Publications by authors named "Donald F Hunt"

197 Publications

Interlaboratory Study for Characterizing Monoclonal Antibodies by Top-Down and Middle-Down Mass Spectrometry.

J Am Soc Mass Spectrom 2020 Sep 19;31(9):1783-1802. Epub 2020 Aug 19.

The University of Edinburgh, EH9 3FJ Edinburgh, United Kingdom.

The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the current state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications. To meet the needs of the rapidly growing therapeutic antibody market, it is important to develop analytical strategies to characterize the heterogeneity of a therapeutic product's primary structure accurately and reproducibly. The major objective of the present study is to determine whether current TD/MD MS technologies and protocols can add value to the more commonly employed bottom-up (BU) approaches with regard to confirming protein integrity, sequencing variable domains, avoiding artifacts, and revealing modifications and their locations. We also aim to gather information on the common TD/MD MS methods and practices in the field. A panel of three mAbs was selected and centrally provided to 20 laboratories worldwide for the analysis: Sigma mAb standard (SiLuLite), NIST mAb standard, and the therapeutic mAb Herceptin (trastuzumab). Various MS instrument platforms and ion dissociation techniques were employed. The present study confirms that TD/MD MS tools are available in laboratories worldwide and provide complementary information to the BU approach that can be crucial for comprehensive mAb characterization. The current limitations, as well as possible solutions to overcome them, are also outlined. A primary limitation revealed by the results of the present study is that the expert knowledge in both experiment and data analysis is indispensable to practice TD/MD MS.
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http://dx.doi.org/10.1021/jasms.0c00036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7539639PMC
September 2020

Sequencing a Bispecific Antibody by Controlling Chain Concentration Effects When Using an Immobilized Nonspecific Protease.

Anal Chem 2020 08 13;92(15):10470-10477. Epub 2020 Jul 13.

Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, United States.

Complete sequence coverage of monospecific antibodies was previously achieved using immobilized aspergillopepsin I in a single LC-MS/MS analysis. Bispecific antibodies are asymmetrical compared to their monospecific antibody counterparts, resulting in a decrease in the concentration of individual subunits. Four standard proteins were used to characterize the effect of a decrease in concentration when using this immobilized enzyme reactor. Low concentration samples resulted in the elimination of large peptide products due to a greater number of enzymatic cleavages. A competitive inhibitor rich in arginine residues reduced the number of enzymatic cleavages to the protein and retained large molecular weight products. The digestion of a bispecific antibody with competitive inhibition of aspergillopepsin I maintained large peptide products better suited for sequence reconstruction, resulting in complete sequence coverage from a single LC-MS/MS analysis.
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http://dx.doi.org/10.1021/acs.analchem.0c01126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8106826PMC
August 2020

Tyrosine Phosphorylation of the Myosin Regulatory Light Chain Controls Non-muscle Myosin II Assembly and Function in Migrating Cells.

Curr Biol 2020 07 4;30(13):2446-2458.e6. Epub 2020 Jun 4.

Molecular Mechanisms Program, Centro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas (CSIC)-University of Salamanca, 37007 Salamanca, Spain. Electronic address:

Active non-muscle myosin II (NMII) enables migratory cell polarization and controls dynamic cellular processes, such as focal adhesion formation and turnover and cell division. Filament assembly and force generation depend on NMII activation through the phosphorylation of Ser19 of the regulatory light chain (RLC). Here, we identify amino acid Tyr (Y) 155 of the RLC as a novel regulatory site that spatially controls NMII function. We show that Y155 is phosphorylated in vitro by the Tyr kinase domain of epidermal growth factor (EGF) receptor. In cells, phosphorylation of Y155, or its phospho-mimetic mutation (Glu), prevents the interaction of RLC with the myosin heavy chain (MHCII) to form functional NMII units. Conversely, Y155 mutation to a structurally similar but non-phosphorylatable amino acid (Phe) restores the more dynamic cellular functions of NMII, such as myosin filament formation and nascent adhesion assembly, but not those requiring stable actomyosin bundles, e.g., focal adhesion elongation or migratory front-back polarization. In live cells, phospho-Y155 RLC is prominently featured in protrusions, where it prevents NMII assembly. Our data indicate that Y155 phosphorylation constitutes a novel regulatory mechanism that contributes to the compartmentalization of NMII assembly and function in live cells.
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http://dx.doi.org/10.1016/j.cub.2020.04.057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7343590PMC
July 2020

Deciphering the Enigma of the Histone H2A.Z-1/H2A.Z-2 Isoforms: Novel Insights and Remaining Questions.

Cells 2020 05 8;9(5). Epub 2020 May 8.

Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC V8W 3P6, Canada.

The replication independent (RI) histone H2A.Z is one of the more extensively studied variant members of the core histone H2A family, which consists of many replication dependent (RD) members. The protein has been shown to be indispensable for survival, and involved in multiple roles from DNA damage to chromosome segregation, replication, and transcription. However, its functional involvement in gene expression is controversial. Moreover, the variant in several groups of metazoan organisms consists of two main isoforms (H2A.Z-1 and H2A.Z-2) that differ in a few (3-6) amino acids. They comprise the main topic of this review, starting from the events that led to their identification, what is currently known about them, followed by further experimental, structural, and functional insight into their roles. Despite their structural differences, a direct correlation to their functional variability remains enigmatic. As all of this is being elucidated, it appears that a strong functional involvement of isoform variability may be connected to development.
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http://dx.doi.org/10.3390/cells9051167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290884PMC
May 2020

MHC-restricted phosphopeptide antigens: preclinical validation and first-in-humans clinical trial in participants with high-risk melanoma.

J Immunother Cancer 2020 05;8(1)

Department of Surgery/Division of Surgical Oncology, University of Virginia School of Medicine, Charlottesville, Virginia, USA.

Background: Phosphorylated peptides presented by MHC molecules represent a new class of neoantigens expressed on cancer cells and recognized by CD8 T-cells. These peptides are promising targets for cancer immunotherapy. Previous work identified an HLA-A*0201-restricted phosphopeptide from insulin receptor substrate 2 (pIRS2) as one such target. The purpose of this study was to characterize a second phosphopeptide, from breast cancer antiestrogen resistance 3 (BCAR3), and to evaluate safety and immunogenicity of a novel immunotherapic vaccine comprising either or both of these phosphorylated peptides.

