Publications by authors named "David P A Kilgour"

30 Publications

  • Page 1 of 1

Rapid Denaturing Organic Digestion Method for Targeted Protein Identification and Characterization.

Anal Chem 2021 03 17;93(12):5046-5053. Epub 2021 Mar 17.

Nottingham Trent University, Clifton Campus, College Drive, Nottingham NG11 8NS, U.K.

Bottom-up mass spectrometry-based protein analysis methods employing protease digestion are routinely used to identify and characterize proteins with high specificity and sensitivity. Method performance is generally measured by sequence coverage capability and the total number of characteristic peptides identified, when compared to predicted databases. Limitations to commonly used solvent-based digestion methods currently employed include long digestion times (18-24 h or more), leading to protease autolysis, which also precludes automation, decreases sensitivity, and increases both intra- and inter-day performance variability. This report describes the development and validation of a simple, 5 min tryptic denaturing organic digestion (DOD) method for use with tandem mass spectrometry in bottom-up protein identification and characterization. It has been evaluated across select protein toxins and diagnostic clinical protein targets, substantially improving digestion performance when compared to other solution-based and enzyme-immobilized methods. The method was compared to two currently used bottom-up methods, the 24 h filter-aided sample prep (FASP) and Flash Digest (1 and 4 h) methods. Single proteins used to compare the methods included the ricin light chain, ricin heavy chain, ricin holotoxin, serotype A toxin, enterotoxin B, ribonuclease A, and thyroglobulin. In tests, across the proteins investigated, the 5 min DOD digestion method resulted in sequence coverages ranging from 55 to 100%, with relatively high reproducibility and precision; results were better than or equal to FASP method results and were greatly enhanced when compared to Flash method results. Importantly, DOD method intra- and inter-day precision was much improved as compared to results for both FASP and Flash digestions. These data indicated that the DOD method, when compared to the FASP and Flash Digest methods, dramatically reduced digestion time, while maintaining or improving the ability to detect and characterize targeted proteins, and reduced analytical variability for tryptic digestion, resulting in markedly faster and more precise analyses.
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http://dx.doi.org/10.1021/acs.analchem.0c04143DOI Listing
March 2021

Improved N- and C-Terminal Sequencing of Proteins by Combining Positive and Negative Ion MALDI In-Source Decay Mass Spectrometry.

Anal Chem 2020 09 1;92(18):12429-12436. Epub 2020 Sep 1.

Center for Proteomics & Metabolomics, Leiden University Medical Center, Albinusdreef 2, Leiden 2333 ZA, The Netherlands.

The development of various ionization and fragmentation techniques has been of key importance for establishing mass spectrometry (MS) as a powerful tool for protein characterization. One example of this is matrix-assisted laser desorption/ionization (MALDI) combined with in-source decay (ISD) fragmentation that allows mapping of N- and C-terminal regions of large proteins without the need for proteolysis. Positive ion mode ISD fragments are commonly assigned in the mass region above / 1000, while MALDI matrix ions generally hamper the detection of smaller singly charged fragments. The ultrahigh resolving power provided by Fourier transform ion cyclotron resonance (FT-ICR) MS partially overcomes this limitation, but to further increase the detection of smaller fragments we have revisited the application of negative ion mode MALDI-ISD and found good coverage of the peptide chain termini starting from c'2 and z'2 fragment ions. For the first time, we demonstrate that the combination of negative and positive ion MALDI FT-ICR MS is a useful tool to improve the characterization of mAbs. The different specificities of the two ion modes allowed us to selectively cover the sequence of the light and heavy chains of mAbs at increased sensitivity. A comprehensive evaluation of positive and negative ion mode MALDI-ISD FT-ICR MS in the / range 46-13 500 showed an increased sequence coverage for three standard proteins, namely, myoglobin, SiLuLite mAb, and NIST mAb. The data obtained in the two ion modes were, in part, complementary.
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http://dx.doi.org/10.1021/acs.analchem.0c02198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7498143PMC
September 2020

Evaluation of Sibling and Twin Fragment Ions Improves the Structural Characterization of Proteins by Top-Down MALDI In-Source Decay Mass Spectrometry.

Anal Chem 2020 04 2;92(8):5871-5881. Epub 2020 Apr 2.

Center for Proteomics & Metabolomics, Leiden University Medical Center, Leiden 2333, ZA, The Netherlands.

Comprehensive determination of primary sequence and identification of post-translational modifications (PTMs) are key elements in protein structural analysis. Various mass spectrometry (MS) based fragmentation techniques are powerful approaches for mapping both the amino acid sequence and PTMs; one of these techniques is matrix-assisted laser desorption/ionization (MALDI), combined with in-source decay (ISD) fragmentation and Fourier-transform ion cyclotron resonance (FT-ICR) MS. MALDI-ISD MS protein analysis involves only minimal sample preparation and does not require spectral deconvolution. The resulting MALDI-ISD MS data is complementary to electrospray ionization-based MS/MS sequencing readouts, providing knowledge on the types of fragment ions is available. In this study, we evaluate the isotopic distributions of ' ions in protein top-down MALDI-ISD FT-ICR mass spectra and show why these distributions can deviate from theoretical profiles as a result of co-occurring and isomeric and -NH ions. Two synthetic peptides, containing either normal or deuterated alanine residues, were used to confirm the presence and unravel the identity of isomeric and -NH fragment ions ("twins"). Furthermore, two reducing MALDI matrices, namely 1,5-diaminonaphthalene and -phenyl--phenylenediamine were applied that yield ISD mass spectra with different fragment ion distributions. This study demonstrates that the relative abundance of isomeric and -NH ions requires consideration for accurate and confident assignments of ' ions in MALDI-ISD FT-ICR mass spectra.
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http://dx.doi.org/10.1021/acs.analchem.9b05683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7178258PMC
April 2020

Isotope Depletion Mass Spectrometry (ID-MS) for Accurate Mass Determination and Improved Top-Down Sequence Coverage of Intact Proteins.

