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  • Using native top-down nESI FTICR-MS to characterize the interaction of tau protein with assembly modulator CLR01 Nshanian, M. AMER CHEMICAL SOC. 2019
  • Native Top-Down Mass Spectrometry and Ion Mobility Spectrometry of the Interaction of Tau Protein with a Molecular Tweezer Assembly Modulator JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY Nshanian, M., Lantz, C., Wongkongkathep, P., Schrader, T., Klaerner, F., Bluemke, A., Despres, C., Ehrmann, M., Smet-Nocca, C., Bitan, G., Loo, J. A. 2019; 30 (1): 16?23

    Abstract

    Native top-down mass spectrometry (MS) and ion mobility spectrometry (IMS) were applied to characterize the interaction of a molecular tweezer assembly modulator, CLR01, with tau, a protein believed to be involved in a number of neurodegenerative disorders, including Alzheimer's disease. The tweezer CLR01 has been shown to inhibit aggregation of amyloidogenic polypeptides without toxic side effects. ESI-MS spectra for different forms of tau protein (full-length, fragments, phosphorylated, etc.) in the presence of CLR01 indicate a primary binding stoichiometry of 1:1. The relatively high charging of the protein measured from non-denaturing solutions is typical of intrinsically disordered proteins, such as tau. Top-down mass spectrometry using electron capture dissociation (ECD) is a tool used to determine not only the sites of post-translational modifications but also the binding site(s) of non-covalent interacting ligands to biomolecules. The intact protein and the protein-modulator complex were subjected to ECD-MS to obtain sequence information, map phosphorylation sites, and pinpoint the sites of inhibitor binding. The ESI-MS study of intact tau proteins indicates that top-down MS is amenable to the study of various tau isoforms and their post-translational modifications (PTMs). The ECD-MS data point to a CLR01 binding site in the microtubule-binding region of tau, spanning residues K294-K331, which includes a six-residue nucleating segment PHF6 (VQIVYK) implicated in aggregation. Furthermore, ion mobility experiments on the tau fragment in the presence of CLR01 and phosphorylated tau reveal a shift towards a more compact structure. The mass spectrometry study suggests a picture for the molecular mechanism of the modulation of protein-protein interactions in tau by CLR01. Graphical Abstract ?.

    View details for DOI 10.1007/s13361-018-2027-6

    View details for Web of Science ID 000454913600004

    View details for PubMedID 30062477

    View details for PubMedCentralID PMC6320309

  • Native and Denaturing MS Protein Deconvolution for Biopharma: Monoclonal Antibodies and Antibody-Drug-Conjugates to Polydisperse Membrane Proteins and Beyond. Analytical chemistry Campuzano, I. D., Robinson, J. H., Hui, J. O., Shi, S. D., Netirojjanakul, C., Nshanian, M., Lippens, J. L., Bagal, D., Egea, P. F., Loo, J. A., Bern, M. 2019

    Abstract

    Electrospray ionization mass spectrometry (ESI-MS) is a ubiquitously used analytical method applied across multiple departments in biopharma, ranging from early Research Discovery to Process Development. Accurate, efficient and consistent protein MS spectral deconvolution across multiple instrument and detector platforms (ToF, Orbitrap, FT-ICR) is essential. When proteins are ionized during the ESI process, a distribution of consecutive multiply charged ions are observed on the m/z scale, either positive (M+nHn+) or negative (M-nHn-) depending on the ionization polarity. The manual calculation of the neutral molecular weight (MW) of single proteins measured by ESI-MS is simple, however algorithmic deconvolution is required for more complex protein mixtures to derive accurate MWs. Multiple deconvolution algorithms have evolved over the past two decades, all of which have their advantages and disadvantages, in terms of speed, user-input parameters (or ideally lack thereof) and whether they perform optimally on proteins analyzed under denatured or native solution and MS conditions. Herein we describe the utility of a parsimonious deconvolution algorithm (explaining the observed spectra with a minimum number of masses) over of a wide range of highly diverse biopharma relevant and research grade proteins and complexes (PEG-GCSF; an IgG1k; IgG1 and IgG2-biotin covalent conjugates; the membrane protein complex AqpZ; a highly polydisperse empty nanodisc, MSP1D1 and the tetradecameric chaperone protein complex GroEL) analysed under native MS, denaturing LC-MS, positive and negative modes of ionization, using multiple instruments and therefore multiple data formats. The implementation of a comb filter and peak sharpening option are also demonstrated to be highly effective for deconvolution of highly polydisperse and enhanced separation of a low level lysine glycation post translational modification (+162.1 Da), partially processed heavy chain lysine resides (+128.1 Da) and loss of N-Acetylglucosamine (GlcNAc; -203.1 Da) respectively.

