15 results on '"Horn, David M."'
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2. Improved Label-Free Quantification of Intact Proteoforms Using Field Asymmetric Ion Mobility Spectrometry.
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Kline, Jake T., Belford, Michael W., Huang, Jingjing, Greer, Joseph B., Bergen, David, Fellers, Ryan T., Greer, Sylvester M., Horn, David M., Zabrouskov, Vlad, Huguet, Romain, Boeser, Cornelia L., Durbin, Kenneth R., and Fornelli, Luca
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- 2023
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3. Improved Label-Free Quantification of Intact Proteoforms Using Field Asymmetric Ion Mobility Spectrometry
- Author
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Kline, Jake T., Belford, Michael W., Huang, Jingjing, Greer, Joseph B., Bergen, David, Fellers, Ryan T., Greer, Sylvester M., Horn, David M., Zabrouskov, Vlad, Huguet, Romain, Boeser, Cornelia L., Durbin, Kenneth R., and Fornelli, Luca
- Abstract
The high-throughput quantification of intact proteoforms using a label-free approach is typically performed on proteins in the 0–30 kDa mass range extracted from whole cell or tissue lysates. Unfortunately, even when high-resolution separation of proteoforms is achieved by either high-performance liquid chromatography or capillary electrophoresis, the number of proteoforms that can be identified and quantified is inevitably limited by the inherent sample complexity. Here, we benchmark label-free quantification of proteoforms of Escherichia coliby applying gas-phase fractionation (GPF) via field asymmetric ion mobility spectrometry (FAIMS). Recent advances in Orbitrap instrumentation have enabled the acquisition of high-quality intact and fragmentation mass spectra without the need for averaging time-domain transients prior to Fourier transform. The resulting speed improvements allowed for the application of multiple FAIMS compensation voltages in the same liquid chromatography–tandem mass spectrometry experiment without increasing the overall data acquisition cycle. As a result, the application of FAIMS to label-free quantification based on intact mass spectra substantially increases the number of both identified and quantified proteoforms without penalizing quantification accuracy in comparison to traditional label-free experiments that do not adopt GPF.
- Published
- 2023
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4. Orbitrap Mass Spectrometry and High-Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Enable the in-Depth Analysis of Human Serum Proteoforms
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Kline, Jake T., Belford, Michael W., Boeser, Cornelia L., Huguet, Romain, Fellers, Ryan T., Greer, Joseph B., Greer, Sylvester M., Horn, David M., Durbin, Kenneth R., Dunyach, Jean-Jacques, Ahsan, Nagib, and Fornelli, Luca
- Abstract
Blood serum and plasma are arguably the most commonly analyzed clinical samples, with dozens of proteins serving as validated biomarkers for various human diseases. Top-down proteomics may provide additional insights into disease etiopathogenesis since this approach focuses on protein forms, or proteoforms, originally circulating in blood, potentially providing access to information about relevant post-translational modifications, truncations, single amino acid substitutions, and many other sources of protein variation. However, the vast majority of proteomic studies on serum and plasma are carried out using peptide-centric, bottom-up approaches that cannot recapitulate the original proteoform content of samples. Clinical laboratories have been slow to adopt top-down analysis, also due to higher sample handling requirements. In this study, we describe a straightforward protocol for intact proteoform sample preparation based on the depletion of albumin and immunoglobulins, followed by simplified protein fractionation via polyacrylamide gel electrophoresis. After molecular weight-based fractionation, we supplemented the traditional liquid chromatography–tandem mass spectrometry (LC-MS2) data acquisition with high-field asymmetric waveform ion mobility spectrometry (FAIMS) to further simplify serum proteoform mixtures. This LC-FAIMS-MS2method led to the identification of over 1000 serum proteoforms < 30 kDa, outperforming traditional LC-MS2data acquisition and more than doubling the number of proteoforms identified in previous studies.