J Am Soc Mass Spectrom 2020 Mar 11;31(3):700-710. Epub 2020 Feb 11.

The EastChem School of Chemistry, University of Edinburgh, Joseph Black Building, Brewster Road, Edinburgh EH9 3FJ, U.K.

Top-down mass spectrometry (MS) is an increasingly important technique for protein characterization. However, in many biological MS experiments, the practicality of applying top-down methodologies is still limited at higher molecular mass. In large part, this is due to the detrimental effect resulting from the partitioning of the mass spectral signal into an increasing number of isotopic peaks as molecular mass increases. Reducing the isotopologue distribution of proteins via depletion of heavy stable isotopes was first reported over 20 years ago (Marshall, A. G.; Senko, M. W.; Li, W.; Li, M.; Dillon, S., Guan, S.; Logan, T. M.. Protein Molecular Mass to 1 Da by C, N Double-Depletion and FT-ICR Mass Spectrometry. 1997, 119, 433-434.) and has been demonstrated for several small proteins. Here we extend this approach, introducing a new highly efficient method for the production of recombinant proteins depleted in C and N and demonstrating its advantages for top-down analysis of larger proteins (up to ∼50 kDa). FT-ICR MS of isotopically depleted proteins reveals dramatically reduced isotope distributions with monoisotopic signal observed up to 50 kDa. In top-down fragmentation experiments, the reduced spectral complexity alleviates fragment-ion signal overlap, the presence of monoisotopic signals allows assignment with higher mass accuracy, and the dramatic increase in signal-to-noise ratio (up to 7-fold) permits vastly reduced acquisition times. These compounding benefits allow the assignment of ∼3-fold more fragment ions than comparable analyses of proteins with natural isotopic abundances. Finally, we demonstrate greatly increased sequence coverage in time-limited top-down experiments-highlighting advantages for top-down LC-MS/MS workflows and top-down proteomics.
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http://dx.doi.org/10.1021/jasms.9b00119DOI Listing
March 2020

Monitoring glycation levels of a bispecific monoclonal antibody at subunit level by ultrahigh-resolution MALDI FT-ICR mass spectrometry.

MAbs 2020 Jan-Dec;12(1):1682403

Leiden University Medical Center, Center for Proteomics and Metabolomics, Leiden, The Netherlands.

Bispecific monoclonal antibodies (BsAbs) are engineered proteins with multiple functionalities and properties. The "bi-specificity" of these complex biopharmaceuticals is a key characteristic for the development of novel and more effective therapeutic strategies. The high structural complexity of BsAbs poses a challenge to the analytical methods needed for their characterization. Modifications of the BsAb structure, resulting from enzymatic and non-enzymatic processes, further complicate the analysis. An important example of the latter type of modification is glycation, which can occur in the manufacturing process, during storage in the formulation or after application of the drug. Glycation affects the structure, function, and stability of monoclonal antibodies, and consequently, a detailed analysis of glycation levels is required. Mass spectrometry (MS) plays a key role in the structural characterization of monoclonal antibodies and top-down, middle-up and middle-down MS approaches are increasingly used for the analysis of modifications. Here, we apply a novel middle-up strategy, based on IdeS digestion and matrix-assisted laser desorption ionization (MALDI) Fourier transform ion cyclotron resonance (FT-ICR) MS, to analyze all six different BsAb subunits in a single high-resolution mass spectrum, namely two light chains, two half fragment crystallizable regions and two Fd' regions, thus avoiding upfront chromatography. This method was used to monitor glycation changes during a 168 h forced-glycation experiment. In addition, hot spot glycation sites were localized using top-down and middle-down MALDI-in-source decay FT-ICR MS, which provided complementary information compared to standard bottom-up MS.
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http://dx.doi.org/10.1080/19420862.2019.1682403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6927770PMC
January 2021

Phase relationships in two-dimensional mass spectrometry.

J Am Soc Mass Spectrom 2019 Dec 15;30(12):2594-2607. Epub 2019 Oct 15.

Department of Chemistry, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, UK.

Two-dimensional mass spectrometry (2D MS) is a data-independent tandem mass spectrometry technique in which precursor and fragment ion species can be correlated without the need for prior ion isolation. The behavior of phase in 2D Fourier transform mass spectrometry is investigated with respect to the calculation of phase-corrected absorption-mode 2D mass spectra. 2D MS datasets have a phase that is defined differently in each dimension. In both dimensions, the phase behavior of precursor and fragment ions is found to be different. The dependence of the phase for both precursor and fragment ion signals on various parameters (e.g., modulation frequency, shape of the fragmentation zone) is discussed. Experimental data confirms the theoretical calculations of the phase in each dimension. Understanding the phase relationships in a 2D mass spectrum is beneficial to the development of possible algorithms for phase correction, which may improve both the signal-to-noise ratio and the resolving power of peaks in 2D mass spectra.
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http://dx.doi.org/10.1007/s13361-019-02308-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6914722PMC
December 2019

A Light-Activated Acyl Carrier Protein "Trap" for Intermediate Capture in Type II Iterative Polyketide Biocatalysis.

