atom of the N-terminal adjacent carbonyl group on the carbonyl carbon atom of the protonated amide, the peptoid ions directly dissociate to form an ion-neutral complex associating an oxazolone ion to the neutral truncated peptoid residue. Dissociation of the ion/neutral complex predominantly produces Y ions due to the high proton affinity of the secondary amide function characteristic of truncated peptoids. Whereas the production of Yx ions from acetylated peptoids also involves the B/Y pathway, the observation of abundant Yx ions from non-acetylated peptoid ions is shown in the present study to arise from an A1-Yx mechanism. The consecutive and competitive characters of the A1-Yx and the B/Y mechanisms are also investigated by drift time-aligned CID experiments.

    更新日期:2019-11-22
  • A First Principle Model of Differential Ion Mobility: the Effect of Ion-Solvent Clustering
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2019-11-21
    Alexander Haack, Jeff Crouse, Femke-Jutta Schlüter, Thorsten Benter, W. Scott Hopkins

    The use of differential mobility spectrometry (DMS) as a separation tool prior to mass analysis has increased in popularity over the years. However, the fundamental principles behind the difference between high- and low-field mobility is still a matter of debate—especially regarding the strong impact of solvent molecules added to the gas phase in chemically modified DMS environments. In this contribution, we aim to present a thorough model for the determination of the ion mobility over a wide range of field strengths and subsequent calculation of DMS dispersion plots. Our model relies on first principle calculations only, incorporating the modeling of the “hard-sphere” mobility, the change in CCS with field strength, and the degree of clustering of solvent molecules to the ion. We show that all three factors have to be taken into account to qualitatively predict dispersion plots. In particular, type A behavior (i.e., strong clustering) in DMS can only be explained by a significant change of the mean cluster size with field strengths. The fact that our model correctly predicts trends between differently strong binding solvents, as well as the solvent concentration and the background gas temperature, highlights the importance of clustering for differential mobility.

    更新日期:2019-11-22
  • Compositional Analysis of Heavy Petroleum Distillates by Comprehensive Two-dimensional Gas Chromatography, Field Ionization and High-resolution Mass Spectrometry
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2019-11-18
    Kuangnan Qian, Frank C. Wang

    We report recent progresses of combining comprehensive two-dimensional gas chromatography (2DGC or GC × GC) separation, field ionization (FI), and time-of-flight mass spectrometry (TOF MS) for the detailed analysis of vacuum gas oil distillation (VGO) cuts. 2DGC separates petroleum molecules by the combination of boiling point and polarity. FI generates molecule ions-only mass spectra. TOF MS allows accurate mass analysis of hydrocarbon molecules. A new data analysis strategy is implemented for compositional analysis. First, all masses were separated into nominal mass classes. Since petroleum homologues have unique Kendrick mass defects (KMD), KMD plots were generated for easy recognition of homologues series within each nominal mass class. Finally, KMD windows were imposed for complete resolution of petroleum molecules. Using this approach, a total of 16 hydrocarbon types, 14 sulfur types, and their carbon number distributions were determined in the three VGO distillation cuts. Two series of geological biomarkers were also revealed by the analysis.

    更新日期:2019-11-18
  • A Decoupled Automation Platform for Hydrogen/Deuterium Exchange Mass Spectrometry Experiments
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2019-11-13
    Alfonso Espada, Ruben Haro, Jesus Castañon, Cristina Sayago, Francisco Perez-Cozar, Leticia Cano, Pablo Redero, Manuel Molina-Martin, Howard Broughton, Ryan E. Stites, Bruce D. Pascal, Patrick R. Griffin, Jeffrey A. Dodge, Michael J. Chalmers

    Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a biophysical technique well suited to the characterization of protein dynamics and protein–ligand interactions. In order to accurately define the rate of exchange, HDX experiments require the repeated measure of deuterium incorporation into the target protein across a range of time points. Accordingly, the HDX-MS experiment is well suited to automation, and a number of automated systems for HDX-MS have been developed. The most widely utilized platforms all operate an integrated design, where robotic liquid handling is interfaced directly with a mass spectrometer. With integrated designs, the exchange samples are prepared and injected into the LC-MS following a “real-time” serial workflow. Here we describe a new HDX-MS platform that is comprised of two complementary pieces of automation that disconnect the sample preparation from the LC-MS analysis. For preparation, a plate-based automation system is used to prepare samples in parallel, followed by immediate freezing and storage. A second piece of automation has been constructed to perform the thawing and LC-MS analysis of frozen samples in a serial mode and has been optimized to maximize the duty cycle of the mass spectrometer. The decoupled configuration described here reduces experiment time, significantly improves capacity, and improves the flexibility of the platform when compared with a fully integrated system.

    更新日期:2019-11-14
  • Hydrogen-Deuterium Exchange and Hydroxyl Radical Footprinting for Mapping Hydrophobic Interactions of Human Bromodomain with a Small Molecule Inhibitor
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2019-11-12
    Ke Sherry Li, Elizabeth T. Schaper Bergman, Brett R. Beno, Richard Y.-C. Huang, Ekaterina Deyanova, Guodong Chen, Michael L. Gross

    Mass spectrometry (MS)–based protein footprinting, a valuable structural tool in mapping protein-ligand interaction, has been extensively applied to protein-protein complexes, showing success in mapping large interfaces. Here, we utilized an integrated footprinting strategy incorporating both hydrogen-deuterium exchange (HDX) and hydroxyl radical footprinting (i.e., fast photochemical oxidation of proteins (FPOP)) for molecular-level characterization of the interaction of human bromodomain-containing protein 4 (BRD4) with a hydrophobic benzodiazepine inhibitor. HDX does not provide strong evidence for the location of the binding interface, possibly because the shielding of solvent by the small molecule is not large. Instead, HDX suggests that BRD4 appears to be stabilized by showing a modest decrease in dynamics caused by binding. In contrast, FPOP points to a critical binding region in the hydrophobic cavity, also identified by crystallography, and, therefore, exhibits higher sensitivity than HDX in mapping the interaction of BRD4 with compound 1. In the absence or under low concentrations of the radical scavenger, FPOP modifications on Met residues show significant differences that reflect the minor change in protein conformation. This problem can be avoided by using a sufficient amount of proper scavenger, as suggested by the FPOP kinetics directed by a dosimeter of the hydroxyl radical.

    更新日期:2019-11-13
  • Enhanced Multiplexing in Fourier Transform Charge Detection Mass Spectrometry by Decoupling Ion Frequency from Mass to Charge Ratio
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2019-11-12
    Conner C. Harper, Evan R. Williams

    Weighing single ions with charge detection mass spectrometry (CDMS) makes it possible to obtain the masses of molecules of essentially unlimited size even in highly heterogeneous samples, but producing a mass histogram that is representative of all of the components in a mixture requires substantial measurement time. Multiple ions can be trapped to reduce analysis time but ion signals can overlap. To determine the maximum gains in analysis speed possible with current instrumentation with multiple ion trapping, simulations calculating the frequency and overlap rate of ions with different mass, charge, and energy ranges were performed. For an analyte with a broad mass distribution, such as long chain polyethylene glycol (PEG, 8 MDa), gains in analysis speed of up to 160 times that of prior CDMS experiments are possible. For signals from homogeneous samples, ions with the same m/z have frequencies that overlap and interfere, reducing the effectiveness of multiplexing in experiments where ions have the same energy per charge. We show that by maximizing the decoupling of ion m/z from frequency using a broad range of ion energies, the rate of signal overlap is significantly reduced making it possible to trap more ions. Under optimum decoupling conditions, a measurement speed nearly 50 times greater than that of prior CDMS experiments is possible for RuBisCO (517 kDa). The reduction in overlap due to decoupling also results in more accurate quantitation in samples that contain multiple analytes with different concentrations.

