Publications by authors named "David C Schriemer"

85 Publications

Improving Spectral Validation Rates in Hydrogen-Deuterium Exchange Data Analysis.

Anal Chem 2021 03 16;93(9):4246-4254. Epub 2021 Feb 16.

Department of Chemistry, University of Calgary, Calgary, Alberta, Canada T2N-4N1.

The data analysis practices associated with hydrogen-deuterium exchange mass spectrometry (HX-MS) lag far behind that of most other MS-based protein analysis tools. A reliance on external tools from other fields and a persistent need for manual data validation restrict this powerful technology to the expert user. Here, we provide an extensive upgrade to the HX data analysis suite available in the Mass Spec Studio in the form of two new apps (HX-PIPE and HX-DEAL), completing a workflow that provides an HX-tailored peptide identification capability, accelerated validation routines, automated spectral deconvolution strategies, and a rich set of exportable graphics and statistical reports. With these new tools, we demonstrate that the peptide identifications obtained from undeuterated samples generated at the start of a project contain information that helps predict and control the extent of manual validation required. We also uncover a large fraction of HX-usable peptides that remains unidentified in most experiments. We show that automated spectral deconvolution routines can identify exchange regimes in a project-wide manner, although they remain difficult to accurately assign in all scenarios. Taken together, these new tools provide a robust and complete solution suitable for the analysis of high-complexity HX-MS data.
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http://dx.doi.org/10.1021/acs.analchem.0c05045DOI Listing
March 2021

The active DNA-PK holoenzyme occupies a tensed state in a staggered synaptic complex.

Structure 2020 Dec 30. Epub 2020 Dec 30.

Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada; Robson DNA Science Centre, Arnie Charbonneau Cancer Institute, University of Calgary, Calgary, AB, Canada; Department of Chemistry, University of Calgary, Calgary, AB, Canada. Electronic address:

In the non-homologous end-joining (NHEJ) of a DNA double-strand break, DNA ends are bound and protected by DNA-PK, which synapses across the break to tether the broken ends and initiate repair. There is little clarity surrounding the nature of the synaptic complex and the mechanism governing the transition to repair. We report an integrative structure of the synaptic complex at a precision of 13.5 Å, revealing a symmetric head-to-head arrangement with a large offset in the DNA ends and an extensive end-protection mechanism involving a previously uncharacterized plug domain. Hydrogen/deuterium exchange mass spectrometry identifies an allosteric pathway connecting DNA end-binding with the kinase domain that places DNA-PK under tension in the kinase-active state. We present a model for the transition from end-protection to repair, where the synaptic complex supports hierarchical processing of the ends and scaffold assembly, requiring displacement of the catalytic subunit and tension release through kinase activity.
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http://dx.doi.org/10.1016/j.str.2020.12.006DOI Listing
December 2020

The substrate specificity of the human TRAPPII complex's Rab-guanine nucleotide exchange factor activity.

Commun Biol 2020 Dec 4;3(1):735. Epub 2020 Dec 4.

Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, V8W 2Y2, Canada.

The TRAnsport Protein Particle (TRAPP) complexes act as Guanine nucleotide exchange factors (GEFs) for Rab GTPases, which are master regulators of membrane trafficking in eukaryotic cells. In metazoans, there are two large multi-protein TRAPP complexes: TRAPPII and TRAPPIII, with the TRAPPII complex able to activate both Rab1 and Rab11. Here we present detailed biochemical characterisation of Rab-GEF specificity of the human TRAPPII complex, and molecular insight into Rab binding. GEF assays of the TRAPPII complex against a panel of 20 different Rab GTPases revealed GEF activity on Rab43 and Rab19. Electron microscopy and chemical cross-linking revealed the architecture of mammalian TRAPPII. Hydrogen deuterium exchange MS showed that Rab1, Rab11 and Rab43 share a conserved binding interface. Clinical mutations in Rab11, and phosphomimics of Rab43, showed decreased TRAPPII GEF mediated exchange. Finally, we designed a Rab11 mutation that maintained TRAPPII-mediated GEF activity while decreasing activity of the Rab11-GEF SH3BP5, providing a tool to dissect Rab11 signalling. Overall, our results provide insight into the GTPase specificity of TRAPPII, and how clinical mutations disrupt this regulation.
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http://dx.doi.org/10.1038/s42003-020-01459-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7719173PMC
December 2020

A substrate binding model for the KEOPS tRNA modifying complex.

Nat Commun 2020 12 4;11(1):6233. Epub 2020 Dec 4.

The Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada.