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- 2023
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5. Interlaboratory Study on Differential Analysis of Protein Glycosylation by Mass Spectrometry: The ABRF Glycoprotein Research Multi-Institutional Study 2012*
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Leymarie, Nancy, Griffin, Paula J., Jonscher, Karen, Kolarich, Daniel, Orlando, Ron, McComb, Mark, Zaia, Joseph, Aguilan, Jennifer, Alley, William R., Altmann, Friederich, Ball, Lauren E., Basumallick, Lipika, Bazemore-Walker, Carthene R., Behnken, Henning, Blank, Michael A., Brown, Kristy J., Bunz, Svenja-Catharina, Cairo, Christopher W., Cipollo, John F., Daneshfar, Rambod, Desaire, Heather, Drake, Richard R., Go, Eden P., Goldman, Radoslav, Gruber, Clemens, Halim, Adnan, Hathout, Yetrib, Hensbergen, Paul J., Horn, David M., Hurum, Deanna, Jabs, Wolfgang, Larson, Göran, Ly, Mellisa, Mann, Benjamin F., Marx, Kristina, Mechref, Yehia, Meyer, Bernd, Möginger, Uwe, Neusüβ, Christian, Nilsson, Jonas, Novotny, Milos V., Nyalwidhe, Julius O., Packer, Nicolle H., Pompach, Petr, Reiz, Bela, Resemann, Anja, Rohrer, Jeffrey S., Ruthenbeck, Alexandra, Sanda, Miloslav, Schulz, Jan Mirco, Schweiger-Hufnagel, Ulrike, Sihlbom, Carina, Song, Ehwang, Staples, Gregory O., Suckau, Detlev, Tang, Haixu, Thaysen-Andersen, Morten, Viner, Rosa I., An, Yanming, Valmu, Leena, Wada, Yoshinao, Watson, Megan, Windwarder, Markus, Whittal, Randy, Wuhrer, Manfred, Zhu, Yiying, and Zou, Chunxia
- Abstract
One of the principal goals of glycoprotein research is to correlate glycan structure and function. Such correlation is necessary in order for one to understand the mechanisms whereby glycoprotein structure elaborates the functions of myriad proteins. The accurate comparison of glycoforms and quantification of glycosites are essential steps in this direction. Mass spectrometry has emerged as a powerful analytical technique in the field of glycoprotein characterization. Its sensitivity, high dynamic range, and mass accuracy provide both quantitative and sequence/structural information. As part of the 2012 ABRF Glycoprotein Research Group study, we explored the use of mass spectrometry and ancillary methodologies to characterize the glycoforms of two sources of human prostate specific antigen (PSA). PSA is used as a tumor marker for prostate cancer, with increasing blood levels used to distinguish between normal and cancer states. The glycans on PSA are believed to be biantennary N-linked, and it has been observed that prostate cancer tissues and cell lines contain more antennae than their benign counterparts. Thus, the ability to quantify differences in glycosylation associated with cancer has the potential to positively impact the use of PSA as a biomarker. We studied standard peptide-based proteomics/glycomics methodologies, including LC-MS/MS for peptide/glycopeptide sequencing and label-free approaches for differential quantification. We performed an interlaboratory study to determine the ability of different laboratories to correctly characterize the differences between glycoforms from two different sources using mass spectrometry methods. We used clustering analysis and ancillary statistical data treatment on the data sets submitted by participating laboratories to obtain a consensus of the glycoforms and abundances. The results demonstrate the relative strengths and weaknesses of top-down glycoproteomics, bottom-up glycoproteomics, and glycomics methods.
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- 2013
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6. Hydrogen Atom Loss in Electron-Capture Dissociation: A Fourier Transform-Ion Cyclotron Resonance Study with Single Isotopomeric Ubiquitin Ions
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Breuker, Kathrin, Oh, HanBin, Cerda, Blas A., Horn, David M., and McLafferty, Fred W.
- Abstract
In electron-capture dissociation (ECD), a multiply-protonated protein ion, trapped in a Fourier transform-ion cyclotron resonance (FT-ICR) cell, captures a low-energy electron at a protonated site. In a major reaction pathway, the resulting hydrogen atom attacks a backbone carbonyl oxygen to form a hypervalent species that immediately dissociates into a complementary c, z•ion pair. For larger proteins, the reduced odd-electron ion (M + nH)(n−1)+•is a major product, as shown here using isotopically isolated precursors. In addition, a hydrogen atom can be lost without further reaction, yielding the [M + (n−1)H](n−1)+even-electron ions. The large effect of charge state on the yield of these ions suggests that the 9+ to 11+ charge states have novel charge-solvated secondary structures.
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- 2002
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7. A novel multifunctional labeling reagent for enhanced protein characterization with mass spectrometry
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Peters, Eric C., Horn, David M., Tully, David C., Brock, Ansgar, and Siuzdak, Gary
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Individual peptides with lysine at the C‐terminus as well as protein tryptic digests were reacted with 2‐methoxy‐4,5‐dihydro‐1H‐imidazole, converting lysine residues to their 4,5‐dihydro‐1H‐imidazol‐2‐yl derivatives. The mass spectra of derivatized digests exhibit a greater number of more intense features than their underivatized counterparts, thus increasing the information obtained in peptide mapping experiments. Additionally, MS/MS spectra of the derivatized peptides are greatly simplified in comparison to their native species, yielding primarily an easily interpretable series of y‐ions. Finally, this novel label also enables differential quantitation studies, as a stable isotopic form containing four deuterium atoms can readily be produced. Copyright © 2001 John Wiley & Sons, Ltd.