Chemistry 2019 Oct 9. Epub 2019 Oct 9.

Department of Chemistry, University of Warwick, Library Road, Coventry, CV4 7AL, UK.

A discrete acyl carrier protein (ACP) bearing a photolabile nonhydrolysable carba(dethia) malonyl pantetheine cofactor was chemoenzymatically prepared and utilised for the trapping of biosynthetic polyketide intermediates following light activation. From the in vitro assembly of the polyketides SEK4 and SEK4b, by the type II actinorhodin "minimal" polyketide synthase (PKS), a range of putative ACP-bound diketides, tetraketides, pentaketides and hexaketides were identified and characterised by FT-ICR-MS, providing direct insights on active site accessibility and substrate processing for this enzyme class.
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http://dx.doi.org/10.1002/chem.201903662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6972679PMC
October 2019

Increased throughput and ultra-high mass resolution in DESI FT-ICR MS imaging through new-generation external data acquisition system and advanced data processing approaches.

Sci Rep 2019 01 9;9(1). Epub 2019 Jan 9.

Maastricht University, Maastricht Multimodal Molecular Imaging Institute (M4I), Maastricht, 6229ER, The Netherlands.

Desorption electrospray ionisation-mass spectrometry imaging (DESI-MSI) is a powerful imaging technique for the analysis of complex surfaces. However, the often highly complex nature of biological samples is particularly challenging for MSI approaches, as options to appropriately address molecular complexity are limited. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) offers superior mass accuracy and mass resolving power, but its moderate throughput inhibits broader application. Here we demonstrate the dramatic gains in mass resolution and/or throughput of DESI-MSI on an FT-ICR MS by developing and implementing a sophisticated data acquisition and data processing pipeline. The presented pipeline integrates, for the first time, parallel ion accumulation and detection, post-processing absorption mode Fourier transform and pixel-by-pixel internal re-calibration. To achieve that, first, we developed and coupled an external high-performance data acquisition system to an FT-ICR MS instrument to record the time-domain signals (transients) in parallel with the instrument's built-in electronics. The recorded transients were then processed by the in-house developed computationally-efficient data processing and data analysis software. Importantly, the described pipeline is shown to be applicable even to extremely large, up to 1 TB, imaging datasets. Overall, this approach provides improved analytical figures of merits such as: (i) enhanced mass resolution at no cost in experimental time; and (ii) up to 4-fold higher throughput while maintaining a constant mass resolution. Using this approach, we not only demonstrate the record 1 million mass resolution for lipid imaging from brain tissue, but explicitly show such mass resolution is required to resolve the complexity of the lipidome.
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http://dx.doi.org/10.1038/s41598-018-36957-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6327097PMC
January 2019

Structural Analysis of Monoclonal Antibodies by Ultrahigh Resolution MALDI In-Source Decay FT-ICR Mass Spectrometry.

Anal Chem 2019 02 8;91(3):2079-2085. Epub 2019 Jan 8.

Center for Proteomics and Metabolomics , Leiden University Medical Center (LUMC) , PO Box 9600, 2300 RC , Leiden , The Netherlands.

The emergence of complex protein therapeutics in general and monoclonal antibodies (mAbs) in particular have stimulated analytical chemists to develop new methods and strategies for their structural characterization. Mass spectrometry plays a key role in providing information on the primary amino acid sequence, post-translational modifications, and other structure characteristics that must be monitored during the manufacturing process and subsequent quality control assessment. In this study, we present a novel method that allows structural characterization of mAbs based on MALDI in-source decay (ISD) fragmentation, coupled with Fourier transform ion cyclotron resonance (FT-ICR) MS. The method benefits from higher resolution of absorption mode FT mass spectra, compared to magnitude mode, which enables simultaneous identification of ISD fragments from both the heavy and light chains with a higher confidence in a wide mass range up to m/ z 13 500. This method was applied to two standard mAbs, namely NIST mAb and trastuzumab, in preparation for method application in an interlaboratory study on mAbs structural analysis coordinated by the Consortium for Top-Down Proteomics. Extensive sequence coverage was obtained from the middle-down analysis (IdeS- and GingisKHAN-digested mAbs) that complemented the top-down analysis of intact mAbs. In addition, MALDI FT-ICR MS of IdeS-digested mAbs allowed isotopic-level profiling of proteoforms with regard to heavy chain N-glycosylation.
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http://dx.doi.org/10.1021/acs.analchem.8b04515DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6365908PMC
February 2019

Perspectives on the future of mass spectrometry.

Rapid Commun Mass Spectrom 2019 07 2;33 Suppl 3:1-2. Epub 2019 Apr 2.

Nottingham Trent University, Nottingham, NG1 4FQ, UK.

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http://dx.doi.org/10.1002/rcm.8346DOI Listing
July 2019

Rapid Food Product Analysis by Surface Acoustic Wave Nebulization Coupled Mass Spectrometry.

Food Anal Methods 2018 Sep 21;11(9):2447-2454. Epub 2018 Mar 21.

Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD 21201, USA.