The KEOPS complex, which is conserved across archaea and eukaryotes, is composed of four core subunits; Pcc1, Kae1, Bud32 and Cgi121. KEOPS is crucial for the fitness of all organisms examined. In humans, pathogenic mutations in KEOPS genes lead to Galloway-Mowat syndrome, an autosomal-recessive disease causing childhood lethality. Kae1 catalyzes the universal and essential tRNA modification N-threonylcarbamoyl adenosine, but the precise roles of all other KEOPS subunits remain an enigma. Here we show using structure-guided studies that Cgi121 recruits tRNA to KEOPS by binding to its 3' CCA tail. A composite model of KEOPS bound to tRNA reveals that all KEOPS subunits form an extended tRNA-binding surface that we have validated in vitro and in vivo to mediate the interaction with the tRNA substrate and its modification. These findings provide a framework for understanding the inner workings of KEOPS and delineate why all KEOPS subunits are essential.
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http://dx.doi.org/10.1038/s41467-020-19990-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7718258PMC
December 2020

Harmonizing structural mass spectrometry analyses in the mass spec studio.

J Proteomics 2020 08 29;225:103844. Epub 2020 May 29.

Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1, Canada; Department of Chemistry, University of Calgary, Calgary, Alberta T2N 4N1, Canada. Electronic address:

Structural Mass Spectrometry (SMS) provides a comprehensive toolbox for the analysis of protein structure and function. It offers multiple sources of structural information that are increasingly useful for integrative structural modeling of complex protein systems. As MS-based structural workflows scale to larger systems, consistent and coherent data interpretation resources are needed to better support modeling. Unlike the proteomics community, practitioners of SMS lack adequate computational tools. Here, we review new developments in the Mass Spec Studio: an expandable ecosystem of workflows for the analysis of complementary SMS techniques with linkages to modeling. Current functionality in the Studio (version 2) supports three major SMS workflows (crosslinking, hydrogen/deuterium exchange and covalent labelling) and two pipelines for structural modeling, with a special focus on data integration. The Mass Spec Studio is an architecture focused on rapid and robust extension of functionality by a community of developers. SIGNIFICANCE: This review surveys the new data analysis capabilities within the Mass Spec Studio, a rich framework for rapid software development specifically targeting the community of structural proteomics and structural mass spectrometry. Updates to crosslinking, hydrogen/deuterium-exchange and covalent labeling apps are provided as well as a utility for translating such analyses into restraints that support integrative structural modeling. These new capabilities, together with the underlying design tools and content, provide the community with a wealth of resources to tackle complex structural problem and design new approaches to data analysis.
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http://dx.doi.org/10.1016/j.jprot.2020.103844DOI Listing
August 2020

Correlation between Labeling Yield and Surface Accessibility in Covalent Labeling Mass Spectrometry.

J Am Soc Mass Spectrom 2020 Feb 8;31(2):207-216. Epub 2020 Jan 8.

Department of Biochemistry and Molecular Biology , University of Calgary , Calgary , Alberta , Canada T2N 4N1.

The functional properties of a protein are strongly influenced by its topography, or the solvent-facing contour map of its surface. Together with crosslinking, covalent labeling mass spectrometry (CL-MS) has the potential to contribute topographical data through the measurement of surface accessibility. However, recent efforts to correlate measures of surface accessibility with labeling yield have been met with mixed success. Most applications of CL-MS involve differential analysis of protein interactions (i.e., footprinting experiments) where such inconsistencies have limited effect. Extending CL-MS into structural analysis requires an improved evaluation of the relationship between labeling and surface exposure. In this study, we applied recently developed diazirine reagents to obtain deep coverage of the large motor domain of Eg5 (a mitotic kinesin), and together with computational methods we correlated labeling yields with accessibility data in a number of ways. We observe that correlations can indeed be seen at a local structural level, but these correlations do not extend across the structure. The lack of correlation arises from the influence of protein dynamics and chemical composition on reagent partitioning and, thus, also on labeling yield. We conclude that our use of CL-MS data should be considered in light of "chemical accessibility" rather than "solvent accessibility" and suggest that CL-MS data would be a useful tool in the fundamental study of protein-solute interactions.
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http://dx.doi.org/10.1021/jasms.9b00083DOI Listing
February 2020

Federating Structural Models and Data: Outcomes from A Workshop on Archiving Integrative Structures.

Structure 2019 12 25;27(12):1745-1759. Epub 2019 Nov 25.

Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158, USA; Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biosciences, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address:

Structures of biomolecular systems are increasingly computed by integrative modeling. In this approach, a structural model is constructed by combining information from multiple sources, including varied experimental methods and prior models. In 2019, a Workshop was held as a Biophysical Society Satellite Meeting to assess progress and discuss further requirements for archiving integrative structures. The primary goal of the Workshop was to build consensus for addressing the challenges involved in creating common data standards, building methods for federated data exchange, and developing mechanisms for validating integrative structures. The summary of the Workshop and the recommendations that emerged are presented here.
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http://dx.doi.org/10.1016/j.str.2019.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7108332PMC
December 2019

SSEThread: Integrative threading of the DNA-PKcs sequence based on data from chemical cross-linking and hydrogen deuterium exchange.