    更新日期:2019-11-13
  • Ion Manipulation in Open Air Using 3D-Printed Electrodes
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2019-11-11
    Kiran Iyer, Brett M. Marsh, Grace O. Capek, Robert L. Schrader, Shane Tichy, R. Graham Cooks

    Ambient ionization techniques provide a way to sample materials via creation of ions in the air. However, transferring and focusing of these ions is typically done in the reduced pressure environment of the mass spectrometer. Spray-based ambient ionization sources require relatively large distances between the source and mass spectrometer inlet for effective desolvation, resulting in a small fraction of the ions being collected. To increase the efficiency of ion transfer from atmosphere to vacuum, 3D-printed focusing devices made of conductive carbon nanotube doped polymers have been designed and evaluated for ion focusing in air. Three main classes of electrodes are considered: (i) conic section electrodes (conical, ellipsoidal, and cylindrical), (ii) simple conductive and non-conductive apertures, and (iii) electrodes with complex geometries (straight, chicane, and curved). Simulations of ion trajectories performed using the statistical diffusion simulation (SDS) model in SIMION showed a measure of agreement with experiment. Cross-sectional images of ion beams were captured using an ion detecting charge-coupled device (IonCCD). After optimization, the best arrangements of electrodes were coupled to an Agilent Ultivo triple quadrupole to record mass spectra. Observations suggest that electrode geometry strongly influences ion trajectories in air. Non-conductive electrodes also assisted in focusing, due to charge buildup from ion deposition. We also observed minimal spreading of the ion packet after exiting the focusing electrodes indicating that atmospheric collisions do not reduce collimation of the beam. The study suggests that high pressures need not be viewed as a hindrance to ion transport, but as a potentially useful force.

    更新日期:2019-11-13
  • Alkyl and aromatic nitrates in atmospheric particles determined by gas chromatography tandem mass spectrometry
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2019-11-11
    Xinhao Yang, Feixian Luo, Junqi Li, Deyang Chen, Ye E., Weili Lin, Jun Jin

    Organic nitrates in the atmosphere are associated with photochemical pollution and are the main components of secondary organic aerosols, which are related to haze. An efficient method for determining organic nitrates in atmospheric fine particles (PM2.5) was established using synthesized standards. Four alkyl (C7–C10) nitrates and three aromatic nitrates (tolyl nitrate, phenethyl nitrate, and p-xylyl nitrate) were synthesized and characterized by 1H and 13C nuclear magnetic resonance spectroscopy. The optimal ions for quantifying and confirming the identities of the analytes were identified by analyzing the standards by gas chromatography tandem mass spectrometry. The tandem mass spectrometer was a triple quadrupole instrument. This method can obtain more accurate information of organic nitrates than on-line methods. Spiked recovery tests were performed using three spike concentrations, and the recoveries were 61.0–111.4 %, and the relative standard deviations were < 8.2% for all of the analytes. Limits of detection and quantification were determined, and the linearity of the method for each analyte was assessed. The applicability of the method was demonstrated by analyzing six PM2.5 samples. Overall, 87% of the analytes were detected in the samples. Phenethyl nitrate, heptyl nitrate, and octyl nitrate were detected in every sample. Phenethyl nitrate was found at a higher mean concentration (3.23 ng/m3) than the other analytes.

    更新日期:2019-11-13
  • Fragmentation Spectra Prediction and DNA Adducts Structural Determination
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2019-11-06
    Andrea Carrà, Veronica Macaluso, Peter W. Villalta, Riccardo Spezia, Silvia Balbo

    In this work, chemical dynamics simulations were optimized and used to predict fragmentation mass spectra for DNA adduct structural determination. O6-methylguanine (O6-Me-G) was used as a simple model adduct to calculate theoretical spectra for comparison with measured high-resolution fragmentation data. An automatic protocol was established to consider the different tautomers accessible at a given energy and obtain final theoretical spectra by insertion of an initial tautomer. In the work reported here, the most stable tautomer was chosen as the initial structure, but in general, any structure could be considered. Allowing for the formation of the various possible tautomers during simulation calculations was found to be important to getting a more complete fragmentation spectrum. The calculated theoretical results reproduce the experimental peaks such that it was possible to determine reaction pathways and product structures. The calculated tautomerization network was crucial to correctly identifying all the observed ion peaks, showing that a mobile proton model holds not only for peptide fragmentation but also for nucleobases. Finally, first principles results were compared to simple machine learning fragmentation models.

    更新日期:2019-11-07
  • Discrimination of Regioisomeric and Stereoisomeric Saponins from Aesculus hippocastanum Seeds by Ion Mobility Mass Spectrometry
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2019-08-26
    Emmanuel Colson, Corentin Decroo, Dale Cooper-Shepherd, Guillaume Caulier, Céline Henoumont, Sophie Laurent, Julien De Winter, Patrick Flammang, Martin Palmer, Jan Claereboudt, Pascal Gerbaux

    Modern mass spectrometry methods provide a huge benefit to saponin structural characterization, especially when combined with collision-induced dissociation experiments to obtain a partial description of the saponin (ion) structure. However, the complete description of the structures of these ubiquitous secondary metabolites remain challenging, especially since isomeric saponins presenting small differences are often present in a single extract. As a typical example, the horse chestnut triterpene glycosides, the so-called escins, comprise isomeric saponins containing subtle differences such as cis-trans ethylenic configuration (stereoisomers) of a side chain or distinct positions of an acetyl group (regioisomers) on the aglycone. In the present paper, the coupling of liquid chromatography and ion mobility mass spectrometry has been used to distinguish regioisomeric and stereoisomeric saponins. Ion mobility arrival time distributions (ATDs) were recorded for the stereoisomeric and regioisomeric saponin ions demonstrating that isomeric saponins can be partially separated using ion mobility on a commercially available traveling wave ion mobility (TWIMS) mass spectrometer. Small differences in the ATD can only be monitored when the isomeric saponins are separated with liquid chromatography prior to the IM-MS analysis. However, gas phase separation between stereoisomeric and regioisomeric saponin ions can be successfully realized, without any LC separation, on a cyclic ion mobility-enabled quadrupole time-of-flight (Q-cIM-oaToF) mass spectrometer. The main outcome of the present paper is that the structural analysis of regioisomeric and stereoisomeric natural compounds that represents a real challenge can take huge advantages of ion mobility experiments but only if increased ion mobility resolution is attainable.

    更新日期:2019-11-04
  • Absolute Quantitation of Oxidizable Peptides by Coulometric Mass Spectrometry
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2019-08-19
    Pengyi Zhao, Richard N. Zare, Hao Chen

    Quantitation methods for peptides using mass spectrometry have advanced rapidly. These methods rely on using standard and/or isotope-labeled peptides, which might be difficult or expensive to synthesize. To tackle this challenge, we present a new approach for absolute quantitation without the use of standards or calibration curves based on coulometry combined with mass spectrometry (MS). In this approach, which we call coulometric mass spectrometry (CMS), the mass spectrum of a target peptide containing one or more tyrosine residues is recorded before and after undergoing electrochemical oxidation. We record the total integrated oxidation current from the electrochemical measurement, which according to the Faraday’s Law of coulometry, provides the number of moles of oxidized peptide. The ion intensity ratio of the target peptide before and after oxidation provides an excellent estimate of the fraction of the peptide that has been oxidized, from which the total amount of peptide is calculated. The striking strength of CMS is that it needs no standard peptide, but CMS does require the peptide to contain a known number of oxidizable groups. To illustrate the power of this method, we analyzed various tyrosine-containing peptides such as GGYR, DRVY, oxytocin, [Arg8]-vasotocin and angiotensinogen 1–14 with a quantification error ranging from − 7.5 to + 2.4%. This approach is also applicable to quantifying phosphopeptides and could be useful in proteomics research.

    更新日期:2019-11-04
  • Bolt: a New Age Peptide Search Engine for Comprehensive MS/MS Sequencing Through Vast Protein Databases in Minutes
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2019-08-26
    Amol Prakash, Shadab Ahmad, Swetaketu Majumder, Conor Jenkins, Ben Orsburn

    Recent increases in mass spectrometry speed, sensitivity, and resolution now permit comprehensive proteomics coverage. However, the results are often hindered by sub-optimal data processing pipelines. In almost all MS/MS peptide search engines, users must limit their search space to a canonical database due to time constraints and q value considerations, but this typically does not reflect the individual genetic variations of the organism being studied. In addition, engines will nearly always assume the presence of only fully tryptic peptides and limit PTMs to a handful. Even on high-performance servers, these search engines are computationally expensive, and most users decide to dial back their search parameters. We present Bolt, a new cloud-based search engine that can search more than 900,000 protein sequences (canonical, isoform, mutations, and contaminants) with 41 post-translation modifications and N-terminal and C-terminal partial tryptic search in minutes on a standard configuration laptop. Along with increases in speed, Bolt provides an additional benefit of improvement in high-confidence identifications. Sixty-one percent of peptides uniquely identified by Bolt may be validated by strong fragmentation patterns, compared with 13% of peptides uniquely identified by SEQUEST and 6% of peptides uniquely identified by Mascot. Furthermore, 30% of unique Bolt identifications were verified by all three software on the longer gradient analysis, compared with only 20% and 27% for SEQUEST and Mascot identifications respectively. Bolt represents, to the best of our knowledge, the first fully scalable, cloud-based quantitative proteomic solution that can be operated within a user-friendly GUI interface. Data are available via ProteomeXchange with identifier PXD012700.