Rapid food product analysis is of great interest for quality control and assurance during the production process. Conventional quality control protocols require time and labor intensive sample preparation for analysis by state-of-the-art analytical methods. To reduce overall cost and facilitate rapid qualitative assessments, food products need to be tested with minimal sample preparation. We present a novel and simple method for assessing food product compositions by mass spectrometry using a novel surface acoustic wave nebulization method. This method provides significant advantages over conventional methods requiring no pumps, capillaries, or additional chemicals to enhance ionization for mass spectrometric analysis. In addition, the surface acoustic wave nebulization - mass spectrometry method is ideal for rapid analysis and to investigate certain compounds by using the mass spectra as a type of species-specific fingerprint analysis. We present for the first time surface acoustic wave nebulization generated mass spectra of a variety of fermented food products from a small selection of vinegars, wines, and beers.
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http://dx.doi.org/10.1007/s12161-018-1232-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6157634PMC
September 2018

Correction to: Top Down Tandem Mass Spectrometric Analysis of a Chemically Modified Rough-Type Lipopolysaccharide Vaccine Candidate.

J Am Soc Mass Spectrom 2018 06;29(6):1230

Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Pharmacy Hall North Room 623, 20 N. Pine St, Baltimore, MD, 21201, USA.

In the preceding article "Top Down Tandem Mass Spectrometric Analysis of a Chemically Modified Rough-Type Lipopolysaccharide Vaccine Candidate" by Oyler et al., an error in the J5 E. coli LPS chemical structure (Figs. 2 and 4) was introduced and propagated into the final revision.
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http://dx.doi.org/10.1007/s13361-018-1960-8DOI Listing
June 2018

Top Down Tandem Mass Spectrometric Analysis of a Chemically Modified Rough-Type Lipopolysaccharide Vaccine Candidate.

J Am Soc Mass Spectrom 2018 06 20;29(6):1221-1229. Epub 2018 Feb 20.

Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Pharmacy Hall North Room 623, 20 N. Pine St, Baltimore, MD, 21201, USA.

Recent advances in lipopolysaccharide (LPS) biology have led to its use in drug discovery pipelines, including vaccine and vaccine adjuvant discovery. Desirable characteristics for LPS vaccine candidates include both the ability to produce a specific antibody titer in patients and a minimal host inflammatory response directed by the innate immune system. However, in-depth chemical characterization of most LPS extracts has not been performed; hence, biological activities of these extracts are unpredictable. Additionally, the most widely adopted workflow for LPS structure elucidation includes nonspecific chemical decomposition steps before analyses, making structures inferred and not necessarily biologically relevant. In this work, several different mass spectrometry workflows that have not been previously explored were employed to show proof-of-principle for top down LPS primary structure elucidation, specifically for a rough-type mutant (J5) E. coli-derived LPS component of a vaccine candidate. First, ion mobility filtered precursor ions were subjected to collision induced dissociation (CID) to define differences in native J5 LPS v. chemically detoxified J5 LPS (dLPS). Next, ultra-high mass resolving power, accurate mass spectrometry was employed for unequivocal precursor and product ion empirical formulae generation. Finally, MS analyses in an ion trap instrument showed that previous knowledge about dissociation of LPS components can be used to reconstruct and sequence LPS in a top down fashion. A structural rationale is also explained for differential inflammatory dose-response curves, in vitro, when HEK-Blue hTLR4 cells were administered increasing concentrations of native J5 LPS v. dLPS, which will be useful in future drug discovery efforts. Graphical Abstract ᅟ.
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http://dx.doi.org/10.1007/s13361-018-1897-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8294406PMC
June 2018

Native-MS Analysis of Monoclonal Antibody Conjugates by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

Anal Chem 2018 01 18;90(1):745-751. Epub 2017 Dec 18.

Department of Chemistry and Biochemistry, and Department of Biological Chemistry, University of California-Los Angeles , Los Angeles, California 90095, United States.

Antibody-drug conjugates (ADCs) are an important class of therapeutic molecule currently being used to treat HER2-positive metastatic breast cancer, relapsed or refractory Hodgkin lymphoma, systemic anaplastic large cell lymphoma, relapsed or refractory B-cell precursor acute lymphoblastic leukemia, and acute myeloid leukemia. An ADC typically consists of a small molecule or peptide-based cytotoxic moiety covalently linked, via lysine or cysteine residues, to a monoclonal antibody (mAb) scaffold. Mass spectrometric (MS) characterization of these molecules affords highly accurate molecular weight (MW) and drug-to-antibody ratio (DAR) determination and is typically performed using orthogonal acceleration time-of-flight (oa-ToF) analyzers and more recently, Orbitrap instruments. Herein we describe for the first time the use of a 15 T solariX Fourier transform ion cyclotron mass spectrometer to characterize an IgG1 mAb molecule conjugated with biotin via native lysine and cysteine residues, under native-MS and solution conditions. The cysteine-biotin conjugates remained fully intact, demonstrating the ability of the FT-ICR to maintain the noncovalent interactions and efficiently transmit labile protein complexes. Native-MS was acquired and is displayed in magnitude mode using a symmetric Hann apodization function. Baseline separation is achieved on all covalent biotin additions, for each charge state, for both the lysine- and cysteine-biotin conjugates. Average DAR values obtained by native-MS for the lysine conjugate are compared to those derived by denaturing reversed phase liquid chromatography using an oa-ToF MS system (1.56 ± 0.02 versus 2.24 ± 0.02 for the 5 equivalent and 3.99 ± 0.09 versus 4.43 ± 0.01 for the 10 equivalent, respectively). Increased DAR value accuracy can be obtained for the higher biotin-load when using standard ESI conditions as opposed to nanoESI native-MS conditions.
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http://dx.doi.org/10.1021/acs.analchem.7b03021DOI Listing
January 2018

Autopiquer - a Robust and Reliable Peak Detection Algorithm for Mass Spectrometry.