Prog Biophys Mol Biol 2019 10 27;147:92-102. Epub 2019 Sep 27.

Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, USA.

X-ray crystallography and electron microscopy maps resolved to 3-8 Å are generally sufficient for tracing the path of the polypeptide chain in space, while often insufficient for unambiguously registering the sequence on the path (i.e., threading). Frequently, however, additional information is available from other biophysical experiments, physical principles, statistical analyses, and other prior models. Here, we formulate an integrative approach for sequence assignment to a partial backbone model as an optimization problem, which requires three main components: the representation of the system, the scoring function, and the optimization method. The method is implemented in the open source Integrative Modeling Platform (IMP) (https://integrativemodeling.org), allowing a number of different terms in the scoring function. We apply this method to localizing the sequence assignment within a 199-residue disordered region of three structured and sequence unassigned helices in the DNA-PKcs crystallographic structure, using chemical crosslinks, hydrogen deuterium exchange, and sequence connectivity. The resulting ensemble of threading models provides two major solutions, one of which suggests that the crucial ABCDE cluster of phosphorylation sites cannot undergo intra-molecular autophosphorylation without a conformational rearrangement. The ensemble of solutions embodies the most accurate and precise sequence threading given the available information.
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http://dx.doi.org/10.1016/j.pbiomolbio.2019.09.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6903780PMC
October 2019

A microtubule crosslinking protocol for integrative structural modeling activities.

Anal Biochem 2019 12 6;586:113416. Epub 2019 Sep 6.

Department of Chemistry, University of Calgary, Calgary, Alberta, Canada; Department of Biochemistry and Molecular Biology, University of Calgary, Alberta, Canada. Electronic address:

Microtubules (MTs) are key components in the cytoskeleton of the eukaryotic cell, and play roles in processes such as intracellular transport and cell division. An improved understanding MT regulation requires structural analysis of the extensive interactions between the MT lattice and its regulatory proteins, but MT interactions are challenging for even the most advanced structural methods to characterize. Integrative methods involving crosslinking mass spectrometry (XL-MS) can extend structural analysis to many interaction classes, but the representation of MTs in crosslinking data-sets has been surprisingly low. Here, we explore the basis for the underrepresentation of the MT lattice and present an enhanced method for mapping MT structural features using an optimized set of reagents, together with fluorescence detection to ensure MT structural integrity. Through the application of stringent identification criteria, 91 unique crosslinks were identified, 78 of which were uniquely matched to 7 distinct structural features of the MT lattice. Of note, 4 crosslinks were detected for the lattice-A protofilament organization. The lattice-A structure defines a "seam" or discontinuity in MTs and is an emerging site of interest for MT regulation. Our methodology should be broadly applicable to integrative structural studies involving any MT-protein interaction.
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http://dx.doi.org/10.1016/j.ab.2019.113416DOI Listing
December 2019

Dipeptidase-1 Is an Adhesion Receptor for Neutrophil Recruitment in Lungs and Liver.

Cell 2019 08;178(5):1205-1221.e17

Arnie Charbonneau Cancer Institute, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada; Department of Oncology, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada. Electronic address:

A hallmark feature of inflammation is the orchestrated recruitment of neutrophils from the bloodstream into inflamed tissue. Although selectins and integrins mediate recruitment in many tissues, they have a minimal role in the lungs and liver. Exploiting an unbiased in vivo functional screen, we identified a lung and liver homing peptide that functionally abrogates neutrophil recruitment to these organs. Using biochemical, genetic, and confocal intravital imaging approaches, we identified dipeptidase-1 (DPEP1) as the target and established its role as a physical adhesion receptor for neutrophil sequestration independent of its enzymatic activity. Importantly, genetic ablation or functional peptide blocking of DPEP1 significantly reduced neutrophil recruitment to the lungs and liver and provided improved survival in models of endotoxemia. Our data establish DPEP1 as a major adhesion receptor on the lung and liver endothelium and identify a therapeutic target for neutrophil-driven inflammatory diseases of the lungs.
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http://dx.doi.org/10.1016/j.cell.2019.07.017DOI Listing
August 2019

Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments.

Nat Methods 2019 07 27;16(7):595-602. Epub 2019 Jun 27.

Department of Pharmacy, University of Copenhagen, Copenhagen, Denmark.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.
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http://dx.doi.org/10.1038/s41592-019-0459-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6614034PMC
July 2019

Quantitative Analysis of Protein Covalent Labeling Mass Spectrometry Data in the Mass Spec Studio.