- Published
- 2001
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8. A novel multifunctional labeling reagent for enhanced protein characterization with mass spectrometry
- Author
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Peters, Eric C., Horn, David M., Tully, David C., and Brock, Ansgar
- Abstract
Individual peptides with lysine at the C-terminus as well as protein tryptic digests were reacted with 2-methoxy-4,5-dihydro-1H-imidazole, converting lysine residues to their 4,5-dihydro-1H-imidazol-2-yl derivatives. The mass spectra of derivatized digests exhibit a greater number of more intense features than their underivatized counterparts, thus increasing the information obtained in peptide mapping experiments. Additionally, MS/MS spectra of the derivatized peptides are greatly simplified in comparison to their native species, yielding primarily an easily interpretable series of y-ions. Finally, this novel label also enables differential quantitation studies, as a stable isotopic form containing four deuterium atoms can readily be produced. Copyright © 2001 John Wiley & Sons, Ltd.
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- 2001
- Full Text
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9. Blackbody infrared radiative dissociation of larger (42 kDa) multiply charged proteins11Dedicated to Professor Nico Nibbering on the occasion of his retirement.
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Ge, Ying, Horn, David M., and McLafferty, Fred W.
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Blackbody infrared radiative dissociation (BIRD), demonstrated originally with ions as large as 17 kDa, has been applied to larger proteins in a 6 T Fourier-transform mass spectrometer. For carbonic anhydrase (29 kDa), ThiF (27 kDa), and thiazole kinase (29 kDa), ion cell temperatures of 60–110 °C give mostly uninformative H2O loss, but 145 °C gives extensive backbone dissociation. For carbonic anhydrase ions at 70 °C, H2O loss continues for >240 s; thermalizing ions for ∼30 s reduces H2O loss eightfold. For thiaminase I (42 kDa), H2O loss is not observed, with backbone dissociation occurring above 150 °C. For these proteins, BIRD has effected cleavages of 34, 41, 23, and 28, respectively, backbone bonds. Although most are the same as those cleaved by infrared multiphoton dissociation and collisionally activated dissociation, some BIRD cleavages do provide additional and complementary sequence information. Carbonic anhydrase also shows extensive H2O loss from its fragment ions that compromises their validity for sequencing.
- Published
- 2001
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10. Charge/radical site initiation versus coulombic repulsion for cleavage of multiply charged ions. Charge solvation in poly(alkene glycol) ions
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Cerda, Blas A., Breuker, Kathrin, Horn, David M., and McLafferty, Fred W.
- Abstract
Electrospray ionization of poly(ethylene glycol) (PEG) followed by separation with Fourier-transform mass spectrometry traps (PEG100+ nH)n+ions. Both collisionally activated dissociation (CAD) and electron capture dissociation (ECD) of these ions (n= 5, 6, 7) produce PEGxfragment ions in which the xvalues correspond closely to those for an equal distribution of charges in the linear polymer ion, e.g., for n= 7, near x= 1, 17, 34, 50, 67, 83, and 100. However, positions intermediate between these charges should represent the maximum coulombic repulsion, so this is not a specific driving force for fragmentation, which is instead consistent with charge site (CAD) or radical site (ECD) initiation. These conclusions were confirmed by studies of a variety of other poly(alkene glycol) polymers. For these, the ECD spectra of the protonated species are consistent with the predicted charge solvation by the ion’s oxygen atoms.
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- 2001
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11. Electron capture dissociation of gaseous multiply charged ions by Fourier-transform ion cyclotron resonance
- Author
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McLafferty, Fred W., Horn, David M., Breuker, Kathrin, Ge, Ying, Lewis, Mark A., Cerda, Blas, Zubarev, Roman A., and Carpenter, Barry K.
- Abstract
Fourier-transform ion cyclotron resonance instrumentation is uniquely applicable to an unusual new ion chemistry, electron capture dissociation (ECD). This causes nonergodic dissociation of far larger molecules (42 kDa) than previously observed (<1 kDa), with the resulting unimolecular ion chemistry also unique because it involves radical site reactions for similarly larger ions. ECD is highly complementary to the well known energetic methods for multiply charged ion dissociation, providing much more extensive protein sequence information, including the direct identification of N- versus C-terminal fragment ions. Because ECD only excites the molecule near the cleavage site, accompanying rearrangements are minimized. Counterintuitively, cleavage of backbone covalent bonds of protein ions is favored over that of noncovalent bonds; larger (>10 kDa) ions give far more extensive ECD if they are first thermally activated. This high specificity for covalent bond cleavage also makes ECD promising for studying the secondary and tertiary structure of gaseous protein ions caused by noncovalent bonding.
- Published
- 2001
- Full Text
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12. Electron capture dissociation of gaseous multiply charged ions by Fourier-transform ion cyclotron resonance
- Author
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McLafferty, Fred W., Horn, David M., Breuker, Kathrin, Ge, Ying, Lewis, Mark A., Cerda, Blas, Zubarev, Roman A., and Carpenter, Barry K.