J Am Soc Mass Spectrom 2017 Feb 6;28(2):253-262. Epub 2016 Dec 6.

Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD, 21201, USA.

We present a simple algorithm for robust and unsupervised peak detection by determining a noise threshold in isotopically resolved mass spectrometry data. Solving this problem will greatly reduce the subjective and time-consuming manual picking of mass spectral peaks and so will prove beneficial in many research applications. The Autopiquer approach uses autocorrelation to test for the presence of (isotopic) structure in overlapping windows across the spectrum. Within each window, a noise threshold is optimized to remove the most unstructured data, whilst keeping as much of the (isotopic) structure as possible. This algorithm has been successfully demonstrated for both peak detection and spectral compression on data from many different classes of mass spectrometer and for different sample types, and this approach should also be extendible to other types of data that contain regularly spaced discrete peaks. Graphical Abstract ᅟ.
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http://dx.doi.org/10.1007/s13361-016-1549-zDOI Listing
February 2017

Norharmane Matrix Enhances Detection of Endotoxin by MALDI-MS for Simultaneous Profiling of Pathogen, Host, and Vector Systems.

Pathog Dis 2016 11 19;74(8). Epub 2016 Sep 19.

Department of Microbial Pathogenesis, School of Dentistry, University of Maryland Baltimore, Baltimore, Maryland, USA

The discovery of novel pathogenic mechanisms engaged during bacterial infections requires the evolution of advanced techniques. Here, we evaluate the dual polarity matrix norharmane (NRM) to improve detection of bacterial lipid A (endotoxin), from host and vector tissues infected with Francisella novicida (Fn). We evaluated NRM for improved detection and characterization of a wide range of lipids in both positive and negative polarities, including lipid A and phospholipids across a range of matrix assisted laser desorption-ionization (MALDI)-coupled applications. NRM matrix improved the limit of detection (LOD) for monophosphoryl lipid A (MPLA) down to picogram-level representing a ten-fold improvement of LOD versus 2,5-dihydroxybenzoic acid (DHB) and 100-fold improvement of LOD versus 9-aminoacridine (9-AA). Improved LOD for lipid A subsequently facilitated detection of the Fn lipid A major ion (m/z 1665) from extracts of infected mouse spleen and the temperature-modified Fn lipid A at m/z 1637 from infected D. variabilis ticks. Finally, we simultaneously mapped bacterial phospholipid signatures within an Fn infected spleen along with exclusively host-derived inositol-based phospholipid (m/z 933) demonstrating co-profiling for the host-pathogen interaction. Expanded use of NRM matrix in other infection models and endotoxin-targeting imaging experiments will improve our understanding of the lipid interactions at the host-pathogen interface.
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http://dx.doi.org/10.1093/femspd/ftw097DOI Listing
November 2016

Surface acoustic wave nebulization device with dual interdigitated transducers improves SAWN-MS performance.

J Mass Spectrom 2016 Jun;51(6):424-9

Deurion, LLC, Seattle, WA, USA.

We compared mass spectrometric (MS) performance of surface acoustic wave nebulization (SAWN) generated by a single interdigitated transducer (IDT) designed to produce a progressive wave (PW) to one with a dual IDT that can in theory generate standing waves (SW). Given that devices using dual IDTs had been shown to produce fewer large size droplets on average, we hypothesized they would improve MS performance by improving the efficiency of desolvation. Indeed, the SW-SAWN chip provided an improved limit of detection of 1 femtomole of peptide placed on chip making it 100× more sensitive than the PW design. However, as measured by high-speed image recording and phase Doppler particle analyzer measurements, there was only a 26% increase in the small diameter (1-10 µm) droplets produced from the new device, precluding a conclusion that the decrease in droplet size was solely responsible for the improvement in MS signal/noise. Given that the dual IDT design produced a more instantaneous plume than the PW design, the more likely contributor to improved MS signal/noise was concluded to be a higher ion flux entering the mass spectrometer for the dual IDT designs. Notably, the dual IDT device allowed production of much higher quality protein mass spectra up to about 20 kDa, compared with the single IDT device. Copyright © 2016 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/jms.3766DOI Listing
June 2016

Glycosylation characterization of therapeutic mAbs by top- and middle-down mass spectrometry.

Data Brief 2016 Mar 24;6:68-76. Epub 2015 Nov 24.

Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD, USA.

A reference monoclonal antibody IgG1 and a fusion IgG protein were analyzed by top- and middle-down mass spectrometry with multiple fragmentation techniques including electron transfer dissociation (ETD) and matrix-assisted laser desorption ionization in-source decay (MALDI-ISD) to investigate heterogeneity of glycosylated protein species. Specifically, glycan structure, sites, relative abundance levels, and termini structural conformation were investigated by use of Fourier transform ion cyclotron resonance (FT-ICR) or high performance liquid chromatography electrospray ionization (HPLC-ESI) linked to an Orbitrap. Incorporating a limited enzymatic digestion by immunoglobulin G-degrading enzyme Streptococcus pyogenes (IdeS) with MALDI-ISD analysis extended sequence coverage of the internal region of the proteins without pre-fractionation. The data in this article is associated with the research article published in Journal of Proteomics (Tran et al., 2015) [1].
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http://dx.doi.org/10.1016/j.dib.2015.11.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4688415PMC
March 2016

An ambient detection system for visualization of charged particles generated with ionization methods at atmospheric pressure.