Methods: Phosphorylated BCAR3 protein was evaluated in melanoma and breast cancer cell lines by Western blot, and recognition by T-cells specific for HLA-A*0201-restricted phosphorylated BCAR3 peptide (pBCAR3) was determined by Cr release assay and intracellular cytokine staining. Human tumor explants were also evaluated by mass spectrometry for presentation of pIRS2 and pBCAR3 peptides. For the clinical trial, participants with resected stage IIA-IV melanoma were vaccinated 6 times over 12 weeks with one or both peptides in incomplete Freund's adjuvant and Hiltonol (poly-ICLC). Adverse events (AEs) were coded based on National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) V.4.03, with provision for early study termination if dose-limiting toxicity (DLT) rates exceeded 33%. The enrollment target was 12 participants evaluable for immune response to each peptide. T-cell responses were assessed by interferon-γ ELISpot assay.

Results: pBCAR3 peptides were immunogenic in vivo in mice, and in vitro in normal human donors, and T-cells specific for pBCAR3 controlled outgrowth of a tumor xenograft. The pIRS2 peptide was identified by mass spectrometry from human hepatocellular carcinoma tumors. In the clinical trial, 15 participants were enrolled. All had grade 1 or 2 treatment-related AEs, but there were no grade 3-4 AEs, DLTs or deaths on study. T-cell responses were induced to the pIRS2 peptide in 5/12 patients (42%, 90% CI 18% to 68%) and to the pBCAR3 peptide in 2/12 patients (17%, 90% CI 3% to 44%).

Conclusion: This study supports the safety and immunogenicity of vaccines containing the cancer-associated phosphopeptides pBCAR3 and pIRS2, and the data support continued development of immune therapy targeting phosphopeptides. Future studies will define ways to further enhance the magnitude and durability of phosphopeptide-specific immune responses.

Trial Registration Number: NCT01846143.
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http://dx.doi.org/10.1136/jitc-2019-000262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7228659PMC
May 2020

Transcription factor binding at Ig enhancers is linked to somatic hypermutation targeting.

Eur J Immunol 2020 03 19;50(3):380-395. Epub 2019 Dec 19.

Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA.

Secondary diversification of the Ig repertoire occurs through somatic hypermutation (SHM), gene conversion (GCV), and class switch recombination (CSR)-three processes that are initiated by activation-induced cytidine deaminase (AID). AID targets Ig genes at orders of magnitude higher than the rest of the genome, but the basis for this specificity is poorly understood. We have previously demonstrated that enhancers and enhancer-like sequences from Ig genes are capable of stimulating SHM of neighboring genes in a capacity distinct from their roles in increasing transcription. Here, we use an in vitro proteomics approach to identify E-box, MEF2, Ets, and Ikaros transcription factor family members as potential binders of these enhancers. ChIP assays in the hypermutating Ramos B cell line confirmed that many of these factors bound the endogenous Igλ enhancer and/or the IgH intronic enhancer (Eμ) in vivo. Further investigation using SHM reporter assays identified binding sites for E2A and MEF2B in Eμ and demonstrated an association between loss of factor binding and decreases in the SHM stimulating activity of Eμ mutants. Our results provide novel insights into trans-acting factors that dictate SHM targeting and link their activity to specific DNA binding sites within Ig enhancers.
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http://dx.doi.org/10.1002/eji.201948357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7202714PMC
March 2020

Murine xenograft bioreactors for human immunopeptidome discovery.

Sci Rep 2019 12 6;9(1):18558. Epub 2019 Dec 6.

Center for Cancer Immunology, Massachusetts General Hospital, Boston, Massachusetts, USA.

The study of peptides presented by MHC class I and class II molecules is limited by the need for relatively large cell numbers, especially when studying post-translationally modified or otherwise rare peptide species. To overcome this problem, we pose the hypothesis that human cells grown as xenografts in immunodeficient mice should produce equivalent immunopeptidomes as cultured cells. Comparing human cell lines grown either in vitro or as murine xenografts, we show that the immunopeptidome is substantially preserved. Numerous features are shared across both sample types, including peptides and proteins featured, length distributions, and HLA-binding motifs. Peptides well-represented in both groups were from more abundant proteins, or those with stronger predicted HLA binding affinities. Samples grown in vivo also recapitulated a similar phospho-immunopeptidome, with common sequences being those found at high copy number on the cell surface. These data indicate that xenografts are indeed a viable methodology for the production of cells for immunopeptidomic discovery.
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http://dx.doi.org/10.1038/s41598-019-54700-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6898210PMC
December 2019

Unambiguous Sequence Characterization of a Monoclonal Antibody in a Single Analysis Using a Nonspecific Immobilized Enzyme Reactor.

Anal Chem 2019 11 16;91(21):13547-13554. Epub 2019 Oct 16.

Department of Chemistry , University of Virginia , Charlottesville , Virginia 22904 , United States.

Accurate sequence characterization is essential for the development of therapeutic antibodies by the pharmaceutical industry. Presented here is a methodology to obtain comprehensive sequence analysis of a monoclonal antibody. An enzyme reactor of immobilized Aspergillopepsin I, a highly stable nonspecific protease, was used to cleave reduced antibody subunits into a peptide profile ranging from 1 to 20 kDa. Utilizing the Thermo Orbitrap Fusion's unique instrument architecture combined with state-of-the-art instrument control software allowed for dynamic instrument methods that optimally characterize eluting peptides based on their size and charge density. Using a data-dependent instrument method, both collisional dissociation and electron transfer dissociation were used to fragment the appropriate charge state of analyte peptides. The instrument layout also allowed for scans to be taken in parallel using both the ion trap and Orbitrap concurrently, thus allowing larger peptides to be analyzed in high resolution using the Orbitrap while simultaneously analyzing tryptic-like peptides using the ion trap. We harnessed these capabilities to develop a custom method to optimally fragment the eluting peptides based on their mass and charge density. Using this approach, we obtained 100% sequence coverage of the total antibody in a single chromatographic analysis, enabling unambiguous sequence assignment of all residues.
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http://dx.doi.org/10.1021/acs.analchem.9b02666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8092443PMC
November 2019

Protamines from liverwort are produced by post-translational cleavage and C-terminal di-aminopropanelation of several male germ-specific H1 histones.