    View details for DOI 10.1021/acs.analchem.9b00062

    View details for PubMedID 31194911

  • Enhancing sensitivity of liquid chromatography-mass spectrometry of peptides and proteins using supercharging agents INTERNATIONAL JOURNAL OF MASS SPECTROMETRY Nshanian, M., Lakshmanan, R., Chen, H., Loo, R., Loo, J. A. 2018; 427: 157?64

    Abstract

    Trifluoroacetic acid (TFA) is often used as a mobile phase modifier to enhance reversed phase chromatographic performance. TFA adjusts solution pH and is an ion-pairing agent, but it is not typically suitable for electrospray ionization-mass spectrometry (ESI-MS) and liquid chromatography/MS (LC/MS) because of its significant signal suppression. Supercharging agents elevate peptide and protein charge states in ESI, increasing tandem MS (MS/MS) efficiency. Here, LC/MS protein supercharging was effected by adding agents to LC mobile phase solvents. Significantly, the ionization suppression generally observed with TFA was, for the most part, rescued by supercharging agents, with improved separation efficiency (higher number of theoretical plates) and lowered detection limits.

    View details for DOI 10.1016/j.ijms.2017.12.006

    View details for Web of Science ID 000429944900021

    View details for PubMedID 29750076

    View details for PubMedCentralID PMC5937529

  • Fixed-Charge Trimethyl Pyrilium Modification for Enabling Enhanced Top-Down Mass Spectrometry Sequencing of Intact Protein Complexes ANALYTICAL CHEMISTRY Polasky, D. A., Lermyte, F., Nshanian, M., Sobott, F., Andrews, P. C., Loo, J. A., Ruotolo, B. T. 2018; 90 (4): 2756?64

    Abstract

    Mass spectrometry of intact proteins and protein complexes has the potential to provide a transformative level of information on biological systems, ranging from sequence and post-translational modification analysis to the structures of whole protein assemblies. This ambitious goal requires the efficient fragmentation of both intact proteins and the macromolecular, multicomponent machines they collaborate to create through noncovalent interactions. Improving technologies in an effort to achieve such fragmentation remains perhaps the greatest challenge facing current efforts to comprehensively analyze cellular protein composition and is essential to realizing the full potential of proteomics. In this work, we describe the use of a trimethyl pyrylium (TMP) fixed-charge covalent labeling strategy aimed at enhancing fragmentation for challenging intact proteins and intact protein complexes. Combining analysis of TMP-modified and unmodified protein complexes results in a greater diversity of regions within the protein that give rise to fragments, and results in an up to 2.5-fold increase in sequence coverage when compared to unmodified protein alone, for protein complexes up to 148 kDa. TMP modification offers a simple and powerful platform to expand the capabilities of existing mass spectrometric instrumentation for the complete characterization of intact protein assemblies.

    View details for DOI 10.1021/acs.analchem.7b04806

    View details for Web of Science ID 000426143100051

    View details for PubMedID 29360341

    View details for PubMedCentralID PMC6340295

  • Native-MS Analysis of Monoclonal Antibody Conjugates by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry ANALYTICAL CHEMISTRY Campuzano, I. G., Netirojjanakul, C., Nshanian, M., Lippens, J. L., Kilgour, D. A., Van Orden, S., Loo, J. A. 2018; 90 (1): 745?51

    Abstract

    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.

    View details for DOI 10.1021/acs.analchem.7b03021

    View details for Web of Science ID 000419419200049

    View details for PubMedID 29193956

  • Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry as a Platform for Characterizing Multimeric Membrane Protein Complexes JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY Lippens, J. L., Nshanian, M., Spahr, C., Egea, P. F., Loo, J. A., Campuzano, I. G. 2018; 29 (1): 183?93

    Abstract

    Membrane protein characterization is consistently hampered by challenges with expression, purification, and solubilization. Among several biophysical techniques employed for their characterization, native-mass spectrometry (MS) has emerged as a powerful tool for the analysis of membrane proteins and complexes. Here, two MS platforms, the FT-ICR and Q-ToF, have been explored to analyze the homotetrameric water channel protein, AquaporinZ (AqpZ), under non-denaturing conditions. This 97 kDa membrane protein complex can be readily liberated from the octylglucoside (OG) detergent micelle under a range of instrument conditions on both MS platforms. Increasing the applied collision energy of the FT-ICR collision cell yielded varying degrees of tetramer (97 kDa) liberation from the OG micelles, as well as dissociation into the trimeric (72 kDa) and monomeric (24 kDa) substituents. Tandem-MS on the Q-ToF yielded higher intensity tetramer signal and, depending on the m/z region selected, the observed monomer signal varied in intensity. Precursor ion selection of an m/z range above the expected protein signal distribution, followed by mild collisional activation, is able to efficiently liberate AqpZ with a high S/N ratio. The tetrameric charge state distribution obtained on both instruments demonstrated superpositioning of multiple proteoforms due to varying degrees of N-terminal formylation. Graphical Abstract ?.