Rapid Commun Mass Spectrom 2016 Feb;30(3):352-8

FOM Institute AMOLF, Science Park 104, 1098 XG, Amsterdam, The Netherlands.

Rationale: With the current state-of-the-art detection of ions only taking place under vacuum conditions, active pixel detectors that operate under ambient conditions are of particular interest. These detectors are ideally suited to study and characterize the charge distributions generated by ambient ionization sources.

Methods: The direct imaging capabilities of the active pixel detector are used to investigate the spatial distributions of charged droplets generated by three ionization sources, named electrospray ionization (ESI), paper spray ionization (PSI) and surface acoustic wave nebulization (SAWN). The ionization spray (ESI/PSI) and ionization plume (SAWN) originating from each source are directly imaged. The effect of source parameters such as spray voltage for ESI and PSI, and the angle of the paper spray tip on the charge distributions, is investigated. Two types of SAWN liquid interface, progressive wave (PW) and standing wave (SW), are studied.

Results: Direct charge detection under ambient conditions is demonstrated using an active pixel detector. Direct charge distributions are obtained of weak, homogeneous/focused and dispersed spray plumes by applying low, intermediate and high spray potentials, respectively, for ESI. Spray plume footprints obtained for various angles of PSI shows the possibility to focus the ion beam as a function of the paper angle. Differences between two designs of the SAWN interface are determined. Droplet charge flux changes are illustrated in a way similar to a total ion chromatogram.

Conclusions: The use of this active pixel detector allows the rapid characterization and optimization of different ambient ionization sources without the actual use of a mass spectrometer. Valuable illustrations are obtained of changes in spatial distribution and number of charges detected for ESI, PSI and SAWN ion plumes. Copyright © 2015 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/rcm.7442DOI Listing
February 2016

Comprehensive glycosylation profiling of IgG and IgG-fusion proteins by top-down MS with multiple fragmentation techniques.

J Proteomics 2016 Feb 17;134:93-101. Epub 2015 Oct 17.

Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD, USA. Electronic address:

Unlabelled: We employed top- and middle-down analyses with multiple fragmentation techniques including electron transfer dissociation (ETD), electron capture dissociation (ECD), and matrix-assisted laser desorption ionization in-source decay (MALDI-ISD) for characterization of a reference monoclonal antibody (mAb) IgG1 and a fusion IgG protein. Fourier transform ion cyclotron resonance (FT-ICR) or high performance liquid chromatography electrospray ionization (HPLC-ESI) on an Orbitrap was employed. These experiments provided a comprehensive view on the protein species; especially for different glycosylation level in these two proteins, which showed good agreement with oligosaccharide profiling. Top- and middle-down MS provided additional information regarding glycosylation sites and different combinational protein species that were not available from oligosaccharide mapping or conventional bottom-up analysis. Finally, incorporating a limited enzymatic digestion by immunoglobulin G-degrading enzyme of Streptococcus pyogene (IdeS) with MALDI-ISD analysis enabled extended sequence coverage of the internal region of protein without pre-fractionation.

Biological Significance: Oligosaccharide profiling together with top- and middle-down methods enabled: 1) detection of heterogeneous glycosylated protein species and sites in intact IgG1 and fusion proteins with high mass accuracy, 2) estimation of relative abundance levels of protein species in the sample, 3) confirmation of the protein termini structural information, and 4) improved sequence coverage by MALDI-ISD analysis for the internal regions of the proteins without sample pre-fractionation.
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http://dx.doi.org/10.1016/j.jprot.2015.10.021DOI Listing
February 2016

Producing absorption mode Fourier transform ion cyclotron resonance mass spectra with non-quadratic phase correction functions.

Rapid Commun Mass Spectrom 2015 Jun;29(11):1087-93

Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne, 1015, Lausanne, Switzerland.

Rationale: Previously described methods for producing absorption mode Fourier transform ion cyclotron resonance (FTICR) mass spectra have all relied on the phase correction function being quadratic. This assumption has been found to be invalid for some instruments and spectra and so it has not been possible to produce absorption mode spectra for these cases.

Methods: The Autophaser algorithm has been adapted to allow nth order polynomial phase correction functions to be optimized. The data was collected on a modified Thermo LTQ FTICR mass spectrometer, using electrospray ionization and a novel ICR cell design (NADEL). Peak assignment and mass calibration were undertaken using the pyFTMS framework.

Results: An nth-order phase correction function has been used to produce an absorption mode mass spectrum of the maltene fraction of a crude oil sample which was not possible using the previous assumption that the phase correction function must be quadratic. Data processing for this spectrum in absorption mode has shown the expected benefits in terms of increasing the number of assigned peaks and also improving the mass accuracy (i.e. confidence) of the assignments.

Conclusions: It is possible to phase-correct time-domain data in FTICRMS to yield absorption mode mass spectra representation even when the data does not correspond to the theoretical quadratic phase correction function predicted by previous studies. This will allow a larger proportion of spectra to be processed in absorption mode.
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http://dx.doi.org/10.1002/rcm.7200DOI Listing
June 2015

Absorption mode Fourier transform mass spectrometry with no baseline correction using a novel asymmetric apodization function.