J Biol Chem 2019 11 16;294(44):16364-16373. Epub 2019 Sep 16.

Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8W 3P6, Canada

Protamines are small, highly-specialized, arginine-rich, and intrinsically-disordered chromosomal proteins that replace histones during spermiogenesis in many organisms. Previous evidence supports the notion that, in the animal kingdom, these proteins have evolved from a primitive replication-independent histone H1 involved in terminal cell differentiation. Nevertheless, a direct connection between the two families of chromatin proteins is missing. Here, we primarily used electron transfer dissociation MS-based analyses, revealing that the protamines in the sperm of the liverwort result from post-translational cleavage of three precursor H1 histones. Moreover, we show that the mature protamines are further post-translationally modified by di-aminopropanelation, and previous studies have reported that they condense spermatid chromatin through a process consisting of liquid-phase assembly likely involving spinodal decomposition. Taken together, our results reveal that the interesting evolutionary ancestry of protamines begins with histone H1 in both the animal and plant kingdoms.
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http://dx.doi.org/10.1074/jbc.RA119.010316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6827293PMC
November 2019

Ion-Ion Proton Transfer and Parallel Ion Parking for the Analysis of Mixtures of Intact Proteins on a Modified Orbitrap Mass Analyzer.

J Am Soc Mass Spectrom 2019 Oct 7;30(10):2163-2173. Epub 2019 Aug 7.

Department of Chemistry, University of Virginia, Charlottesville, VA, 22904, USA.

We have enabled parallel ion parking on a modified Orbitrap Elite™ as a way to control ion-ion proton transfer reactions via selective activation of a range of ions. The result is the concentration of the majority of ion current from multiple charge states of each precursor proteoform into a single charge state, maximizing signal intensity and increasing effective sensitivity compared to conventional MS1 spectra. These techniques were applied in an on-line HPLC, data-dependent MS/MS analysis of intact E. coli ribosomal proteins with HCD fragmentation. With one injection, all but two ribosomal proteins were selected for fragmentation and subsequently identified. The techniques described facilitate rapid identification of intact proteins in complex mixtures and an enhanced ability to observe proteins of low abundance.
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http://dx.doi.org/10.1007/s13361-019-02290-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6805958PMC
October 2019

O-GlcNAc Site Mapping by Using a Combination of Chemoenzymatic Labeling, Copper-Free Click Chemistry, Reductive Cleavage, and Electron-Transfer Dissociation Mass Spectrometry.

Anal Chem 2019 02 4;91(4):2620-2625. Epub 2019 Feb 4.

Department of Biological Chemistry , The Johns Hopkins University School of Medicine , Baltimore , Maryland 21205 , United States.

As a dynamic post-translational modification, O-linked β- N-acetylglucosamine ( O-GlcNAc) modification (i.e., O-GlcNAcylation) of proteins regulates many biological processes involving cellular metabolism and signaling. However, O-GlcNAc site mapping, a prerequisite for site-specific functional characterization, has been a challenge since its discovery. Herein we present a novel method for O-GlcNAc enrichment and site mapping. In this method, the O-GlcNAc moiety on peptides was labeled with UDP-GalNAz followed by copper-free azide-alkyne cycloaddition with a multifunctional reagent bearing a terminal cyclooctyne, a disulfide bridge, and a biotin handle. The tagged peptides were then released from NeutrAvidin beads upon reductant treatment, alkylated with (3-acrylamidopropyl)trimethylammonium chloride, and subjected to electron-transfer dissociation mass spectrometry analysis. After validation by using standard synthetic peptide gCTD and model protein α-crystallin, such an approach was applied to the site mapping of overexpressed TGF-β-activated kinase 1/MAP3K7 binding protein 2 (TAB2), with four O-GlcNAc sites unambiguously identified. Our method provides a promising tool for the site-specific characterization of O-GlcNAcylation of important proteins.
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http://dx.doi.org/10.1021/acs.analchem.8b05688DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6756848PMC
February 2019

OGT (-GlcNAc Transferase) Selectively Modifies Multiple Residues Unique to Lamin A.

Cells 2018 May 17;7(5). Epub 2018 May 17.

Department of Cell Biology, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.

The gene encodes lamins A and C with key roles in nuclear structure, signaling, gene regulation, and genome integrity. Mutations in cause over 12 diseases ('laminopathies'). Lamins A and C are identical for their first 566 residues. However, they form separate filaments in vivo, with apparently distinct roles. We report that lamin A is β--linked -acetylglucosamine--GlcNAc)-modified in human hepatoma (Huh7) cells and in mouse liver. In vitro assays with purified -GlcNAc transferase (OGT) enzyme showed robust -GlcNAcylation of recombinant mature lamin A tails (residues 385⁻646), with no detectable modification of lamin B1, lamin C, or 'progerin' (Δ50) tails. Using mass spectrometry, we identified 11 -GlcNAc sites in a 'sweet spot' unique to lamin A, with up to seven sugars per peptide. Most sites were unpredicted by current algorithms. Double-mutant (S612A/T643A) lamin A tails were still robustly -GlcNAc-modified at seven sites. By contrast, -GlcNAcylation was undetectable on tails bearing deletion Δ50, which causes Hutchinson⁻Gilford progeria syndrome, and greatly reduced by deletion Δ35. We conclude that residues deleted in progeria are required for substrate recognition and/or modification by OGT in vitro. Interestingly, deletion Δ35, which does not remove the majority of identified -GlcNAc sites, does remove potential OGT-association motifs (lamin A residues 622⁻625 and 639⁻645) homologous to that in mouse Tet1. These biochemical results are significant because they identify a novel molecular pathway that may profoundly influence lamin A function. The hypothesis that lamin A is selectively regulated by OGT warrants future testing in vivo, along with two predictions: genetic variants may contribute to disease by perturbing OGT-dependent regulation, and nutrient or other stresses might cause OGT to misregulate wildtype lamin A.
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http://dx.doi.org/10.3390/cells7050044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5981268PMC
May 2018

Phosphorylation coexists with O-GlcNAcylation in a plant virus protein and influences viral infection.