    更新日期:2019-11-04
  • Design Characteristics to Eliminate the Need for Parameter Optimization in Nanoflow ESI-MS
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2019-08-15
    Yang Kang, Bradley B. Schneider, Leigh Bedford, Thomas R. Covey

    The sampling efficiency in electrospray ionization-mass spectrometry (ESI-MS) can be improved by decreasing the liquid flow rate to the nanoflow regime, where it is possible to draw a large fraction of the ESI plume into the mass spectrometer. This mode of operation is typically more difficult than ESI-MS at higher flow rates because it requires careful optimization of a number of parameters to achieve optimal sampling efficiency. In this work, we screened the relative impact on signal intensity and spray stability of factors that included sprayer position, spray electrode protrusion, sprayer tip shape, spray angle relative to the MS inlet, nebulizer gas flow rate, ESI potential, and means for generating the electric field to initiate electrospray. Based on the screening results, we explore the possibility of providing fixed optimal values for many of the key source parameters to eliminate much of the tuning that is required for conventional nanoflow sources. This approach has potential to greatly simplify nanoflow ESI-MS, while providing optimized sensitivity, stability, and robustness, with decreased variability between analyses.

    更新日期:2019-11-04
  • Ion-Ion Interactions in Charge Detection Mass Spectrometry
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2019-11-01
    Daniel Y. Botamanenko, Martin F. Jarrold

    Charge detection mass spectrometry (CDMS) is a single-particle technique where the masses of individual ions are determined by simultaneously measuring their mass-to-charge ratio (m/z) and charge. Ions are usually trapped inside an electrostatic linear ion trap (ELIT) where they oscillate back and forth through a detection cylinder, generating a periodic signal that is analyzed by fast Fourier transforms. The oscillation frequency is related to the ion’s m/z, and the magnitude is related to the ion’s charge. In early work, multiple ion trapping events were discarded because there was a question about whether ion-ion interactions affected the results. Here, we report trajectory calculations performed to assess the influence of ion-ion interactions when multiple highly charged ions are simultaneously trapped in an ELIT. Ion-ion interactions cause trajectory and energy fluctuations that lead to variations in the oscillation frequencies that in turn degrade the precision and accuracy of the m/z measurements. The peak shapes acquire substantial high and low m/z tails, and the average m/z shifts to a higher value as the number of trapped ions increases. The effects of the ion-ion interactions are proportional to the product of the charges and the square root of the number of trapped ions and depend on the ions’ m/z distribution. For the ELIT design examined here, ion-ion interactions limit the m/z resolving power to several hundred for a typical homogeneous ion population.

    更新日期:2019-11-01
  • 1,4-Benzoquinone as a Highly Efficient Dopant for Enhanced Ionization and Detection of Nitramine Explosives on a Single-Quadrupole Mass Spectrometer Fitted with a Helium-Plasma Ionization (HePI) Source
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2019-10-31
    Julius Pavlov, David Douce, Steve Bajic, Athula B. Attygalle

    Previous investigations have evaluated the efficacy of anions such as NO3−, Cl−, Br−, CH3COO−, and CF3COO− as additives to generate or enhance mass spectrometric signals from explosives under plasma ionization conditions. The results of this study demonstrate that for detecting nitramine-class explosives, such as 1,3,5-trinitroperhydro-1,3,5-triazine (RDX) and 1,3,5,7-tetranitro-1,3,5,7-tetrazacyclooctane (HMX), 1,4-benzoquinone (BQ) is a highly effective and efficient dopant. When used in conjunction with ambient-pressure negative-ion helium-plasma ionization (HePI), 1,4-benzoquinone readily captures an electron, forming an abundant molecular anion (m/z 108), which upon exposure to vapors of RDX and HMX generates adduct ions of m/z 330 and 404, respectively. The signal level recorded for RDX upon adduction to the radical anion of 1,4-benzoquinone under our experimental conditions was significantly higher than that realized by chloride adduction using dichloromethane (DCM) as the dopant.

    更新日期:2019-11-01
  • A Scoring Algorithm for the Automated Analysis of Glycosaminoglycan MS/MS Data
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2019-10-31
    Jiana Duan, Lauren Pepi, I. Jonathan Amster

    The role of glycosaminoglycans (GAGs) in major biological functions is numerous and diverse, yet structural characterization of them by mass spectrometric techniques proves to be challenging. Characterization of GAG structure from tandem mass spectrometry is a tedious and time-consuming process but one that can be automated in a database-independent, high-throughput fashion through the assistance of software implementing a genetic algorithm (J. Am. Soc. Mass Spectrom. 29, 1802–1911, 2018). This work presents the manner in which this data is interpreted by the software, specifically addressing the development of a scoring algorithm. The significance of glycosidic and cross-ring fragment ions and the implications that specific fragments provide for assigning the positions of modifications are discussed. The scoring algorithm is tested for statistical merit using the widely accepted expectation value as the criterion for quality. Using MS/MS data for well-characterized standards, this scoring approach is shown to assign the correct structure, with a low likelihood (1 in 1012 chances) that the assigned structure matches the data due to random chance. The integrated software that automates the structure assignment is called Glycosaminoglycan-Unambiguous Identification Technology (G-UNIT).

    更新日期:2019-11-01
  • Electron Ionization of Imidazole and Its Derivative 2-Nitroimidazole
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2019-10-30
    Rebecca Meißner, Linda Feketeová, Anita Ribar, Katharina Fink, Paulo Limão-Vieira, Stephan Denifl

    Imidazole (IMI) is a basic building block of many biologically important compounds. Thus, its electron ionization properties are of major interest and essential for the comparison with other molecular targets containing its elemental structure. 2-Nitroimidazole (2NI) contains the imidazole ring together with nitrogen dioxide bound to the C2 position, making it a radiosensitizing compound in hypoxic tumors. In the present study, we investigated electron ionization of IMI and 2NI and determined the mass spectra, the ionization energies, and appearance energies of the most abundant fragment cations. The experiments were complemented by quantum chemical calculations on the thermodynamic thresholds and potential energy surfaces, with particular attention to the calculated transition states for the most important dissociation reactions. In the case of IMI, substantially lower threshold values (up to ~ 1.5 eV) were obtained in the present work compared to the only available previous electron ionization study. Closer agreement was found with recent photon ionization values, albeit the general trend of slightly higher values for the case of electron ionization. The only exception for imidazole was found in the molecular cation at m/z 40 which is tentatively assigned to the quasi-linear HCCNH+/ HCNCH+. Electron ionization of 2NI leads to analogous fragment cations as in imidazole, yet different dissociation pathways must be operative due to the presence of the NO2 group. Regarding the potential radiosensitization properties of 2NI, electron ionization is characterized by dominant parent cation formation and release of the neutral NO radical.

    更新日期:2019-11-01
  • 更新日期:2019-11-01
  • Cooperative Formation of Icosahedral Proline Clusters from Dimers.
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2017-11-12
    Alexander D Jacobs,K V Jovan Jose,Rachel Horness,Krishnan Raghavachari,Megan C Thielges,David E Clemmer

    Ion mobility spectrometry-mass spectrometry and Fourier transform infrared spectroscopy (FTIR) techniques were combined with quantum chemical calculations to examine the origin of icosahedral clusters of the amino acid proline. When enantiopure proline solutions are electrosprayed (using nanospray) from 100 mM ammonium acetate, only three peaks are observed in the mass spectrum across a concentration range of five orders of magnitude: a monomer [Pro+H]+ species, favored from 0.001 to 0.01 mM proline concentrations; a dimer [2Pro+H]+ species, the most abundant species for proline concentrations above 0.01 mM; and, the dimer and dodecamer [12Pro+2H]2+ for 1.0 mM and more concentrated proline solutions. Electrospraying racemic D/L-proline solutions from 100 mM ammonium acetate leads to a monomer at low proline concentrations (0.001 to 0.1 mM), and a dimer at higher concentrations (>0.09 mM), as well as a very small population of 8 to 15 Pro clusters that comprise <0.1% of the total ion signals even at the highest proline concentration. Solution FTIR studies show unique features that increase in intensity in the enantiopure proline solutions, consistent with clustering, presumably from the icosahedral geometry in bulk solution. When normalized for the total proline, these results are indicative of a cooperative formation of the enantiopure 12Pro species from 2Pro. Graphical Abstract.