    View details for DOI 10.1007/s13361-017-1799-4

    View details for Web of Science ID 000423390800021

    View details for PubMedID 28971338

    View details for PubMedCentralID PMC5786498

  • Top-down/Bottom-up Mass Spectrometry Workflow Using Dissolvable Polyacrylamide Gels ANALYTICAL CHEMISTRY Takemori, N., Takemori, A., Wongkongkathep, P., Nshanian, M., Loo, R., Lermyte, F., Loo, J. A. 2017; 89 (16): 8244?50

    Abstract

    Biologists' preeminent toolbox for separating, analyzing, and visualizing proteins is SDS-PAGE, yet recovering the proteins embedded in these polyacrylamide media as intact species is a long-standing challenge for mass spectrometry. In conventional workflows, protein mixtures from crude biological samples are electrophoretically separated at high-resolution within N,N'-methylene-bis-acrylamide cross-linked polyacrylamide gels to reduce sample complexity and facilitate sensitive characterization. However, low protein recoveries, especially for high molecular weight proteins, often hinder characterization by mass spectrometry. We describe a workflow for top-down/bottom-up mass spectrometric analyses of proteins in polyacrylamide slab gels using dissolvable, bis-acryloylcystamine-cross-linked polyacrylamide, enabling high-resolution protein separations while recovering intact proteins over a broad size range efficiently. The inferior electrophoretic resolution long associated with reducible gels has been overcome, as demonstrated by SDS-PAGE of crude tissue extracts. This workflow elutes intact proteins efficiently, supporting MS and MS/MS from proteins resolved on biologists' preferred separation platform.

    View details for DOI 10.1021/acs.analchem.71300357

    View details for Web of Science ID 000407988600011

    View details for PubMedID 28723075

    View details for PubMedCentralID PMC5590889

  • Inhibition of Huntingtin Exon-1 Aggregation by the Molecular Tweezer CLR01 JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Voepel, T., Bravo-Rodriguez, K., Mittal, S., Vachharajani, S., Gnutt, D., Sharma, A., Steinhof, A., Fatoba, O., Ellrichmann, G., Nshanian, M., Heid, C., Loo, J. A., Klaener, F., Schrader, T., Bitan, G., Wanker, E. E., Ebbinghaus, S., Sanchez-Garcia, E. 2017; 139 (16): 5640?43

    Abstract

    Huntington's disease is a neurodegenerative disorder associated with the expansion of the polyglutamine tract in the exon-1 domain of the huntingtin protein (htte1). Above a threshold of 37 glutamine residues, htte1 starts to aggregate in a nucleation-dependent manner. A 17-residue N-terminal fragment of htte1 (N17) has been suggested to play a crucial role in modulating the aggregation propensity and toxicity of htte1. Here we identify N17 as a potential target for novel therapeutic intervention using the molecular tweezer CLR01. A combination of biochemical experiments and computer simulations shows that binding of CLR01 induces structural rearrangements within the htte1 monomer and inhibits htte1 aggregation, underpinning the key role of N17 in modulating htte1 toxicity.

    View details for DOI 10.1021/jacs.6b11039

    View details for Web of Science ID 000400321500002

    View details for PubMedID 28406616

    View details for PubMedCentralID PMC5506490

  • Open-tubular capillary electrochromatography of small polar molecules using etched, chemically modified capillaries ELECTROPHORESIS Pesek, J. J., Matyska, M. T., Nshanian, M. 2011; 32 (13): 1728?34

    Abstract

    The migration characteristics of small polar molecules are evaluated on etched, chemically modified capillaries with four different moieties (C5, C18, diol and cholesterol) bonded onto a silica hydride surface. The effects of pH on migration are used to determine the possible contributions of eletrophoretic mobility, electroosmotic flow (EOF) and analyte/bonded phase interactions. The EOF on etched capillaries is more complicated than on ordinary fused capillaries because it changes from anodic to cathodic as the pH is raised. A mixture of neurotransmitters and related compounds is used to further evaluate the effects of the bonded moiety on the separation properties of this particular electrophoretic format.

    View details for DOI 10.1002/elps.201000614

    View details for Web of Science ID 000292971000021

    View details for PubMedID 21604283

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