Mol Plant Pathol 2018 06 30;19(6):1427-1443. Epub 2018 Jan 30.

Department of Plant Molecular Genetics, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Madrid 28049, Spain.

Phosphorylation and O-GlcNAcylation are two widespread post-translational modifications (PTMs), often affecting the same eukaryotic target protein. Plum pox virus (PPV) is a member of the genus Potyvirus which infects a wide range of plant species. O-GlcNAcylation of the capsid protein (CP) of PPV has been studied extensively, and some evidence of CP phosphorylation has also been reported. Here, we use proteomics analyses to demonstrate that PPV CP is phosphorylated in vivo at the N-terminus and the beginning of the core region. In contrast with the 'yin-yang' mechanism that applies to some mammalian proteins, PPV CP phosphorylation affects residues different from those that are O-GlcNAcylated (serines Ser-25, Ser-81, Ser-101 and Ser-118). Our findings show that PPV CP can be concurrently phosphorylated and O-GlcNAcylated at nearby residues. However, an analysis using a differential proteomics strategy based on iTRAQ (isobaric tags for relative and absolute quantitation) showed a significant enhancement of phosphorylation at Ser-25 in virions recovered from O-GlcNAcylation-deficient plants, suggesting that crosstalk between O-GlcNAcylation and phosphorylation in PPV CP takes place. Although the preclusion of phosphorylation at the four identified phosphotarget sites only had a limited impact on viral infection, the mimicking of phosphorylation prevents PPV infection in Prunus persica and weakens infection in Nicotiana benthamiana and other herbaceous hosts, prompting the emergence of potentially compensatory second mutations. We postulate that the joint action of phosphorylation and O-GlcNAcylation in the N-proximal segment of CP allows a fine-tuning of protein stability, providing the amount of CP required in each step of viral infection.
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http://dx.doi.org/10.1111/mpp.12626DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5895533PMC
June 2018

The antigenic identity of human class I MHC phosphopeptides is critically dependent upon phosphorylation status.

Oncotarget 2017 Aug 8;8(33):54160-54172. Epub 2017 Apr 8.

Cancer Immunology and Immunotherapy Centre, Institute of Immunology and Immunotherapy, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.

Dysregulated post-translational modification provides a source of altered self-antigens that can stimulate immune responses in autoimmunity, inflammation, and cancer. In recent years, phosphorylated peptides have emerged as a group of tumour-associated antigens presented by MHC molecules and recognised by T cells, and represent promising candidates for cancer immunotherapy. However, the impact of phosphorylation on the antigenic identity of phosphopeptide epitopes is unclear. Here we examined this by determining structures of MHC-bound phosphopeptides bearing canonical position 4-phosphorylations in the presence and absence of their phosphate moiety, and examining phosphopeptide recognition by the T cell receptor (TCR). Strikingly, two peptides exhibited major conformational changes upon phosphorylation, involving a similar molecular mechanism, which focussed changes on the central peptide region most critical for T cell recognition. In contrast, a third epitope displayed little conformational alteration upon phosphorylation. In addition, binding studies demonstrated TCR interaction with an MHC-bound phosphopeptide was both epitope-specific and absolutely dependent upon phosphorylation status. These results highlight the critical influence of phosphorylation on the antigenic identity of naturally processed class I MHC epitopes. In doing so they provide a molecular framework for understanding phosphopeptide-specific immune responses, and have implications for the development of phosphopeptide antigen-specific cancer immunotherapy approaches.
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http://dx.doi.org/10.18632/oncotarget.16952DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5589570PMC
August 2017

Shared peptide binding of HLA Class I and II alleles associate with cutaneous nevirapine hypersensitivity and identify novel risk alleles.

Sci Rep 2017 08 17;7(1):8653. Epub 2017 Aug 17.

Institute for Immunology and Infectious Diseases, Murdoch University, Murdoch, WA, 6150, Australia.

Genes of the human leukocyte antigen (HLA) system encode cell-surface proteins involved in regulation of immune responses, and the way drugs interact with the HLA peptide binding groove is important in the immunopathogenesis of T-cell mediated drug hypersensitivity syndromes. Nevirapine (NVP), is an HIV-1 antiretroviral with treatment-limiting hypersensitivity reactions (HSRs) associated with multiple class I and II HLA alleles. Here we utilize a novel analytical approach to explore these multi-allelic associations by systematically examining HLA molecules for similarities in peptide binding specificities and binding pocket structure. We demonstrate that primary predisposition to cutaneous NVP HSR, seen across ancestral groups, can be attributed to a cluster of HLA-C alleles sharing a common binding groove F pocket with HLA-C*04:01. An independent association with a group of class II alleles which share the HLA-DRB1-P4 pocket is also observed. In contrast, NVP HSR protection is afforded by a cluster of HLA-B alleles defined by a characteristic peptide binding groove B pocket. The results suggest drug-specific interactions within the antigen binding cleft can be shared across HLA molecules with similar binding pockets. We thereby provide an explanation for multiple HLA associations with cutaneous NVP HSR and advance insight into its pathogenic mechanisms.
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http://dx.doi.org/10.1038/s41598-017-08876-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5561238PMC
August 2017

Front-End Electron Transfer Dissociation Coupled to a 21 Tesla FT-ICR Mass Spectrometer for Intact Protein Sequence Analysis.

J Am Soc Mass Spectrom 2017 09 18;28(9):1787-1795. Epub 2017 Jul 18.

Department of Chemistry, University of Virginia, McCormick Road, P.O. Box 400319, Charlottesville, VA, 22904-4319, USA.