    更新日期:2019-11-01
  • Chiral Differentiation of Non-Covalent Diastereomers Based on Multichannel Dissociation Induced by 213-nm Ultraviolet Photodissociation.
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2019-08-15
    Yingying Shi,Min Zhou,Kailin Zhang,Lifu Ma,Xianglei Kong

    Here we present the implementation of 213-nm ultraviolet photodissociation (UVPD) in a FT-ICR mass spectrometer for chiral differentiation in the gas phase. The L/D amino acid-substituted serine octamer ions were selected as examples of diastereoisomers for chiral analysis. Several kinds of fragment ions were observed in these experiments, including fragment ions that are similar to the ones observed in corresponding collision-activated dissociation (CAD) experiments, fragment ions generated with different protonation sites by only destroying non-covalent bonds, and unique non-covalent cluster radical ions. The latter two kinds of fragment ions are found to be more sensitive to the chirality of the substituted units. Further experiments suggest that the formation of radical ions is mainly affected by chromophores on side chains of the substituted units and micro surroundings of the characterized non-covalent diastereoisomers. A comparing experiment performed by only changing the wavelength of UV laser to 266 nm shows that the 213-nm UV laser has the priority in the diversity of fragmentation pathways and potential of further application in chiral differentiation experiments.

    更新日期:2019-11-01
  • Identification of anionic supramolecular complexes of sulfonamide receptors with Cl-, NO3-, Br-, and I- by APCI-MS.
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2005-08-01
    Konstantinos Kavallieratos,Alberto J Sabucedo,Amanda T Pau,Johanna M Rodriguez

    As part of a mass spectrometric investigation of the binding properties of sulfonamide anion receptors, an atmospheric pressure chemical ionization mass spectrometric (APCI-MS) method involving direct infusion followed by thermal desorption was employed for identification of anionic supramolecular complexes in dichloromethane (CH2Cl2). Specifically, the dansylamide derivative of tris(2-aminoethyl)amine (tren) (1), the chiral 1,3-benzenesulfonamide derivatives of (1R,2S)-(+)-cis-1-amino-2-indanol (2), and (R)-(+)-bornylamine, (3), were shown to bind halide and nitrate ions in the presence of (n-Bu)4N+X- (X- = Cl-, NO3-, Br-, I-). Solutions of receptors and anions in CH2Cl2 were combined to form the anionic supramolecular complexes, which were subsequently introduced into the mass spectrometer via direct infusion followed by thermal desorption. The anionic supramolecular complexes [M + X]-, (M = 1-3, X- = Cl-, NO3-, Br-, I-) were observed in negative mode APCI-MS along with the deprotonated receptors [M - H]-. Full ionization energy of the APCI corona pin (4.5 kV) was necessary for obtaining mass spectra with the best signal-to-noise ratios.

    更新日期:2019-11-01
  • 更新日期:2019-11-01
  • Structural and Energetic Effects of O2'-Ribose Methylation of Protonated Pyrimidine Nucleosides.
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2019-08-23
    C C He,L A Hamlow,Y Zhu,Y-W Nei,L Fan,C P McNary,P Maître,V Steinmetz,B Schindler,I Compagnon,P B Armentrout,M T Rodgers

    The 2'-substituents distinguish DNA from RNA nucleosides. 2'-O-methylation occurs naturally in RNA and plays important roles in biological processes. Such 2'-modifications may alter the hydrogen-bonding interactions of the nucleoside and thus may affect the conformations of the nucleoside in an RNA chain. Structures of the protonated 2'-O-methylated pyrimidine nucleosides were examined by infrared multiple photon dissociation (IRMPD) action spectroscopy, assisted by electronic structure calculations. The glycosidic bond stabilities of the protonated 2'-O-methylated pyrimidine nucleosides, [Nuom+H]+, were also examined and compared to their DNA and RNA nucleoside analogues via energy-resolved collision-induced dissociation (ER-CID). The preferred sites of protonation of the 2'-O-methylated pyrimidine nucleosides parallel their canonical DNA and RNA nucleoside analogues, [dNuo+H]+ and [Nuo+H]+, yet their nucleobase orientation and sugar puckering differ. The glycosidic bond stabilities of the protonated pyrimidine nucleosides follow the order: [dNuo+H]+ < [Nuo+H]+ < [Nuom+H]+. The slightly altered structures help explain the stabilization induced by 2'-O-methylation of the pyrimidine nucleosides.

    更新日期:2019-11-01
  • A Case Study to Identify the Drug Conjugation Site of a Site-Specific Antibody-Drug-Conjugate Using Middle-Down Mass Spectrometry.
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2019-08-21
    Oscar Hernandez-Alba,Stéphane Houel,Steve Hessmann,Stéphane Erb,David Rabuka,Romain Huguet,Jonathan Josephs,Alain Beck,Penelope M Drake,Sarah Cianférani

    Middle-down mass spectrometry (MD MS) has emerged as a promising alternative to classical bottom-up approaches for protein characterization. Middle-level experiments after enzymatic digestion are routinely used for subunit analysis of monoclonal antibody (mAb)-related compounds, providing information on drug load distribution and average drug-to-antibody ratio (DAR). However, peptide mapping is still the gold standard for primary amino acid sequence assessment, post-translational modifications (PTM), and drug conjugation identification and localization. However, peptide mapping strategies can be challenging when dealing with more complex and heterogeneous mAb formats, like antibody-drug conjugates (ADCs). We report here, for the first time, MD MS analysis of a third-generation site-specific DAR4 ADC using different fragmentation techniques, including higher-energy collisional- (HCD), electron-transfer (ETD) dissociation and 213 nm ultraviolet photodissociation (UVPD). UVPD used as a standalone technique for ADC subunit analysis afforded, within the same liquid chromatography-MS/MS run, enhanced performance in terms of primary sequence coverage compared to HCD- or ETD-based MD approaches, and generated substantially more MS/MS fragments containing either drug conjugation or glycosylation site information, leading to confident drug/glycosylation site identification. In addition, our results highlight the complementarity of ETD and UVPD for both primary sequence validation and drug conjugation/glycosylation site assessment. Altogether, our results highlight the potential of UVPD for ADC MD MS analysis for drug conjugation/glycosylation site assessment, and indicate that MD MS strategies can improve structural characterization of empowered next-generation mAb-based formats, especially for PTMs and drug conjugation sites validation.

    更新日期:2019-11-01
  • Structural differentiation of uronosyl substitution patterns in acidic heteroxylans by electrospray tandem mass spectrometry.
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2003-12-31
    Ana Reis,Paula Pinto,M A Coimbra,Dmitry V Evtuguin,Carlos P Neto,A J Ferrer Correia,M Rosário M Domingues

    The structures of two oligomers of acidic xylo-oligosaccharides (XOS) of the same molecular weight (634 Da), Xyl(2)MeGlcAHex and Xyl(2)GlcA(2) were differentiated by electrospray tandem mass spectrometry (ESI-MS/MS). These oligomers were present in a mixture of XOS obtained by acid hydrolysis of heteroxylans extracted from Eucalyptus globulus wood (Xyl(2)MeGlcAHex) and Olea europaea olive fruit (Xyl(2)GlcA(2)). In the ESI-MS spectra of the XOS, ions at m/z 657 and 652 were observed and assigned to [M + Na](+) and [M + NH(4)](+), respectively. The ESI-MS/MS spectrum of [M + Na](+) ion of Xyl(2)MeGlcAHex showed the loss of Hex residue from the reducing end followed by the loss of MeGlcA moiety. Simultaneously, the loss of a Xyl residue from either the reducing or the non-reducing ends was detected. On the other hand, the fragmentation of Xyl(2)GlcA(2) occurs mainly by the loss of one and two GlcA residues or by the loss of the GlcAXyl moiety, due to the glycosidic bond cleavage between the two Xyl residues. Loss of one and two CO(2) molecules was only observed for this oligomer, where the GlcA are in vicinal Xyl residues. The ESI-MS/MS spectra of [M + NH(4)](+) of both oligomers showed the loss of NH(3), resulting in the protonated molecule, where the presence of ions assigned as protonated molecules of aldobiuronic acid residues, [MeGlcA - Xyl + H](+) and [GlcA - Xyl + H](+), are diagnostic ions of the presence of MeGlcA and GlcA moieties in XOS. Since these structures occur in small amounts in complex acidic XOS mixtures and are very difficult, if possible, to isolate, tandem mass spectrometry revealed to be a powerful tool for the characterization of these types of substitution patterns present in heteroxylans.