Rapid Commun Mass Spectrom 2015 Jun;29(11):1009-18

Bruker Daltonics Inc, Billerica, MA, USA.

Rationale: Absorption mode Fourier transform ion cyclotron resonance (FTICR) mass spectra offer significant benefits in terms of spectral resolution, signal-to-noise (S/N) ratio and measured mass accuracy. However, to date, methods for producing absorption mode spectra have created an undesirable baseline deviation as a consequence of FFT artifacts, resulting in interference of the frequency side-lobes of intense peaks. Methods for fitting and removing this deviation have been developed, but these are computationally intensive, slow and can be unreliable in practice.

Methods: We have developed an approach for producing FTICR mass spectra which uses a new apodization approach to produce spectra which do not exhibit baseline deviation, whilst maintaining all the normal absorption mode benefits. This method involves the use of 'full' apodization function, replacing the more common Hann or half Hann functions, and where the user can control the position of the function maximum expressed as a fraction (F) of the transient length.

Results: Absorption mode spectra produced using the new apodization function we propose provide all the normal benefits but do not exhibit baseline deviation that must be corrected prior to spectral interpretation. Additionally, varying the value of the F parameter allows users additional control over the compromise between the spectral resolving power and the S/N ratio. This is particularly beneficial in spectra with pronounced amplitude changes during the recording of the transient (detection).

Conclusions: The use of a 'full' apodization function, which may be asymmetric, prior to zero-padding and Fourier transformation, allows the production of absorption mode spectra which do not suffer from baseline deviation. Hence, it is no longer necessary to apply a baseline deviation correction in post processing, providing a significant performance advantage.
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http://dx.doi.org/10.1002/rcm.7190DOI Listing
June 2015

Producing Isotopic Distribution Models for Fully Apodized Absorption Mode FT-MS.

Anal Chem 2015 Jun 15;87(11):5797-801. Epub 2015 May 15.

†Mass Spectrometry Center, Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland 21201, United States.

Isotopic distributions are frequently used as part of the peak assignment process in the processing of mass spectra. The best methods for producing accurate peak assignments must account for the peak shape and resolving power. In other words, the full profile of the isotopic distribution is important. Conventional methods for modeling isotopic distributions generally assume a peak profile that is not applicable to fully apodized absorption mode spectra because the peak shapes in these spectra are distinctly different from those seen in normal (i.e., magnitude mode) spectra. We present results illustrating this problem and describe a method for producing more accurate isotopic distribution models for this class of spectra.
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http://dx.doi.org/10.1021/acs.analchem.5b01032DOI Listing
June 2015

Absorption mode FTICR mass spectrometry imaging.

Anal Chem 2013 Dec 14;85(23):11180-4. Epub 2013 Nov 14.

FOM Institute AMOLF , Science Park 104, Amsterdam, North Holland, 1098 XG The Netherlands.

Fourier transform ion cyclotron resonance mass spectrometry offers the highest mass resolving power for molecular imaging experiments. This high mass resolving power ensures that closely spaced peaks at the same nominal mass are resolved for proper image generation. Typically higher magnetic fields are used to increase mass resolving power. However, a gain in mass resolving power can also be realized by phase correction of the data for absorption mode display. In addition to mass resolving power, absorption mode offers higher mass accuracy and signal-to-noise ratio over the conventional magnitude mode. Here, we present the first use of absorption mode for Fourier transform ion cyclotron resonance mass spectrometry imaging. The Autophaser algorithm is used to phase correct each spectrum (pixel) in the image, and then, these parameters are used by the Chameleon work-flow based data processing software to generate absorption mode "Datacubes" for image and spectral viewing. Absorption mode reveals new mass and spatial features that are not resolved in magnitude mode and results in improved selected ion image contrast.
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http://dx.doi.org/10.1021/ac403039tDOI Listing
December 2013

Improved optimization of the Fourier transform ion cyclotron resonance mass spectrometry phase correction function using a genetic algorithm.

Rapid Commun Mass Spectrom 2013 Sep;27(17):1977-82

Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK.

Rationale: Fourier Transform Ion Cyclotron Resonance mass spectra exhibit improved resolving power, mass accuracy and signal-to-noise ratio when presented in absorption mode; a process which requires calculation of a phase correction function. Mass spectrometric images can contain many thousands of pixels; hence methods of decreasing the time required to solve for a phase correction function will result in significant improvements in this application.

Methods: A genetic algorithm approach for optimizing the phase correction function has been developed and compared with a previously described convergent iteration technique.

Results: The genetic algorithm method has been shown to offer a five-fold improvement in processing speed compared with the previous iterative approach used in the Autophaser algorithm, while maintaining the levels of accuracy. This translates to an 11 hour improvement in processing for a 20 000 pixel mass spectrometric image.

Conclusions: The genetic algorithm method described in this manuscript offers significant processing speed advantages over the previously described convergent iteration technique. This improvement is key to allowing the future routine use of absorption mode mass spectrometric images.
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http://dx.doi.org/10.1002/rcm.6658DOI Listing
September 2013

Absorption-mode Fourier transform mass spectrometry: the effects of apodization and phasing on modified protein spectra.

J Am Soc Mass Spectrom 2013 Jun 9;24(6):828-34. Epub 2013 Apr 9.

Department of Chemistry, University of Warwick, Coventry, UK.