High resolution mass spectrometry is a key technology for in-depth protein characterization. High-field Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) enables high-level interrogation of intact proteins in the most detail to date. However, an appropriate complement of fragmentation technologies must be paired with FTMS to provide comprehensive sequence coverage, as well as characterization of sequence variants, and post-translational modifications. Here we describe the integration of front-end electron transfer dissociation (FETD) with a custom-built 21 tesla FT-ICR mass spectrometer, which yields unprecedented sequence coverage for proteins ranging from 2.8 to 29 kDa, without the need for extensive spectral averaging (e.g., ~60% sequence coverage for apo-myoglobin with four averaged acquisitions). The system is equipped with a multipole storage device separate from the ETD reaction device, which allows accumulation of multiple ETD fragment ion fills. Consequently, an optimally large product ion population is accumulated prior to transfer to the ICR cell for mass analysis, which improves mass spectral signal-to-noise ratio, dynamic range, and scan rate. We find a linear relationship between protein molecular weight and minimum number of ETD reaction fills to achieve optimum sequence coverage, thereby enabling more efficient use of instrument data acquisition time. Finally, real-time scaling of the number of ETD reactions fills during method-based acquisition is shown, and the implications for LC-MS/MS top-down analysis are discussed. Graphical Abstract ᅟ.
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http://dx.doi.org/10.1007/s13361-017-1702-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5711562PMC
September 2017

Canonical and Cross-reactive Binding of NK Cell Inhibitory Receptors to HLA-C Allotypes Is Dictated by Peptides Bound to HLA-C.

Front Immunol 2017 14;8:193. Epub 2017 Mar 14.

Lung Immunology Group, Department of Medicine, Imperial College London , London , UK.

Background: Human natural killer (NK) cell activity is regulated by a family of killer cell immunoglobulin-like receptors (KIRs) that bind human leukocyte antigen (HLA) class I. Combinations of KIR and HLA genotypes are associated with disease, including susceptibility to viral infection and disorders of pregnancy. KIR2DL1 binds HLA-C alleles of group C2 (Lys). KIR2DL2 and KIR2DL3 bind HLA-C alleles of group C1 (Asn). However, this model cannot explain HLA-C allelic effects in disease or the impact of HLA-bound peptides. The goal of this study was to determine the extent to which the endogenous HLA-C peptide repertoire can influence the specific binding of inhibitory KIR to HLA-C allotypes.

Results: The impact of HLA-C bound peptide on inhibitory KIR binding was investigated taking advantage of the fact that HLA-C*05:01 (HLA-C group 2, C2) and HLA-C*08:02 (HLA-C group 1, C1) have identical sequences apart from the key KIR specificity determining epitope at residues 77 and 80. Endogenous peptides were eluted from HLA-C*05:01 and used to test the peptide dependence of KIR2DL1 and KIR2DL2/3 binding to HLA-C*05:01 and HLA-C*08:02 and subsequent impact on NK cell function. Specific binding of KIR2DL1 to the C2 allotype occurred with the majority of peptides tested. In contrast, KIR2DL2/3 binding to the C1 allotype occurred with only a subset of peptides. Cross-reactive binding of KIR2DL2/3 with the C2 allotype was restricted to even fewer peptides. Unexpectedly, two peptides promoted binding of the C2 allotype-specific KIR2DL1 to the C1 allotype. We showed that presentation of endogenous peptides or HIV Gag peptides by HLA-C can promote KIR cross-reactive binding.

Conclusion: KIR2DL2/3 binding to C1 is more peptide selective than that of KIR2DL1 binding to C2, providing an explanation for KIR2DL3-C1 interactions appearing weaker than KIR2DL1-C2. In addition, cross-reactive binding of KIR is characterized by even higher peptide selectivity. We demonstrate a hierarchy of functional peptide selectivity of KIR-HLA-C interactions with relevance to NK cell biology and human disease associations. This selective peptide sequence-driven binding of KIR provides a potential mechanism for pathogen as well as self-peptide to modulate NK cell activation through altering levels of inhibition.
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http://dx.doi.org/10.3389/fimmu.2017.00193DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5348643PMC
March 2017

Peptide-binding motifs of two common equine class I MHC molecules in Thoroughbred horses.

Immunogenetics 2017 05 18;69(5):351-358. Epub 2017 Mar 18.

Department of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA, 92037, USA.

Quantitative peptide-binding motifs of MHC class I alleles provide a valuable tool to efficiently identify putative T cell epitopes. Detailed information on equine MHC class I alleles is still very limited, and to date, only a single equine MHC class I allele, Eqca-1*00101 (ELA-A3 haplotype), has been characterized. The present study extends the number of characterized ELA class I specificities in two additional haplotypes found commonly in the Thoroughbred breed. Accordingly, we here report quantitative binding motifs for the ELA-A2 allele Eqca-16*00101 and the ELA-A9 allele Eqca-1*00201. Utilizing analyses of endogenously bound and eluted ligands and the screening of positional scanning combinatorial libraries, detailed and quantitative peptide-binding motifs were derived for both alleles. Eqca-16*00101 preferentially binds peptides with aliphatic/hydrophobic residues in position 2 and at the C-terminus, and Eqca-1*00201 has a preference for peptides with arginine in position 2 and hydrophobic/aliphatic residues at the C-terminus. Interestingly, the Eqca-16*00101 motif resembles that of the human HLA A02-supertype, while the Eqca-1*00201 motif resembles that of the HLA B27-supertype and two macaque class I alleles. It is expected that the identified motifs will facilitate the selection of candidate epitopes for the study of immune responses in horses.
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http://dx.doi.org/10.1007/s00251-017-0978-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5555743PMC
May 2017

Identification of Glycopeptides as Posttranslationally Modified Neoantigens in Leukemia.

Cancer Immunol Res 2017 05 17;5(5):376-384. Epub 2017 Mar 17.

Department of Clinical Immunology, University of Birmingham, Birmingham, United Kingdom.

Leukemias are highly immunogenic, but they have a low mutational load, providing few mutated peptide targets. Thus, the identification of alternative neoantigens is a pressing need. Here, we identify 36 MHC class I-associated peptide antigens with O-linked β--acetylglucosamine (-GlcNAc) modifications as candidate neoantigens, using three experimental approaches. Thirteen of these peptides were also detected with disaccharide units on the same residues and two contain either mono- and/or di-methylated arginine residues. A subset were linked with key cancer pathways, and these peptides were shared across all of the leukemia patient samples tested (5/5). Seven of the -GlcNAc peptides were synthesized and five (71%) were shown to be associated with multifunctional memory T-cell responses in healthy donors. An -GlcNAc-specific T-cell line specifically killed autologous cells pulsed with the modified peptide, but not the equivalent unmodified peptide. Therefore, these posttranslationally modified neoantigens provide logical targets for cancer immunotherapy. .
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http://dx.doi.org/10.1158/2326-6066.CIR-16-0280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5508727PMC
May 2017

The Arabidopsis O-fucosyltransferase SPINDLY activates nuclear growth repressor DELLA.