    更新日期:2019-11-01
  • Development of LC-IMS-CID-TOFMS techniques: analysis of a 256 component tetrapeptide combinatorial library.
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2003-12-04
    Amy E Hilderbrand,Sunnie Myung,Catherine A Srebalus Barnes,David E Clemmer

    Recent improvements in ion mobility/time-of-flight mass spectrometry techniques have made it possible to incorporate nano-flow liquid chromatography and collision induced dissociation techniques. This combination of approaches provides a new strategy for detailed characterization of complex systems--such as, combinatorial libraries. Our work uses this technology to provide a detailed analysis of a tetrapeptide library having the general form Xxx1-Xxx2-Xxx3-Xxx4 where Xxx1 = Glu, Phe, Val, Asn; Xxx2 = Glu, Phe, Val, Tyr; Xxx3 = Glu, Phe, Val, Thr; and Xxx4 = Glu, Phe, Val, Leu--a system that is expected to contain 256 different peptide sequences. The results corroborate the presence of many expected peptide sequences and indicate that some synthetic steps appear to have failed. Particularly interesting is the observation of a t-butyl protecting group on the tyrosine (Tyr) residue. It appears that most Tyr containing peptides that have this t-butyl group attached favor formation of [2M + 2H]2+ dimers, which can be readily distinguished from [M + H]+ monomers based on differences in their gas-phase mobilities. In this case, we demonstrate the use of the mobility differences between [2M + 2H]2+ and [M + H]+ ions as a signature for a failure of a synthetic step.

    更新日期:2019-11-01
  • Detection and characterization by mass spectrometry of radical adducts produced by linoleic acid oxidation.
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2003-11-05
    Ana Reis,M Rosário M Domingues,Francisco M L Amado,A J V Ferrer-Correia,Pedro Domingues

    The formation of linoleic acid radical species under the oxidative conditions of the Fenton reaction (using hydrogen peroxide and Fe (II)) was monitored by FAB-MS and ES-MS using the spin trap 5,5-dimethyl-1-pyrrolidine-N-oxide, DMPO. Both the FAB and ES mass spectra were very similar and showed the presence of ions corresponding to carbon- and oxygen centered spin adducts (DMPO/L*, DMPO/LO*, and DMPO/LOO*). Cyclic structures, formed between the DMPO oxygen and the neighboring carbon of the fatty acid, were also observed. Electrospray tandem mass spectrometry of these ions was performed to confirm the proposed structure of these adducts. All MS/MS spectra showed an ion at m/z 114, correspondent to the [DMPO + H]+, and a fragment ion due to loss of DMPO (loss of 113 Da), confirming that they are DMPO adducts. ES-MS/MS spectra of alkoxyl radical adducts (DMPO/LO*) showed an additional ion at m/z 130 [DMPO - O + H]+, while ES MS/MS of peroxyl radical adducts (DMPO/LOO*) showed a fragment ion at m/z 146 [DMPO - OO + H]+, confirming both structures. Other fragment ions were observed, such as alkyl acylium radical ions, formed by cleavage of the alkyl chain after loss of water and the DMPO molecule. The identification of fragment ions observed in the MS/MS spectra of the different DMPO adducts suggests the occurrence of structural isomers containing the DMPO moiety both at C9 and C13. The use of ES tandem mass spectrometry, associated with spin trapping experiments, has been shown to be a valuable tool for the structural characterization of carbon and oxygen-centered spin adducts of lipid radicals.

    更新日期:2019-11-01
  • Ion-molecule reactions of gas-phase chromium oxyanions. CrxOyHz-+ O2.
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2003-10-08
    A K Gianotto,B D M Hodges,P de B Harrington,A D Appelhans,J E Olson,G S Groenewold

    Chromium oxyanions, Cr(x)O(y)H(z)(-), were generated in the gas-phase using a quadrupole ion trap secondary ion mass spectrometer (IT-SIMS), where they were reacted with O(2). Only CrO(2)(-) of the Cr(1)O(y)H(z)(-) envelope was observed to react with oxygen, producing primarily CrO(3)(-). The rate constant for the reaction of CrO(2)(-) with O(2) was approximately 38% of the Langevin collision constant at 310 K. CrO(3)(-), CrO(4)(-), and CrO(4)H(-) were unreactive with O(2) in the ion trap. In contrast, Cr(2)O(4)(-) was observed to react with O(2) producing CrO(3)(-) + CrO(3) via oxidative degradation at a rate that was approximately 15% efficient. The presence of background water facilitated the reaction of Cr(2)O(4)(-) + H(2)O to form Cr(2)O(5)H(2)(-); the hydrated product ion Cr(2)O(5)H(2)(-) reacted with O(2) to form Cr(2)O(6)(-) (with concurrent elimination of H(2)O) at a rate that was 6% efficient. Cr(2)O(5)(-) also reacted with O(2) to form Cr(2)O(7)(-) (4% efficient) and Cr(2)O(6)(-) + O (2% efficient); these reactions proceeded in parallel. By comparison, Cr(2)O(6)(-) was unreactive with O(2), and in fact, no further O(2) addition could be observed for any of the Cr(2)O(6)H(z)(-) anions. Generalizing, Cr(x)O(y)H(z)(-) species that have low coordinate, low oxidation state metal centers are susceptible to O(2) oxidation. However, when the metal coordination is >3, or when the formal oxidation state is > or =5, reactivity stops.

    更新日期:2019-11-01
  • Identification of 2D-gel proteins: a comparison of MALDI/TOF peptide mass mapping to mu LC-ESI tandem mass spectrometry.
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2003-09-05
    Hanjo Lim,Jimmy Eng,John R Yates,Sandra L Tollaksen,Carol S Giometti,James F Holden,Michael W W Adams,Claudia I Reich,Gary J Olsen,Lara G Hays

    A comparative analysis of protein identification for a total of 162 protein spots separated by two-dimensional gel electrophoresis from two fully sequenced archaea, Methanococcus jannaschii and Pyrococcus furiosus, using MALDI-TOF peptide mass mapping (PMM) and mu LC-MS/MS is presented. 100% of the gel spots analyzed were successfully matched to the predicted proteins in the two corresponding open reading frame databases by mu LC-MS/MS while 97% of them were identified by MALDI-TOF PMM. The high success rate from the PMM resulted from sample desalting/concentrating with ZipTip(C18) and optimization of several PMM search parameters including a 25 ppm average mass tolerance and the application of two different protein molecular weight search windows. By using this strategy, low-molecular weight (<23 kDa) proteins could be identified unambiguously with less than 5 peptide matches. Nine percent of spots were identified as containing multiple proteins. By using mu LC-MS/MS, 50% of the spots analyzed were identified as containing multiple proteins. mu LC-MS/MS demonstrated better protein sequence coverage than MALDI-TOF PMM over the entire mass range of proteins identified. MALDI-TOF and PMM produced unique peptide molecular weight matches that were not identified by mu LC-MS/MS. By incorporating amino acid sequence modifications into database searches, combined sequence coverage obtained from these two complimentary ionization methods exceeded 50% for approximately 70% of the 162 spots analyzed. This improved sequence coverage in combination with enzymatic digestions of different specificity is proposed as a method for analysis of post-translational modification from 2D-gel separated proteins.

    更新日期:2019-11-01
  • Use of ProteinChip array surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) to identify thymosin beta-4, a differentially secreted protein from lymphoblastoid cell lines.
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2003-07-03
    Deborah L Diamond,Yanni Zhang,Alexander Gaiger,Molly Smithgall,Thomas S Vedvick,Darrick Carter

    The identification of proteins differentially expressed between cancer and normal cells is vital for the development of cancer diagnostics, therapeutics and vaccines. Using a ProteinChip Biomarker System (Ciphergen Biosystems, Fremont, CA) which combines ProteinChip technology with time-of-flight mass spectrometry, we have developed a simple method to screen and identify differentially secreted proteins from tumor cell lines. Mass spectra of the range of proteins secreted from normal B-cells were generated along with those secreted from Epstein-Barr virus transformed B-cells. A mass peak at m/z = 4972.1 that was highly over-represented in the transformed B-cell line was chosen for identification and purified by reversed phase chromatography with concomitant monitoring of fractions by SELDI-TOF MS. The resulting purified protein was digested with trypsin and the peptide masses derived from the SELDI-TOF spectrum were used to search the public databases for protein identification. Fragment matching of the resulting peptides identified the protein as thymosin beta-4. Using LC-electrospray ionization MS/MS, the identity of this protein was confirmed. Thymosin beta-4 is a known marker in LCLs establishing the utility of this method to discover and identify proteins differentially expressed between cancers and their matched normal counterparts.