- Abstract
Fourier-transform ion cyclotron resonance instrumentation is uniquely applicable to an unusual new ion chemistry, electron capture dissociation (ECD). This causes nonergodic dissociation of far larger molecules (42 kDa) than previously observed (<1 kDa), with the resulting unimolecular ion chemistry also unique because it involves radical site reactions for similarly larger ions. ECD is highly complementary to the well known energetic methods for multiply charged ion dissociation, providing much more extensive protein sequence information, including the direct identification of N- versus C-terminal fragment ions. Because ECD only excites the molecule near the cleavage site, accompanying rearrangements are minimized. Counterintuitively, cleavage of backbone covalent bonds of protein ions is favored over that of noncovalent bonds; larger (>10 kDa) ions give far more extensive ECD if they are first thermally activated. This high specificity for covalent bond cleavage also makes ECD promising for studying the secondary and tertiary structure of gaseous protein ions caused by noncovalent bonding.
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- 2001
- Full Text
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13. Automated reduction and interpretation of
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Horn, David M., Zubarev, Roman A., and McLafferty, Fred W.
- Abstract
Here a fully automated computer algorithm is applied to complex mass spectra of peptides and proteins. This method uses a subtractive peak finding routine to locate possible isotopic clusters in the spectrum, subjecting these to a combination of the previous Fourier transform/ Patterson method for primary charge determination and the method for least-squares fitting to a theoretically derived isotopic abundance distribution for m/zdetermination of the most abundant isotopic peak, and the statistical reliability of this determination. If a predicted protein sequence is available, each such m/zvalue is checked for assignment as a sequence fragment. A new signal-to-noise calculation procedure has been devised for accurate determination of baseline and noise width for spectra with high peak density. In 2 h, the program identified 824 isotopic clusters representing 581 mass values in the spectrum of a GluC digest of a 191 kDa protein; this is \s>50% more than the number of mass values found by the extremely tedious operator-applied methodology used previously. The program should be generally applicable to classes of large molecules, including DNA and polymers. Thorough high resolution analysis of spectra by Horn (THRASH) is proposed as the program’s verb.
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- 2000
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14. Electron Capture Dissociation of Multiply-Charged Oxygenated Cations. A Nonergodic Process
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Cerda, Blas A., Horn, David M., Breuker, Kathrin, Carpenter, Barry K., and McLafferty, Fred W.
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Several mechanistic aspects have been proposed as important in causing the unusual ion chemistry induced in multiply-charged protein cations by electron capture. The 5–7 eV energy released by neutralization appears to induce cleavage before energy randomization (nonergodic), and the electron forms radical species whose activation energies for dissociation should be much lower. In contrast, electron capture by [HO(C2H4O)24H + 2H]2+ions from polyethylene glycol yields no radical ions, losing H•consistent with the lower H•affinity of the hydroxyl and ether groups vs the amide and S–S functionalities of proteins. However, the dominant product ions, [HO(C2H4O)24–nH + H]+(n= 2 to 8), do appear to be formed by nonergodic dissociation of the hypervalent (M + 2H)1+•intermediate. The expected complementary alkoxy radical ion product is not found, possibly due to an energetic Franck–Condon relaxation. Precursors ionized with (NH4)22+and Na22+yield ECD products that are analogous but of different size (nvalues). Those for Na22+can be rationalized with structures proposed by Bowers and coworkers. ECD spectra of polyethers should be useful for sequencing.
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- 1999
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15. Simultaneous measurement of flight time and energy of large matrix-assisted laser desorption ionization ions with a superconducting tunnel junction detector
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Banner, W. Henry, Horn, David M., Jaklevic, Joseph M., Frank, Matthias, Mears, Carl, Labov, Simon, and Barfknecht, A. T.
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We evaluated a cryogenically cooled superconducting Nb-Al2O3-Nb tunnel junction (STJ) for use as a molecular ion detector in a matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometer. The STJ responds to ion energy and theoretically should detect large molecular ions with a velocity-independent efficiency approaching 100%. The STJ detector produces pulses whose heights are approximately proportional to ion energy, thus the height of a pulse generated by the impact of a doubly charged ion is about twice the height of a singly charged ion pulse. Measurements were performed by bombarding the STJ with human serum albumin (HSA) (66,000 Da) and immunoglobulin (150,000 Da) ions. We demonstrate that pulse height analysis of STJ signals provides a way to distinguish with good discrimination HSA+from 2HSA2+, whose flight times are coincident. The rise time of STJ detector pulses allows ion flight times to be determined with a precision better than 200 ns, which is a value smaller than the flight time variation typically observed for large isobaric MALDI ions detected with conventional microchannel plate (MCP) detectors. Deflection plates in the flight tube of the mass spectrometer provided a way to aim ions alternatively at a MCP ion detector.
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- 1997
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