Nat Chem Biol 2017 05 28;13(5):479-485. Epub 2017 Feb 28.

Department of Biology, Duke University, Durham, North Carolina, USA.

Plant development requires coordination among complex signaling networks to enhance the plant's adaptation to changing environments. DELLAs, transcription regulators originally identified as repressors of phytohormone gibberellin signaling, play a central role in integrating multiple signaling activities via direct protein interactions with key transcription factors. Here, we found that DELLA is mono-O-fucosylated by the novel O-fucosyltransferase SPINDLY (SPY) in Arabidopsis thaliana. O-fucosylation activates DELLA by promoting its interaction with key regulators in brassinosteroid- and light-signaling pathways, including BRASSINAZOLE-RESISTANT1 (BZR1), PHYTOCHROME-INTERACTING-FACTOR3 (PIF3) and PIF4. Moreover, spy mutants displayed elevated responses to gibberellin and brassinosteroid, and increased expression of common target genes of DELLAs, BZR1 and PIFs. Our study revealed that SPY-dependent protein O-fucosylation plays a key role in regulating plant development. This finding may have broader importance because SPY orthologs are conserved in prokaryotes and eukaryotes, thus suggesting that intracellular O-fucosylation may regulate a wide range of biological processes in diverse organisms.
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http://dx.doi.org/10.1038/nchembio.2320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5391292PMC
May 2017

A Dual Inhibitory Mechanism Sufficient to Maintain Cell-Cycle-Restricted CENP-A Assembly.

Mol Cell 2017 Jan 22;65(2):231-246. Epub 2016 Dec 22.

Instituto Gulbenkian de Ciência, 2780-156 Oeiras, Portugal. Electronic address:

Chromatin featuring the H3 variant CENP-A at the centromere is critical for its mitotic function and epigenetic maintenance. Assembly of centromeric chromatin is restricted to G1 phase through inhibitory action of Cdk1/2 kinases in other phases of the cell cycle. Here, we identify the two key targets sufficient to maintain cell-cycle control of CENP-A assembly. We uncovered a single phosphorylation site in the licensing factor M18BP1 and a cyclin A binding site in the CENP-A chaperone, HJURP, that mediated specific inhibitory phosphorylation. Simultaneous expression of mutant proteins lacking these residues results in complete uncoupling from the cell cycle. Consequently, CENP-A assembly is fully recapitulated under high Cdk activities, indistinguishable from G1 assembly. We find that Cdk-mediated inhibition is exerted by sequestering active factors away from the centromere. Finally, we show that displacement of M18BP1 from the centromere is critical for the assembly mechanism of CENP-A.
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http://dx.doi.org/10.1016/j.molcel.2016.11.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5250512PMC
January 2017

Identification and Characterization of Complex Glycosylated Peptides Presented by the MHC Class II Processing Pathway in Melanoma.

J Proteome Res 2017 01 13;16(1):228-237. Epub 2016 Sep 13.

Department of Surgery, Johns Hopkins University School of Medicine , Baltimore, Maryland 21205, United States.

The MHC class II (MHCII) processing pathway presents peptides derived from exogenous or membrane-bound proteins to CD4+ T cells. Several studies have shown that glycopeptides are necessary to modulate CD4+ T cell recognition, though glycopeptide structures in these cases are generally unknown. Here, we present a total of 93 glycopeptides from three melanoma cell lines and one matched EBV-transformed line with most found only in the melanoma cell lines. The glycosylation we detected was diverse and comprised 17 different glycoforms. We then used molecular modeling to demonstrate that complex glycopeptides are capable of binding the MHC and may interact with complementarity determining regions. Finally, we present the first evidence of disulfide-bonded peptides presented by MHCII. This is the first large scale study to sequence glyco- and disulfide bonded MHCII peptides from the surface of cancer cells and could represent a novel avenue of tumor activation and/or immunoevasion.
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http://dx.doi.org/10.1021/acs.jproteome.6b00496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5218890PMC
January 2017

Analyses of Histone Proteoforms Using Front-end Electron Transfer Dissociation-enabled Orbitrap Instruments.

Mol Cell Proteomics 2016 Mar 19;15(3):975-88. Epub 2016 Jan 19.

From the ‡Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904; the ¶Department of Pathology, University of Virginia, Charlottesville, Virginia 22908

Histones represent a class of proteins ideally suited to analyses by top-down mass spectrometry due to their relatively small size, the high electron transfer dissociation-compatible charge states they exhibit, and the potential to gain valuable information concerning combinatorial post-translational modifications and variants. We recently described new methods in mass spectrometry for the acquisition of high-quality MS/MS spectra of intact proteins (Anderson, L. C., English, A. M., Wang, W., Bai, D. L., Shabanowitz, J., and Hunt, D. F. (2015) Int. J. Mass Spectrom. 377, 617-624). Here, we report an extension of these techniques. Sequential ion/ion reactions carried out in a modified Orbitrap Velos Pro/Elite(TM) capable of multiple fragment ion fills of the C-trap, in combination with data-dependent and targeted HPLC-MS experiments, were used to obtain high resolution MS/MS spectra of histones from butyrate-treated HeLa cells. These spectra were used to identify several unique intact histone proteoforms with up to 81% sequence coverage. We also demonstrate that parallel ion parking during ion/ion proton transfer reactions can be used to separate species of overlapping m/z that are not separated chromatographically, revealing previously indiscernible signals. Finally, we characterized several truncated forms of H2A and H2B found within the histone fractions analyzed, achieving up to 93% sequence coverage by electron transfer dissociation MS/MS. Results of follow-up in vitro experiments suggest that some of the truncated histone H2A proteoforms we observed can be generated by cathepsin L, an enzyme known to also catalyze clipping of histone H3.
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http://dx.doi.org/10.1074/mcp.O115.053843DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4813714PMC
March 2016

O-GlcNAcylation of master growth repressor DELLA by SECRET AGENT modulates multiple signaling pathways in Arabidopsis.