    更新日期:2019-11-01
  • On-line strong cation exchange micro-HPLC-ESI-MS/MS for protein identification and process optimization.
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2003-07-03
    Thierry Le Bihan,Henry S Duewel,Daniel Figeys

    We have developed an on-line strong cation exchange (SCX)-ESI-MS/MS platform for the rapid identification of proteins contained in mixtures. This platform consists of a SCX precolumn followed by a nanoflow SCX column on-line with an electrospray ion trap mass spectrometer. We also used this platform to study the dynamics of peptide separation/extraction by SCX, in particular to understand the parameters affecting the performance of SCX in multidimensional chromatography. For example, we have demonstrated that the buffer typically used for tryptic digestion of protein mixtures can have a detrimental effect on the chromatographic behaviour of peptides during SCX separations, thereby affecting certain peptide quantitation approaches that rely on reproducible peptide fractionation. We have also demonstrated that band broadening results when a step (discontinuous) gradient approach is used to displace peptides from the SCX precolumn, reducing the separation power of SCX in multidimensional chromatography. In contrast, excellent chromatographic peak shapes are observed when a defined (continuous) gradient is used. Finally, using a tryptic digest of a protein extract derived from human K562 cells, we observed that larger molecular weight peptides are identified using this on-line SCX approach compared to the more conventional reverse phase (RP) LC/MS approach. Both methods used in tandem complement each other and can lead to a greater number of peptide identifications from a given sample.

    更新日期:2019-11-01
  • Comparative ESI-MS study of approximately 2.2 MDa native hemocyanins from deep-sea and shore crabs: from protein oligomeric state to biotope.
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2003-05-15
    Sarah Sanglier,Emmanuelle Leize,Alain Van Dorsselaer,Franck Zal

    In the past years, the potential of electrospray ionization mass spectrometry (ESI-MS) for the observation of intact weak interactions, such as non-covalent protein-ligand, protein-protein, protein-DNA complexes, has spread out. The coupling of ESI with time-of-flight (TOF) and quadrupole-time-of-flight (Q-TOF) analyzers has even enabled the detection of larger complexes with molecular weights greatly higher than 200 kDa. In this paper, we report a comparative ESI-MS study on the protein quaternary structure of native hemocyanins (Hc) from crabs living in different biotopes: a shore crab (Carcinus maenas) and two deep-sea crabs (Segonzacia mesatlantica and Bythograea thermydron). Hc is an extracellular blood protein, composed of several protein chains which can associate in large multimers. The goal of this study is to point out that the oligomerization state of native Hcs is biotope-dependent. Depending on the crab, ESI-MS analyses under non-denaturing conditions reveal different oligomeric forms present in equilibrium in solution. Molecular weights up to 2,235 kDa were measured for the associations of 30 subunits of the Bythograea thermydron Hc. Thanks to ESI-MS analyses, it could be concluded for the first time that the oligomerization state of native Hcs is dependent on the crab environment. The investigation of these different non-covalent self-assemblies is very important for the life history of crabs, since they are directly related with different oxygen binding abilities and thus, with their ability to colonize habitats with different oxygen contents.

    更新日期:2019-11-01
  • Identification of an in vitro insulin receptor substrate-1 phosphorylation site by negative-ion muLC/ES-API-CID-MS hybrid scan technique.
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2003-04-11
    Alexander Beck,Klaus Moeschel,Martin Deeg,Hans Ulrich Häring,Wolfgang Voelter,Erwin D Schleicher,Rainer Lehmann

    Recently, we reported a fast on-line alkaline micro-liquid chromatography/electrospray-atmospheric pressure ionization/collision-induced dissociation/mass spectrometric approach for sensitive phosphopeptide screening of a tryptic digested protein and subsequent characterization of the identified phosphopeptide. Based on this study, we now applied an improved method for the identification of phosphorylation sites in insulin receptor substrate 1, an important mediator in insulin signal transduction which was phosphorylated in vitro by protein kinase C-zeta. The approach consists of an on-line alkaline negative-ion micro-liquid chromatography/electrospray-atmospheric pressure ionization/collision-induced dissociation/mass spectrometric hybrid scan experiment using a triple-quadrupole mass spectrometer with fractionation and subsequent off-line nanoES-MS (ion trap) analysis of the phosphopeptide-containing fractions. During the liquid chromatography (LC)/ES-MS experiment, the phosphopeptides of the enzymatic digest mixture of the studied insulin receptor substrate 1 fragment were detected under high skimmer potential (API-CID) using phosphorylation-specific m/z 79 marker ions as well as the intact m/z-values of the peptides which were recorded under low skimmer potential. Subsequently, the targeted fractions were analyzed by off-line nanoES-MS/MS and MS(3). Using this approach, serine 318 was clearly identified as a major in vitro protein kinase C-zeta phosphorylation site in the insulin receptor substrate -1 fragment. Together, our results indicate that the applied strategy is useful for unequivocal and fast analysis of phosphorylation sites in low abundant signaling proteins.

    更新日期:2019-11-01
  • Top down characterization of secreted proteins from Mycobacterium tuberculosis by electron capture dissociation mass spectrometry.
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2003-03-22
    Ying Ge,Mariam El-Naggar,Siu Kwan Sze,Han Bin Oh,Tadhg P Begley,Fred W McLafferty,Helena Boshoff,Clifton E Barry

    Secreted proteins of Mycobacterium tuberculosis are implicated in its disease pathogenesis and so are considered as potential diagnostic and vaccine candidates. The search for these has been slow, even though the entire genome sequence of M. tuberculosis is now available; of the 620 protein spots resolved by 2-D gel electrophoresis, 114 secreted proteins have been identified, but for only 13 has the primary structure been partly characterized. For comparison, in this top down mass spectrometry (MS) approach the secreted proteins were precipitated from cell culture filtrate, resuspended, and examined directly by electrospray ionization (ESI) Fourier transform MS. The ESI spectra of three precipitates showed 93, 535, and 369 molecular weight (M(r)) values, for a total of 689 different values. However, only approximately 10% of these values matched (+/-1 Da) the DNA predicted M(r) values, but these identifications were unreliable. Of nine molecular ions characterized by MS/MS, only one protein match was confirmed, and its isotopic molecular ions were overlapped by those of another protein. MS/MS identified a total of ten proteins by sequence tag search, of which three were unidentified previously. The low success of M(r) matching was due to unusually extensive posttranslational modifications, including loss of a signal sequence, loss of the N-terminal residue, proteolytic degradation, oxidation, and glycosylation. Although in eubacteria the latter is relatively rare, a 9 kDa protein showed 7 hexose attachments and two 20 kDa proteins each had 20 attachments. For MS/MS, electron capture dissociation was especially effective.

    更新日期:2019-11-01
  • Experimental and theoretical study of the gas-phase interaction between ionized nitrile sulfides and pyridine.
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2003-03-22
    Pascal Gerbaux,Yves Van Haverbeke,Robert Flammang

    The gas-phase reactivity of ionized nitrile sulfides, R-C[triple bond]N(+)-S*, towards neutral pyridine was studied both experimentally (six sector hybrid mass spectrometer) and theoretically (density functional theory and Møller-Plesset ab initio calculations). An ionized sulfur atom transfer and a cycloaddition process respectively yielding ionized pyridine N-thioxide and a thiazolopyridinium cation were observed. Whereas the very efficient S*+ transfer reaction probably involves the intermediacy of several ion-molecule complexes, the thiazolopyridinium ion formation is likely to be initiated by an electrophilic attack of the R-C[triple bond]N(+)-S* ion on the nitrogen atom of pyridine; the resulting intermediate then undergo an intramolecular substitution of an alpha-hydrogen atom by the sulfur atom.