Anal Chem 2019 07 14;91(13):8492-8499. Epub 2019 Jun 14.

Covalent labeling with mass spectrometry (CL-MS) provides a direct measure of the chemical and structural features of proteins with the potential for resolution at the amino-acid level. Unfortunately, most applications of CL-MS are limited to narrowly defined differential analyses, where small numbers of residues are compared between two or more protein states. Extending the utility of high-resolution CL-MS for structure-based applications requires more robust computational routines and the development of methodology capable of reporting of labeling yield accurately. Here, we provide a substantial improvement in the analysis of CL-MS data with the development of an extended plug-in built within the Mass Spec Studio development framework (MSS-CLEAN). All elements of data analysis-from database search to site-resolved and normalized labeling output-are accommodated, as illustrated through the nonselective labeling of the human kinesin Eg5 with photoconverted 3,3'-azibutan-1-ol. In developing the new features within the CL-MS plug-in, we identified additional complexities associated with the application of CL reagents, arising primarily from digestion-induced bias in yield measurements and ambiguities in site localization. A strategy is presented involving the use of redundant site labeling data from overlapping peptides, the imputation of missing data, and a normalization routine to determine relative protection factors. These elements together provide for a robust structural interpretation of CL-MS/MS data while minimizing the over-reporting of labeling site resolution. Finally, to minimize bias, we recommend that digestion strategies for the generation of useful overlapping peptides involve the application of complementary enzymes that drive digestion to completion.
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http://dx.doi.org/10.1021/acs.analchem.9b01625DOI Listing
July 2019

First Community-Wide, Comparative Cross-Linking Mass Spectrometry Study.

Anal Chem 2019 06 22;91(11):6953-6961. Epub 2019 May 22.

Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Charles Tanford Protein Center , Martin Luther University Halle-Wittenberg , Kurt-Mothes-Strasse 3a , 06120 Halle/Saale , Germany.

The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein-protein interactions has increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats. Such consensus formats should become accepted in the field and be shown to lead to reproducible results. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. Our study therefore builds the framework for establishing best practice guidelines to conduct cross-linking experiments, perform data analysis, and define reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results.
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http://dx.doi.org/10.1021/acs.analchem.9b00658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6625963PMC
June 2019

The CHD6 chromatin remodeler is an oxidative DNA damage response factor.

Nat Commun 2019 01 16;10(1):241. Epub 2019 Jan 16.

Robson DNA Science Centre, Arnie Charbonneau Cancer Institute, Departments of Biochemistry & Molecular Biology and/or Oncology, Cumming School of Medicine, University of Calgary, Calgary, AB, T2N 4N1, Canada.

Cell survival after oxidative DNA damage requires signaling, repair and transcriptional events often enabled by nucleosome displacement, exchange or removal by chromatin remodeling enzymes. Here, we show that Chromodomain Helicase DNA-binding protein 6 (CHD6), distinct to other CHD enzymes, is stabilized during oxidative stress via reduced degradation. CHD6 relocates rapidly to DNA damage in a manner dependent upon oxidative lesions and a conserved N-terminal poly(ADP-ribose)-dependent recruitment motif, with later retention requiring the double chromodomain and central core. CHD6 ablation increases reactive oxygen species persistence and impairs anti-oxidant transcriptional responses, leading to elevated DNA breakage and poly(ADP-ribose) induction that cannot be rescued by catalytic or double chromodomain mutants. Despite no overt epigenetic or DNA repair abnormalities, CHD6 loss leads to impaired cell survival after chronic oxidative stress, abnormal chromatin relaxation, amplified DNA damage signaling and checkpoint hypersensitivity. We suggest that CHD6 is a key regulator of the oxidative DNA damage response.
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http://dx.doi.org/10.1038/s41467-018-08111-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6335469PMC
January 2019

Photo-Cross-Linking Mass Spectrometry and Integrative Modeling Enables Rapid Screening of Antigen Interactions Involving Bacterial Transferrin Receptors.

J Proteome Res 2019 03 11;18(3):934-946. Epub 2019 Jan 11.