The method of phasing broadband Fourier transform ion cyclotron resonance (FT-ICR) spectra allows plotting the spectra in the absorption-mode; this new approach significantly improves the quality of the data at no extra cost. Herein, an internal calibration method for calculating the phase function has been developed and successfully applied to the top-down spectra of modified proteins, where the peak intensities vary by 100×. The result shows that the use of absorption-mode spectra allows more peaks to be discerned within the recorded data, and this can reveal much greater information about the protein and modifications under investigation. In addition, noise and harmonic peaks can be assigned immediately in the absorption-mode.
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http://dx.doi.org/10.1007/s13361-013-0600-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4024093PMC
June 2013

Autophaser: an algorithm for automated generation of absorption mode spectra for FT-ICR MS.

Anal Chem 2013 Apr 26;85(8):3903-11. Epub 2013 Mar 26.

Department of Chemistry, University of Warwick, Coventry, CV4 7AL, United Kingdom.

Phase correction of Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry data allows the spectra to be presented in absorption mode. Absorption mode spectra offer superior mass resolving power (up to a factor of 2), mass accuracy, and sensitivity over the conventional magnitude mode. Hitherto, the use of absorption mode in FT-ICR mass spectrometry has required either specially adapted instrumentation or a manually intensive process of phase correction or has ignored the potentially significant effects of image charge and the associated frequency shifts. Here we present an algorithm that allows spectra recorded on unadapted FT-ICR mass spectrometers to be phase corrected, their baseline deviations removed, and then an absorption mode spectrum presented in an automated manner that requires little user interaction.
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http://dx.doi.org/10.1021/ac303289cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3712280PMC
April 2013

Use of an artificial immune system derived method for the charge state assignment of small-molecule mass spectra.

Anal Chem 2012 Sep 15;84(17):7436-9. Epub 2012 Aug 15.

Department of Chemistry, University of Warwick, Coventry, UK.

Knowing the charge state of an ion in a mass spectrum is crucial to being able to assign a formula to it. For many small-molecule peaks in complex mass spectra, the intensities of the isotopic peaks are too low to allow the charge state to be calculated from isotopic spacings, which is the basis of the conventional method of determining the charge state of an ion. A novel artificial intelligence derived method for identifying the charge state of ions, in the absence of any isotopic information or a series of charge states, has been developed using an artificial immune system approach. This technique has been tested against synthetic and real data sets and has proven successful in identifying the majority of multiply charged ions, thereby significantly improving the peak assignment rate and confidence.
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http://dx.doi.org/10.1021/ac3013576DOI Listing
September 2012

Appropriate degree of trust: deriving confidence metrics for automatic peak assignment in high-resolution mass spectrometry.

Anal Chem 2012 Sep 21;84(17):7431-5. Epub 2012 Aug 21.

Department of Chemistry, University of Warwick, Coventry, UK.

Techniques for deriving confidence metrics for the reliability of automatically assigned elemental formulas in complex spectra, from high-resolution mass spectrometers, are described. These metrics can help an analyst to place an appropriate degree of trust in the results obtained from automated spectral analysis of, for example, natural organic materials. To provide these metrics of confidence, common mass spectrometric tests for reliability of peak assignment (mass accuracy/error, relative ion abundance, and rings-plus-double-bonds equivalence) are combined with novel confidence metrics based on the interconnectivity and consistency of a mass difference or mass defect based peak inference network and on the confidence of the initial library matches. These are shown to provide improved peak assignment confidence over manual or simple automatic assignment methods.
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http://dx.doi.org/10.1021/ac301339dDOI Listing
September 2012

Improved detection of low vapor pressure compounds in air by serial combination of single-sided membrane introduction with fiber introduction mass spectrometry (SS-MIMS-FIMS).

Analyst 2005 May 18;130(5):679-86. Epub 2005 Mar 18.

Chemistry Department, Purdue University, West Lafayette, IN 47907, USA.

The use of two methods in tandem, single-sided membrane introduction mass spectrometry (SS-MIMS) and fiber introduction mass spectrometry (FIMS), is presented as a technique for field analysis. The combined SS-MIMS-FIMS technique was employed in both a modified commercial mass spectrometer and a miniature mass spectrometer for the selective preconcentration of the explosive simulant o-nitrotoluene (ONT) and the chemical warfare agent simulant, methyl salicylate (MeS), in air. A home-built FIMS inlet was fabricated to allow introduction of the solid-phase microextraction (SPME) fiber into the mass spectrometer chamber and subsequent desorption of the trapped compounds using resistive heating. The SS-MIMS preconcentration system was also home-built from commercial vacuum parts. Optimization experiments were done separately for each preconcentration system to achieve the best extraction conditions prior to use of the two techniques in combination. Improved limits of detection, in the low ppb range, were observed for the combination compared to FIMS alone, using several SS-MIMS preconcentration cycles. The SS-MIMS-FIMS response for both instruments was found to be linear over the range 50 to 800 ppb. Other parameters studied were absorption time profiles, effects of sample flow rate, desorption temperature, fiber background, memory effects, and membrane fatigue. This simple, sensitive, accurate, robust, selective, and rapid sample preconcentration and introduction technique shows promise for field analysis of low vapor pressure compounds, where analyte concentrations will be extremely low and the compounds are difficult to extract from a matrix like air.
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http://dx.doi.org/10.1039/b417791fDOI Listing
May 2005
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