Genes Dev 2016 Jan;30(2):164-76

Department of Biology, Duke University, Durham, North Carolina 27708, USA;

The DELLA family of transcription regulators functions as master growth repressors in plants by inhibiting phytohormone gibberellin (GA) signaling in response to developmental and environmental cues. DELLAs also play a central role in mediating cross-talk between GA and other signaling pathways via antagonistic direct interactions with key transcription factors. However, how these crucial protein-protein interactions can be dynamically regulated during plant development remains unclear. Here, we show that DELLAs are modified by the O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) SECRET AGENT (SEC) in Arabidopsis. O-GlcNAcylation of the DELLA protein REPRESSOR OF ga1-3 (RGA) inhibits RGA binding to four of its interactors-PHYTOCHROME-INTERACTING FACTOR3 (PIF3), PIF4, JASMONATE-ZIM DOMAIN1, and BRASSINAZOLE-RESISTANT1 (BZR1)-that are key regulators in light, jasmonate, and brassinosteroid signaling pathways, respectively. Consistent with this, the sec-null mutant displayed reduced responses to GA and brassinosteroid and showed decreased expression of several common target genes of DELLAs, BZR1, and PIFs. Our results reveal a direct role of OGT in repressing DELLA activity and indicate that O-GlcNAcylation of DELLAs provides a fine-tuning mechanism in coordinating multiple signaling activities during plant development.
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http://dx.doi.org/10.1101/gad.270587.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4719307PMC
January 2016

Characterization of the peptide binding specificity of the HLA class I alleles B*38:01 and B*39:06.

Immunogenetics 2016 Mar 11;68(3):231-6. Epub 2016 Jan 11.

La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA, 92037, USA.

B*38:01 and B*39:06 are present with phenotypic frequencies <2% in the general population, but are of interest as B*39:06 is the B allele most associated with type 1 diabetes susceptibility and 38:01 is most protective. A previous study derived putative main anchor motifs for both alleles based on peptide elution data. The present study has utilized panels of single amino acid substitution peptide libraries to derive detailed quantitative motifs accounting for both primary and secondary influences on peptide binding. From these analyses, both alleles were confirmed to utilize the canonical position 2/C-terminus main anchor spacing. B*38:01 preferentially bound peptides with the positively charged or polar residues H, R, and Q in position 2 and the large hydrophobic residues I, F, L, W, and M at the C-terminus. B*39:06 had a similar preference for R in position 2, but also well-tolerated M, Q, and K. A more dramatic contrast between the two alleles was noted at the C-terminus, where the specificity of B*39:06 was clearly for small residues, with A as most preferred, followed by G, V, S, T, and I. Detailed position-by-position and residue-by-residue coefficient values were generated from the panels to provide detailed quantitative B*38:01 and B*39:06 motifs. It is hoped that these detailed motifs will facilitate the identification of T cell epitopes recognized in the context of two class I alleles associated with dramatically different dispositions towards type 1 diabetes, offering potential avenues for the investigation of the role of CD8 T cells in this disease.
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http://dx.doi.org/10.1007/s00251-015-0898-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4760861PMC
March 2016

Identification of the Post-translational Modifications Present in Centromeric Chromatin.

Mol Cell Proteomics 2016 Mar 18;15(3):918-31. Epub 2015 Dec 18.

From the ‡Department of Cell Biology, University of Virginia, Charlottesville, Virginia, 22908; ‖Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia, 22908; **Department of Biochemistry and Molecular Genetics, Northwestern University, Feinberg School of Medicine, Chicago Illinois 60611

The centromere is the locus on the chromosome that acts as the essential connection point between the chromosome and the mitotic spindle. A histone H3 variant, CENP-A, defines the location of the centromere, but centromeric chromatin consists of a mixture of both CENP-A-containing and H3-containing nucleosomes. We report a surprisingly uniform pattern of primarily monomethylation on lysine 20 of histone H4 present in short polynucleosomes mixtures of CENP-A and H3 nucleosomes isolated from functional centromeres. Canonical H3 is not a component of CENP-A-containing nucleosomes at centromeres, so the H3 we copurify from these preparations comes exclusively from adjacent nucleosomes. We find that CENP-A-proximal H3 nucleosomes are not uniformly modified but contain a complex set of PTMs. Dually modified K9me2-K27me2 H3 nucleosomes are observed at the centromere. Side-chain acetylation of both histone H3 and histone H4 is low at the centromere. Prior to assembly at centromeres, newly expressed CENP-A is sequestered for a large portion of the cell cycle (late S-phase, G2, and most of mitosis) in a complex that contains its partner, H4, and its chaperone, HJURP. In contrast to chromatin associated centromeric histone H4, we show that prenucleosomal CENP-A-associated histone H4 lacks K20 methylation and contains side-chain and α-amino acetylation. We show HJURP displays a complex set of serine phosphorylation that may potentially regulate the deposition of CENP-A. Taken together, our findings provide key information regarding some of the key components of functional centromeric chromatin.
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http://dx.doi.org/10.1074/mcp.M115.053710DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4813710PMC
March 2016

Analysis of Monoclonal Antibody Sequence and Post-translational Modifications by Time-controlled Proteolysis and Tandem Mass Spectrometry.

Mol Cell Proteomics 2016 Apr 29;15(4):1479-88. Epub 2015 Nov 29.

From the ‡Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904;

Methodology for sequence analysis of ∼150 kDa monoclonal antibodies (mAb), including location of post-translational modifications and disulfide bonds, is described. Limited digestion of fully denatured (reduced and alkylated) antibody was accomplished in seconds by flowing a sample in 8murea at a controlled flow rate through a micro column reactor containing immobilized aspergillopepsin I. The resulting product mixture containing 3-9 kDa peptides was then fractionated by capillary column liquid chromatography and analyzed on-line by both electron-transfer dissociation and collisionally activated dissociation mass spectrometry (MS). This approach enabled identification of peptides that cover the complete sequence of a murine mAb. With customized tandem MS and ProSightPC Biomarker search, we verified 95% amino acid residues of this mAb and identified numerous post-translational modifications (oxidized methionine, pyroglutamylation, deamidation of Asn, and several forms ofN-linked glycosylation). For disulfide bond location, native mAb is subjected to the same procedure but with longer digestion times controlled by sample flow rate through the micro column reactor. Release of disulfide containing peptides from accessible regions of the folded antibody occurs with short digestion times. Release of those in the interior of the molecule requires longer digestion times. The identity of two peptides connected by a disulfide bond is determined using a combination of electron-transfer dissociation and ion-ion proton transfer chemistry to read the two N-terminal and two C-terminal sequences of the connected peptides.
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http://dx.doi.org/10.1074/mcp.O115.056721DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4824869PMC
April 2016

Site-Specific Zwitterionic Polymer Conjugates of a Protein Have Long Plasma Circulation.