Structure-based approaches to the delineation of immunogens for vaccine development have a throughput requirement that is difficult to meet in practice with conventional methods of structure determination. Here we present a strategy for rapid and accurate structure generation in support of antigen engineering programs. The approach is developed around the modeling of interactions between host transferrin (Tf) and the bacterial vaccine target transferrin binding protein B (TbpB) from Gram-negative pathogens such as Neisseria meningitidis. Using an approach based solely on cross-linking mass spectrometry (XL-MS) data, monomeric structural models, and the Integrative Modeling Platform (IMP), we demonstrate that converged representations of the Tf:TbpB interactions can be returned that accurately reflect the binding interface and the relative orientation of the monomeric units, with the capacity to scale to the analysis of interactions from any number of additional strains. We show that a key element to accurate modeling involves the application of hetero-bifunctional cross-linkers incorporating fast-acting photoactivatable diazirines coupled with conventional amine-targeting N-hydroxysuccinimide esters, and we demonstrate that conventional homo-bifunctional reagents used in cross-linking kinetically trap dynamic states in the ensemble. Therefore, the application of both classes of cross-linker provides an opportunity to empirically detect protein dynamics during integrative structural modeling.
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http://dx.doi.org/10.1021/acs.jproteome.8b00629DOI Listing
March 2019

Lysine Propionylation To Boost Sequence Coverage and Enable a "Silent SILAC" Strategy for Relative Protein Quantification.

Anal Chem 2018 08 20;90(15):9077-9084. Epub 2018 Jul 20.

Quantification in proteomics largely relies on the incorporation of stable isotopes, with protocols that either introduce the label through metabolic incorporation or chemical tagging. Most methods rely on the use of trypsin and/or LysC to generate labeled peptides. Although alternative proteases can enhance proteome coverage, generic quantitative methods that port over to such enzymes are lacking. Here we describe a quantification strategy amenable to most proteases, which involves propionylation of metabolically labeled lysine, using a "silent stable isotope labeling by amino acids in cell culture (SILAC)" strategy that reveals isotopic labels on second-stage mass spectrometry (MS2) fragmentation in a tandem mass tag (TMT)-like manner. We selectively propionylated lysine residues prior to digestion to generate pure ArgC-like digestion for trypsin and novel ArgN-like digestions for LysargiNase, by restricting digestion at lysine. The modification offers highly complementary sequence coverage, and even enhanced protein identification rates in certain situations (GluC digestion). Propionylated lysine residues were present in the majority of identified peptides generated from digests of cell lysates and led to the consistent release of an intense cyclic imine reporter ion at mass-to-charge ratio ( m/ z) 140 using higher-energy collisional dissociation. We grew A549 cells in media containing either l-1-C-lysine or l-6-C-lysine, to generate proteins that share the same accurate mass when paired. Peptides were indistinguishable on the first-stage mass spectrometry (MS1) level and, upon fragmentation, released reporter ions at m/ z 140 and m/ z 141, without otherwise affecting sequence ion mass. The quantification approach is independent of the number of peptide lysines and offers a new strategy for quantitative proteomics.
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http://dx.doi.org/10.1021/acs.analchem.8b01403DOI Listing
August 2018

Simultaneous Proteoform Analysis of Histones H3 and H4 with a Simplified Middle-Down Proteomics Method.

Anal Chem 2018 03 15;90(5):3083-3090. Epub 2018 Feb 15.

Dynamic post-translational modifications of histones regulate transcriptional gene expression in eukaryotes. Unique combinations of modifications, almost exclusively displayed at the flexible N-terminal tails on histones, create distributions of proteoforms that need to be characterized in order to understand the complexity of gene regulation and how aberrant modification patterns influence disease. Although mass spectrometry is a preferred method for the analysis of histone modifications, information is lost when using conventional trypsin-based histone methods. Newer "middle-down" protocols may retain a greater fraction of the full proteoform distribution. We describe a strategy for the simultaneous characterization of histones H3 and H4 with near-complete retention of proteoform distributions, using a conventional proteomics liquid chromatography-tandem mass spectrometry (LC-MS/MS) configuration. The selective prolyl endoprotease neprosin generates convenient peptide lengths for retention and dispersion of modified H3 and H4 peptides on reversed-phase chromatography, offering an alternative to the hydrophilic interaction liquid chromatography typically used in middle-down methods. No chemical derivatizations are required, presenting a significant advantage over the trypsin-based protocol. Over 200 proteoforms can be readily profiled in a single analysis of histones from HeLa S3 cells. An in-gel digestion protocol provides additional options for effective histone analysis.
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http://dx.doi.org/10.1021/acs.analchem.7b03948DOI Listing
March 2018

Amino Acid Insertion Frequencies Arising from Photoproducts Generated Using Aliphatic Diazirines.

J Am Soc Mass Spectrom 2017 10 10;28(10):2011-2021. Epub 2017 Aug 10.

Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, T2N 4N1, Canada.