    更新日期:2019-11-01
  • ESI-mass spectrometry analysis of unsubstituted and disubstituted beta-cyclodextrins: fragmentation mode and identification of the AB, AC, AD regioisomers.
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2003-02-15
    Stefano Sforza,Gianni Galaverna,Roberto Corradini,Arnaldo Dossena,Rosangela Marchelli

    The study of unsubstituted and disubstituted beta-cyclodextrins (beta-CDs) by ESI-mass spectrometry is reported, applying a cone-induced fragmentation in the presence of a twofold excess of sodium chloride, in order to gain information about the fragmentation of the different regioisomers. On the basis of the fragmentation pattern observed for the unsusbstituted beta-CD, a statistical model shows that the fragments generated by every regioisomer of a disubstituted CD (AB, AC, and AD) are expected to differ in their relative intensity and, therefore, they can be used for correctly identifying the three different regioisomers. The model was tested on the three regioisomeric (AB, AC, and AD) diamino-beta-CDs and ditosyl-beta-CD and on the AC and AD regioisomers of dimesitylenesulphonyl-beta-CD, allowing in every case through statistical analysis of the fragmentation pattern the correct assignment of every regioisomer on the basis of an ESI mass spectrum (single quadrupole analyzer, high cone voltage) of the pure compounds. The absolute intensities of the fragmentation peaks were voltage-dependent but their ratios was voltage-independent, indicating that no mass bias in peak ratios is introduced by the analyzer. Given the fast time of analysis and its general applicability, independently from the substituents, we propose our method as an easy way to identify the regioisomers of disubstituted beta-CDs.

    更新日期:2019-11-01
  • Molecular mass determination of saturated hydrocarbons: reactivity of eta5-cyclopentadienylcobalt ion (CpCo*+) and linear alkanes up to C-30.
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2002-12-31
    H C Michelle Byrd,Charles M Guttman,Douglas P Ridge

    The present study demonstrates the feasibility of the eta5-cyclopentadienylcobalt ion (CpCo*+) as a suitable cationization reagent for saturated hydrocarbon analysis by mass spectrometry. Ion/molecule reactions of CpCo*+ and three medium chain-length n-alkanes were examined using Fourier-transform ion cyclotron resonance mass spectrometry. Second-order rate constants and reaction efficiencies were determined for the reactions studied. Loss of two hydrogen molecules from the CpCo-alkane ion complex was found to dominate all reactions ( > or = 80%). Furthermore, this dehydrogenation reaction efficiency increases with increasing chain length. These preliminary results suggest that the CpCo*+ ion may be a promising cationization reagent of longer chain saturated hydrocarbons and polyolefins.

    更新日期:2019-11-01
  • Alkali cation attachment to derivatized fullerenes studied by matrix-assisted laser desorption/ionization.
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2002-12-18
    Dorina Fati,Victoria Leeman,Yury V Vasilév,Thomas Drewello,Bernard Leyh,Hartmut Hungerbühler

    The complexation of alkali metal ions with amphiphilic fullerene derivatives has been investigated by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry. The formation of analyte ions occurs via two competing mechanisms including electron transfer from matrix-derived ions and metal ion attachment. The interplay of these processes has been examined by laser fluence dependent sample activation and by variation of the target composition. The attachment of metal ions has been established as the gentler and thus more efficient route towards the formation of intact analyte ions. Investigations into the metal ion complexation have been conducted to reveal the reactivity order of the alkali metals in these reactions and to elucidate the influence of structural differences of the analytes, as well as to unravel effects caused by the anionic counter ion of the metal. The experimental data have been derived by two complementary approaches. Competing reactants were either studied simultaneously, so that the product distribution would provide direct insight into the reactivity pattern, and/or product distributions were obtained in a large variety of separate experiments and normalized for reliable comparison. It has been found that the extent to which complexation is observed follows the charge density order of the alkali metal ions. The structural features of the fullerene-attached ligands were of profound influence on the attachment of the metal ion, inducing enhanced selectivity for the complexation with less reactive metals. The metal ion attachment is reduced with the use of smaller anionic counter ions. Rationalization of these findings is provided within the framework of the mechanisms of ion formation in MALDI.

    更新日期:2019-11-01
  • Unexpected fragmentation of beta-substituted meso-tetraphenylporphyrins induced by high-energy collisional activation.
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2002-12-18
    M Rosario M Domingues,M Graça O S Marques,Cristina M A Alonso,M Graça P M S Neves,J A S Cavaleiro,A J Ferrer-Correia,Olga V Nemirovskiy,Michael L Gross

    The protonated molecules and radical cations of meso-tetraphenylporphyrins with beta-pyrrolic substituents, when formed by fast atom bombardment (FAB) and subjected to high-energy collisions, give rise to unexpected fragment ions. The reaction involves hydrogen migration from the ortho position of the phenyl ring to the a atom of the substituent, with formation of an intramolecular, six-membered ring. The process is analogous to condensed-phase cyclizations described for the same type of compounds. The fragmentation requires the presence of a double bond in the substituent group attached to the pyrrolic ring. A rearrangement process involving anchimeric assistance by the phenyl group (analogous to an ortho effect) is proposed for the formation of these ions.

    更新日期:2019-11-01
  • 更新日期:2019-11-01
  • CH3CH+* formation from some C3H6O+* isomers according to theory.
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2002-10-22
    Charles E Hudson,David J McAdoo,John C Traeger

    Dissociations to alkane ions in gas phase ion chemistry are rare and poorly characterized. Therefore, the pathways to CH3CH3+* + CO from *CH2CH2O+=CH2 and some of its isomers are investigated by theory. The pathway found for this reaction is *CH2CH2O+=CH2 --> CH3CH2O+=CH* --> [CH3CH2- -H- -CO]+* --> CH3CH+* + CO. The crucial intermediate in this pathway is the stable hydrogen-bonded ion-neutral complex [CH3CH2- -H- -CO]+*, a species held together by a strong hydrogen bond. CH3CH3+* + CO rather than CH3CH3 + CO+* is formed from *CH2CH2O+=CH2 and other C3H6O+* ions because the former pair is much more stable than the latter. The photoionization appearance energies of CH3CH3+* from CH3CH2CHO+* and from CH3CH2CO2H+* demonstrate that the onsets of these reactions are at to just above their thermochemical thresholds, consistent with the intermediacy of ion-neutral complexes. We also found transition states for interconversion of CH3CH2CHO+* and CH3CH2O+=CH* and transformation of CH3CH2C:=OH+* to CH3CH2CHO+*; the latter reaction occurs by a 1,2-H-shift from O to C.

    更新日期:2019-11-01
  • On the Competition Between Electron Autodetachment and Dissociation of Molecular Anions.
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2019-05-30
    Gerd Marowsky,Jürgen Troe,Albert A Viggiano

    We treat the competition between autodetachment of electrons and unimolecular dissociation of excited molecular anions as a rigid-/loose-activated complex multichannel reaction system. To start, the temperature and pressure dependences under thermal excitation conditions are represented in terms of falloff curves of separated single-channel processes within the framework of unimolecular reaction kinetics. Channel couplings, caused by collisional energy transfer and "rotational channel switching" due to angular momentum effects, are introduced afterward. The importance of angular momentum considerations is stressed in addition to the usual energy treatment. Non-thermal excitation conditions, such as typical for chemical activation and complex-forming bimolecular reactions, are considered as well. The dynamics of excited SF6- anions serves as the principal example. Other anions such as CF3- and POCl3- are also discussed.

    更新日期:2019-11-01
  • Quantitative TOF-SIMS analysis of oligomeric degradation products at the surface of biodegradable poly(alpha-hydroxy acid)s.
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2002-09-27
    Joo-Woon Lee,Joseph A Gardella

    This paper reports the development of a new method for quantification of the hydrolytic surface degradation kinetics of biodegradable poly(alpha-hydroxy acid)s using time-of-flight secondary ion mass spectrometry (TOF-SIMS). We report results from static SIMS spectra of a series of poly(alpha-hydroxy acid)s including poly(glycolic acid), poly(L-lactic acid), and random poly(D,L-lactic acid-co-glycolic acid) hydrolyzed in various buffer systems. The distribution of the most intense peak intensities of ions generated in high mass range of the spectrum reflects the intact degradation products (oligomeric hydrolysis products) of each biodegradable polymer. First, a detailed analysis of the oligomeric ions is given based on rearrangement of the intact hydrolysis products. The pattern of ions can distinguish both degradation-generated intact oligomers and their fragment ion peaks with a variety of combinations of each repeat unit. Then, the integration and summation of the area of all ion peaks with the same number of repeat units is proposed as a measurement that provides a more accurate MW average than the typically used method which counts only the most intense peak. The multiple ion summation method described in this paper would be practical in the improvement of quantitative TOF-SIMS studies as a better data reduction method, especially in the surface degradation kinetics of biodegradable polymers.