Chembiochem 2015 Nov 20;16(17):2451-5. Epub 2015 Oct 20.

Department of Biomedical Engineering, Duke University, 101 Science Drive, Durham, NC, 27708, USA.

Many proteins suffer from suboptimal pharmacokinetics (PK) that limit their utility as drugs. The efficient synthesis of polymer conjugates of protein drugs with tunable PK to optimize their in vivo efficacy is hence critical. We report here the first study of the in vivo behavior of a site-specific conjugate of a zwitterionic polymer and a protein. To synthesize the conjugate, we first installed an initiator for atom-transfer radical polymerization (ATRP) at the N terminus of myoglobin (Mb-N-Br). Subsequently, in situ ATRP was carried out in aqueous buffer to grow an amine-functionalized polymer from Mb-N-Br. The cationic polymer was further derivatized to two zwitterionic polymers by treating the amine groups of the cationic polymer with iodoacetic acid to obtain poly(carboxybetaine methacrylate) with a one-carbon spacer (PCBMA; C1 ), and sequentially with 3-iodopropionic acid and iodoacetic acid to obtain PCBMA(mix) with a mixture of C1 and C2 spacers. The Mb-N-PCBMA polymer conjugates had a longer in vivo plasma half-life than a PEG-like comb polymer conjugate of similar molecular weights (MW). The structure of the zwitterion plays a role in controlling the in vivo behavior of the conjugate, as the PCBMA conjugate with a C1 spacer had significantly longer plasma circulation than the conjugate with a mixture of C1 and C2 spacers.
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http://dx.doi.org/10.1002/cbic.201500439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4802966PMC
November 2015

Pepsin-Containing Membranes for Controlled Monoclonal Antibody Digestion Prior to Mass Spectrometry Analysis.

Anal Chem 2015 Nov 22;87(21):10942-9. Epub 2015 Oct 22.

Department of Chemistry, Michigan State University , East Lansing, Michigan 48824, United States.

Monoclonal antibodies (mAbs) are the fastest growing class of therapeutic drugs, because of their high specificities to target cells. Facile analysis of therapeutic mAbs and their post-translational modifications (PTMs) is essential for quality control, and mass spectrometry (MS) is the most powerful tool for antibody characterization. This study uses pepsin-containing nylon membranes as controlled proteolysis reactors for mAb digestion prior to ultrahigh-resolution Orbitrap MS analysis. Variation of the residence times (from 3 ms to 3 s) of antibody solutions in the membranes yields "bottom-up" (1-2 kDa) to "middle-down" (5-15 kDa) peptide sizes within less than 10 min. These peptides cover the entire sequences of Trastuzumab and a Waters antibody, and a proteolytic peptide comprised of 140 amino acids from the Waters antibody contains all three complementarity determining regions on the light chain. This work compares the performance of "bottom-up" (in-solution tryptic digestion), "top-down" (intact protein fragmentation), and "middle-down" (in-membrane digestion) analysis of an antibody light chain. Data from tandem MS show 99%, 55%, and 99% bond cleavage for "bottom-up", "top-down", and "middle-down" analyses, respectively. In-membrane digestion also facilitates detection of PTMs such as oxidation, deamidation, N-terminal pyroglutamic acid formation, and glycosylation. Compared to "bottom-up" and "top-down" approaches for antibody characterization, in-membrane digestion uses minimal sample preparation time, and this technique also yields high peptide and sequence coverage for the identification of PTMs.
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http://dx.doi.org/10.1021/acs.analchem.5b02739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5016144PMC
November 2015

The common equine class I molecule Eqca-1*00101 (ELA-A3.1) is characterized by narrow peptide binding and T cell epitope repertoires.

Immunogenetics 2015 Nov 23;67(11-12):675-89. Epub 2015 Sep 23.

Department of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA, 92037, USA.

Here we describe a detailed quantitative peptide-binding motif for the common equine leukocyte antigen (ELA) class I allele Eqca-1*00101, present in roughly 25 % of Thoroughbred horses. We determined a preliminary binding motif by sequencing endogenously bound ligands. Subsequently, a positional scanning combinatorial library (PSCL) was used to further characterize binding specificity and derive a quantitative motif involving aspartic acid in position 2 and hydrophobic residues at the C-terminus. Using this motif, we selected and tested 9- and 10-mer peptides derived from the equine herpesvirus type 1 (EHV-1) proteome for their capacity to bind Eqca-1*00101. PSCL predictions were very efficient, with an receiver operating characteristic (ROC) curve performance of 0.877, and 87 peptides derived from 40 different EHV-1 proteins were identified with affinities of 500 nM or higher. Quantitative analysis revealed that Eqca-1*00101 has a narrow peptide-binding repertoire, in comparison to those of most human, non-human primate, and mouse class I alleles. Peripheral blood mononuclear cells from six EHV-1-infected, or vaccinated but uninfected, Eqca-1*00101-positive horses were used in IFN-γ enzyme-linked immunospot (ELISPOT) assays. When we screened the 87 Eqca-1*00101-binding peptides for T cell reactivity, only one Eqca-1*00101 epitope, derived from the intermediate-early protein ICP4, was identified. Thus, despite its common occurrence in several horse breeds, Eqca-1*00101 is associated with a narrow binding repertoire and a similarly narrow T cell response to an important equine viral pathogen. Intriguingly, these features are shared with other human and macaque major histocompatibility complex (MHC) molecules with a similar specificity for D in position 2 or 3 in their main anchor motif.
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http://dx.doi.org/10.1007/s00251-015-0872-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4783141PMC
November 2015