Mapping proteins with chemical reagents and mass spectrometry can generate a measure of accessible surface area, which in turn can be used to support the modeling and refinement of protein structures. Photolytically generated carbenes are a promising class of reagent for this purpose. Substituent effects appear to influence surface mapping properties, allowing for a useful measure of design control. However, to use carbene labeling data in a quantitative manner for modeling activities, we require a better understanding of their inherent amino acid reactivity, so that incorporation data can be normalized. The current study presents an analysis of the amino acid insertion frequency of aliphatic carbenes generated by the photolysis of three different diazirines: 3,3'-azibutyl-1-ammonium, 3,3'-azibutan-1-ol, and 4,4'-azipentan-1-oate. Leveraging an improved photolysis system for single-shot labeling of sub-microliter frozen samples, we used EThCD to localize insertion products in a large population of labeled peptides. Counting statistics were drawn from data-dependent LC-MS experiments and used to estimate the frequencies of insertion as a function of amino acid. We observed labeling of all 20 amino acids over a remarkably narrow range of insertion frequencies. However, the nature of the substituent could influence relative insertion frequencies, within a general preference for larger polar amino acids. We confirm a large (6-fold) increase in labeling yield when carbenes were photogenerated in the solid phase (77 K) relative to the liquid phase (293 K), and we suggest that carbene labeling should always be conducted in the frozen state to avoid information loss in surface mapping experiments. Graphical Abstract ᅟ.
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http://dx.doi.org/10.1007/s13361-017-1730-zDOI Listing
October 2017

Structural and functional characterization of the PNKP-XRCC4-LigIV DNA repair complex.

Nucleic Acids Res 2017 Jun;45(10):6238-6251

Department of Biochemistry, University of Alberta, Edmonton, AB T6G-2H7, Canada.

Non-homologous end joining (NHEJ) repairs DNA double strand breaks in non-cycling eukaryotic cells. NHEJ relies on polynucleotide kinase/phosphatase (PNKP), which generates 5΄-phosphate/3΄-hydroxyl DNA termini that are critical for ligation by the NHEJ DNA ligase, LigIV. PNKP and LigIV require the NHEJ scaffolding protein, XRCC4. The PNKP FHA domain binds to the CK2-phosphorylated XRCC4 C-terminal tail, while LigIV uses its tandem BRCT repeats to bind the XRCC4 coiled-coil. Yet, the assembled PNKP-XRCC4-LigIV complex remains uncharacterized. Here, we report purification and characterization of a recombinant PNKP-XRCC4-LigIV complex. We show that the stable binding of PNKP in this complex requires XRCC4 phosphorylation and that only one PNKP protomer binds per XRCC4 dimer. Small angle X-ray scattering (SAXS) reveals a flexible multi-state complex that suggests that both the PNKP FHA and catalytic domains contact the XRCC4 coiled-coil and LigIV BRCT repeats. Hydrogen-deuterium exchange indicates protection of a surface on the PNKP phosphatase domain that may contact XRCC4-LigIV. A mutation on this surface (E326K) causes the hereditary neuro-developmental disorder, MCSZ. This mutation impairs PNKP recruitment to damaged DNA in human cells and provides a possible disease mechanism. Together, this work unveils multipoint contacts between PNKP and XRCC4-LigIV that regulate PNKP recruitment and activity within NHEJ.
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http://dx.doi.org/10.1093/nar/gkx275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5449630PMC
June 2017

Neprosin, a Selective Prolyl Endoprotease for Bottom-up Proteomics and Histone Mapping.

Mol Cell Proteomics 2017 06 12;16(6):1162-1171. Epub 2017 Apr 12.

From the ‡Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N4N1, Canada;

Trypsin dominates bottom-up proteomics, but there are reasons to consider alternative enzymes. Improving sequence coverage, exposing proteomic "dark matter," and clustering post-translational modifications in different ways and with higher-order drive the pursuit of reagents complementary to trypsin. Additionally, enzymes that are easy to use and generate larger peptides that capitalize upon newer fragmentation technologies should have a place in proteomics. We expressed and characterized recombinant neprosin, a novel prolyl endoprotease of the DUF239 family, which preferentially cleaves C-terminal to proline residues under highly acidic conditions. Cleavage also occurs C-terminal to alanine with some frequency, but with an intriguingly high "skipping rate." Digestion proceeds to a stable end point, resulting in an average peptide mass of 2521 units and a higher dependence upon electron-transfer dissociation for peptide-spectrum matches. In contrast to most proline-cleaving enzymes, neprosin effectively degrades proteins of any size. For 1251 HeLa cell proteins identified in common using trypsin, Lys-C, and neprosin, almost 50% of the neprosin sequence contribution is unique. The high average peptide mass coupled with cleavage at residues not usually modified provide new opportunities for profiling clusters of post-translational modifications. We show that neprosin is a useful reagent for reading epigenetic marks on histones. It generates peptide 1-38 of histone H3 and peptide 1-32 of histone H4 in a single digest, permitting the analysis of co-occurring post-translational modifications in these important N-terminal tails.
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http://dx.doi.org/10.1074/mcp.M116.066803DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5461545PMC
June 2017

Lactoferrin binding protein B - a bi-functional bacterial receptor protein.