    更新日期:2019-11-01
  • In vivo labeling: a glimpse of the dynamic proteome and additional constraints for protein identification.
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2002-08-01
    Rachel R Ogorzalek Loo,Joseph A Loo,Ping Du,Tod Holler

    Identities ascribed to the intact protein ions detected in MALDI-MS of whole bacterial cells or from other complex mixtures are often ambiguous. Isolation of candidate proteins can establish that they are of correct molecular mass and sufficiently abundant, but by itself is not definitive. An in vivo labeling strategy replacing methionine with selenomethionine has been employed to deliver an additional constraint for protein identification, i.e., number of methionine residues, derived from the shift in mass of labeled versus unlabeled proteins. By stressing a culture and simultaneously labeling, it was possible to specifically image the cells' response to the perturbation. Because labeled protein is only synthesized after application of the stress, it provides a means to view dynamic changes in the cellular proteome. These methods have been applied to identify a 15,879 Da protein ion from E. coli that was induced by an antibacterial agent with an unknown mechanism of action as SpY, a stress protein produced abundantly in spheroplasts. It has also allowed us to propose protein identities (and eliminate others from consideration) for many of the ions observed in MALDI (and ESI-MS) whole cell profiling at a specified growth condition.

    更新日期:2019-11-01
  • Gas-phase ion-molecule reactions of transition metal complexes: the effect of different coordination spheres on complex reactivity.
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2002-08-01
    Marianny Y Combariza,Richard W Vachet

    Using a modified quadrupole ion trap mass spectrometer, a series of metal complex ions have been reacted with acetonitrile in the gas phase. Careful control of the coordination number and the type of coordinating functionality in diethylenetriamine-substituted ligands enable the effects of the coordination sphere on metal complex reactivity to be examined. The association reaction kinetics of acetonitrile with these pentacoordinate complexes are followed in order to obtain information about the starting complexes and the reaction dynamics. The kinetics and thermodynamics of acetonitrile addition to the metal complex ions are strongly affected by the chemical environment around the metal center such that significant differences in reactivity are observed for Co(II) and Cu(II) complexes with various coordination spheres. When thiophene, furan, or benzene moieties are present in the coordination sphere of the complex, addition of two acetonitrile molecules is readily observed. In contrast, ligands with better sigma donors react mainly to add one acetonitrile molecule. Among the ligands with good sigma donors, a clear trend in reactivity is observed in which complexes with nitrogen-containing ligands are the least reactive, sulfur-containing complexes are more reactive, and oxygen-containing complexes are the most reactive. In general, equilibrium and reaction rate constants seem to be consistent with the hard and soft acid and base (HSAB) principle. Interestingly, the presence of certain groups (e.g., pyridine and imidazole) in the coordination sphere clearly can change the acid character of the metal as seen by their effect on the binding properties of other functional groups in the same ligand. Finally, we conclude that because complexes with different coordination spheres react to noticeably different extents, ion-molecule (I-M) reactions may be potentially useful for obtaining coordination structure information for transition metal complexes.

    更新日期:2019-11-01
  • Correlations of chemical mass shifts of para-substituted acetophenones and benzophenones with Brown's sigma constants.
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2002-06-12
    Yanan Peng,Wolfgang R Plass,R Graham Cooks

    Relationships between chemical mass shifts and physiochemical properties of ions are sought by examining substituted acetophenones, benzophenones, and pyridines in a modified ion trap mass spectrometer. Systematic changes in chemical mass shift occur with changes in substituent in the acetophenones and the benzophenones. Brown's sigma+ constant, which is a measure of electronic effects of substituents in reactions that involve positive charge development, is shown to correlate linearly with chemical mass shifts in para-substituted acetophenones and benzophenones. Brown's sigma+ constant also correlates with the ease of dissociation of the ions via a correlation with ionization energy. It is suggested that ease of dissociation is the underlying factor in determining chemical mass shifts. The experimental results also suggest that dissociative collisions between ions and buffer gas make a much greater contribution to chemical mass shifts than do elastic collisions.

    更新日期:2019-11-01
  • 更新日期:2019-11-01
  • Gas-phase fragmentation of the Ag+-phenylalanine complex: cation-pi interactions and radical cation formation.
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2002-04-16
    Tamer Shoeib,Alwin Cunje,Alan C Hopkinson,K W Michael Siu

    Collision-induced dissociation experiments on the Ag+-phenylalanine complex using several collision energies were shown to yield ten different fragment ions. Unambiguous assignment of these fragment ions were made by careful analysis of deuterium labeling experiments. The losses of H2O, CO, CO2, and AgH were commonly observed; also encountered were the losses of H2, Ag, and H. Deuterium labeling experiments and density functional calculations have been employed to probe fragmentation mechanisms that account for all experimental results.

    更新日期:2019-11-01
  • Desorption/ionization of biomolecules from aqueous solutions at atmospheric pressure using an infrared laser at 3 microm.
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2002-04-16
    Victor V Laiko,Nelli I Taranenko,Vadym D Berkout,Mikhail A Yakshin,Coorg R Prasad,H Sang Lee,Vladimir M Doroshenko

    A new atmospheric pressure (AP) infrared (IR) matrix-assisted laser desorption/ionization (MALDI) ion source was developed and interfaced with a Thermo Finnigan LCQ ion trap mass spectrometer. The source utilized a miniature all-solid-state optical parametric oscillator (OPO)-based IR laser system tunable in the lambda = 1.5-4 microm spectral range and a nitrogen ultraviolet (UV) laser (lambda = 337 nm) for use in comparative studies. The system demonstrated comparable performance at 3 microm and 337 nm wavelengths if UV matrices were used. However, AP IR-MALDI using a 3 microm wavelength showed good performance with a much broader choice of matrices including glycerol and liquid water. AP IR-MALDI mass spectra of peptides in the mass range up to 2000 Da were obtained directly from aqueous solutions at atmospheric conditions for the first time. A potential use of the new AP IR-MALDI ion source includes direct MS analysis of biological cells and tissues in a normal atmospheric environment as well as on-line coupling of mass spectrometers with liquid separation techniques.

    更新日期:2019-11-01
  • Characterization of the elusive disulfide bridge forming human Hb variant: Hb Ta-Li beta83 (EF7)Gly --> Cys by electrospray mass spectrometry.
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2002-02-12
    Dilip K Rai,Britta Landin,William J Griffiths,Gunvor Alvelius,Brian N Green

    An electrospray mass spectrometric approach to the identification of a human hemoglobin (Hb) variant involving a Cys residue incorporation is presented. In Hb Ta-Li (beta83Gly --> Cys), Cys83 forms inter-molecular disulfide bridges. Routine analysis of the denatured Hb showed the presence of a minor beta chain variant whose mass apparently was 1 Da less than the expected mass difference of 46 Da for a Gly --> Cys substitution. Reduction of the globin chains with dithiothreitol gave an intense monomer with the expected mass difference for the Gly --> Cys substitution. After reprocessing the original raw data from the denatured Hb and taking into account the possibility of dimer formation, a component was revealed whose mass was consistent with a disulfide linked dimer of Ta-Li beta globins. The mutation was localized to peptide betaT10 by analysis of a tryptic digest. Tandem mass spectrometry and DNA sequencing confirmed the Gly --> Cys substitution occurred at residue 83 of the beta chain. Problems encountered in identifying the components in mixtures of monomers and dimers are discussed.

    更新日期:2019-11-01
  • Detailed analysis of alpha,omega-bis(4-hydroxybutyl) poly(dimethylsiloxane) using GPC-MALDI TOF mass spectrometry.
    J. Am. Soc. Mass Spectrom. (IF 3.202) Pub Date : 2002-02-12
    E Peter Maziarz,X Michael Liu,Edmond T Quinn,Yu-Chin Lai,Daniel M Ammon,George L Grobe

    In this study the prepolymer alpha,omega-bis(4-hydroxybutyl) poly(dimethylsiloxane), used in the formulation of oxygen permeable films, is evaluated by gel permeation chromatography (GPC) combined with matrix assisted laser desorption ionization (MALDI) time of flight (TOF) mass spectrometry (MS). Two unexpected mass distributions are observed in the mass spectra. Reaction schemes for the formation of these distributions are proposed. A solution phase trimethylsilane end group modification was performed on the prepolymer to determine whether the unexpected mass distributions occur as impurities from synthesis or as artifacts from the MS process. Evaluation of the TMS modified prepolymer indicates the unexpected mass distributions indeed occur as impurities from the synthetic procedure. Average molecular weight values are determined by traditional GPC, direct MALDI-TOF MS, and GPC-MALDI-TOF MS methods and the results are compared.

    更新日期:2019-11-01
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