PLoS Pathog 2017 03 3;13(3):e1006244. Epub 2017 Mar 3.

Department of Microbiology & Infectious Diseases, University of Calgary, Calgary, Alberta, Canada.

Lactoferrin binding protein B (LbpB) is a bi-lobed outer membrane-bound lipoprotein that comprises part of the lactoferrin (Lf) receptor complex in Neisseria meningitidis and other Gram-negative pathogens. Recent studies have demonstrated that LbpB plays a role in protecting the bacteria from cationic antimicrobial peptides due to large regions rich in anionic residues in the C-terminal lobe. Relative to its homolog, transferrin-binding protein B (TbpB), there currently is little evidence for its role in iron acquisition and relatively little structural and biophysical information on its interaction with Lf. In this study, a combination of crosslinking and deuterium exchange coupled to mass spectrometry, information-driven computational docking, bio-layer interferometry, and site-directed mutagenesis was used to probe LbpB:hLf complexes. The formation of a 1:1 complex of iron-loaded Lf and LbpB involves an interaction between the Lf C-lobe and LbpB N-lobe, comparable to TbpB, consistent with a potential role in iron acquisition. The Lf N-lobe is also capable of binding to negatively charged regions of the LbpB C-lobe and possibly other sites such that a variety of higher order complexes are formed. Our results are consistent with LbpB serving dual roles focused primarily on iron acquisition when exposed to limited levels of iron-loaded Lf on the mucosal surface and effectively binding apo Lf when exposed to high levels at sites of inflammation.
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http://dx.doi.org/10.1371/journal.ppat.1006244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5352143PMC
March 2017

Nanospray HX-MS configuration for structural interrogation of large protein systems.

Analyst 2017 Mar;142(6):904-910

Department of Chemistry, University of Calgary, Calgary, AB T2N 1N4, Canada. and Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB T2N 1N4, Canada.

Hydrogen-deuterium exchange mass spectrometry (HX-MS) has made important contributions to the study of protein structure and function. Unfortunately, it is not known for low limits of detection, when compared with other forms of peptide-based or bottom-up protein MS methods. Systems perform poorly on sub-pmol quantities of protein states with greater than 300 kDa of unique sequences. The HX-MS analysis of complex protein states would be possible if proteomics-grade configurations could be used reliably, but temperature and temporal constraints have proven to be significant design challenges. Here, we describe an integrated HX-MS ion source operating on a vented-column geometry, which brings regulated column cooling right to the spray tip. The design offers chromatographic peak widths of 2-6 s (FWHM). It provides stable operation at 500 nL min, while retaining deuteration levels comparable to conventional geometries. We demonstrate at least a 50-fold improvement in protein consumption levels, and illustrate robustness by measuring peptide-averaged protection factors for 90% of DNA-PKcs, a 469 kDa protein, from 0.5 pmol injections.
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http://dx.doi.org/10.1039/c6an02707eDOI Listing
March 2017

Novel Allosteric Pathway of Eg5 Regulation Identified through Multivariate Statistical Analysis of Hydrogen-Exchange Mass Spectrometry (HX-MS) Ligand Screening Data.

Mol Cell Proteomics 2017 03 5;16(3):428-437. Epub 2017 Jan 5.

From the ‡Department of Chemistry, University of Calgary, Calgary, Alberta, Canada;

The mitotic kinesin Eg5 is an important target in cancer chemotherapy. A structurally diverse collection of canonical loop L5 inhibitors engage an allosteric pathway that includes elements of its microtubule binding region. However, recent evidence suggests that Eg5 may permit alternative allosteric mechanisms. Terpendole E, a natural-product Eg5 inhibitor, is active against mutants resistant to canonical loop L5 inhibitors and appears to offer a unique mode of inhibition. To investigate the variety of inhibitor responses, the structure-function properties of eighteen kinesin inhibitors were quantified with hydrogen-exchange mass spectrometry (HX-MS), functional analysis and molecular modeling. A unique strategy for high-density data analysis was implemented, based on a scalable multivariate statistical method, as current HX-MS routines have a limited capacity to guide a characterization of ligands when additional functional data is available. Inhibitor evaluation was achieved using orthogonal partial least squares projection to latent structures discriminant analysis (OPLS-DA). The strategy generated a model that identified functionally-significant conformational elements involved in kinesin inhibition, confirming the canonical allosteric pathway and identifying a novel response pathway. Terpendole E is demonstrated to be an atypical L5 site inhibitor, where binding induces an allosteric effect mediated by a destabilization in the β-sheet core of the molecular motor, an element involved in mechanochemical coupling for structurally-related kinesins. The analysis suggests that a different approach to inhibitor development may be fruitful.
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http://dx.doi.org/10.1074/mcp.M116.064246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5341003PMC
March 2017

Supporting metabolomics with adaptable software: design architectures for the end-user.

Curr Opin Biotechnol 2017 02 18;43:110-117. Epub 2016 Nov 18.

Department of Biochemistry and Molecular Biology, University of Calgary, Alberta T2N 1N4, Canada; Department of Chemistry, University of Calgary, Alberta T2N 1N4, Canada. Electronic address:

Large and disparate sets of LC-MS data are generated by modern metabolomics profiling initiatives, and while useful software tools are available to annotate and quantify compounds, the field requires continued software development in order to sustain methodological innovation. Advances in software development practices allow for a new paradigm in tool development for metabolomics, where increasingly the end-user can develop or redeploy utilities ranging from simple algorithms to complex workflows. Resources that provide an organized framework for development are described and illustrated with LC-MS processing packages that have leveraged their design tools. Full access to these resources depends in part on coding experience, but the emergence of workflow builders and pluggable frameworks strongly reduces the skill level required. Developers in the metabolomics community are encouraged to use these resources and design content for uptake and reuse.
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http://dx.doi.org/10.1016/j.copbio.2016.11.001DOI Listing
February 2017

Addressing proteolytic efficiency in enzymatic degradation therapy for celiac disease.

Sci Rep 2016 08 2;6:30980. Epub 2016 Aug 2.

Department of Biochemistry and Molecular Biology and the Southern Alberta Cancer Research Institute, University of Calgary, Calgary, Alberta, Canada.

Celiac disease is triggered by partially digested gluten proteins. Enzyme therapies that complete protein digestion in vivo could support a gluten-free diet, but the barrier to completeness is high. Current options require enzyme amounts on the same order as the protein meal itself. In this study, we evaluated proteolytic components of the carnivorous pitcher plant (Nepenthes spp.) for use in this context. Remarkably low doses enhance gliadin solubilization rates, and degrade gliadin slurries within the pH and temporal constraints of human gastric digestion. Potencies in excess of 1200:1 (substrate-to-enzyme) are achieved. Digestion generates small peptides through nepenthesin and neprosin, the latter a novel enzyme defining a previously-unknown class of prolyl endoprotease. The digests also exhibit reduced TG2 conversion rates in the immunogenic regions of gliadin, providing a twin mechanism for evading T-cell recognition. When sensitized and dosed with enzyme-treated gliadin, NOD/DQ8 mice did not show intestinal inflammation, when compared to mice challenged with only pepsin-treated gliadin. The low enzyme load needed for effective digestion suggests that gluten detoxification can be achieved in a meal setting, using metered dosing based on meal size. We demonstrate this by showing efficient antigen processing at total substrate-to-enzyme ratios exceeding 12,000:1.
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http://dx.doi.org/10.1038/srep30980DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4969619PMC
August 2016

Carnivorous Nutrition in Pitcher Plants (Nepenthes spp.) via an Unusual Complement of Endogenous Enzymes.

J Proteome Res 2016 09 1;15(9):3108-17. Epub 2016 Aug 1.

Department of Biochemistry and Molecular Biology and the Southern Alberta Cancer Research Institute, University of Calgary , Calgary, Alberta T2N 4N1, Canada.

Plants belonging to the genus Nepenthes are carnivorous, using specialized pitfall traps called "pitchers" that attract, capture, and digest insects as a primary source of nutrients. We have used RNA sequencing to generate a cDNA library from the Nepenthes pitchers and applied it to mass spectrometry-based identification of the enzymes secreted into the pitcher fluid using a nonspecific digestion strategy superior to trypsin in this application. This first complete catalog of the pitcher fluid subproteome includes enzymes across a variety of functional classes. The most abundant proteins present in the secreted fluid are proteases, nucleases, peroxidases, chitinases, a phosphatase, and a glucanase. Nitrogen recovery involves a particularly rich complement of proteases. In addition to the two expected aspartic proteases, we discovered three novel nepenthensins, two prolyl endopeptidases that we name neprosins, and a putative serine carboxypeptidase. Additional proteins identified are relevant to pathogen-defense and secretion mechanisms. The full complement of acid-stable enzymes discovered in this study suggests that carnivory in the genus Nepenthes can be sustained by plant-based mechanisms alone and does not absolutely require bacterial symbiosis.
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http://dx.doi.org/10.1021/acs.jproteome.6b00224DOI Listing
September 2016

High Sensitivity Crosslink Detection Coupled With Integrative Structure Modeling in the Mass Spec Studio.

Mol Cell Proteomics 2016 09 13;15(9):3071-80. Epub 2016 Jul 13.

From the ‡Department of Biochemistry and Molecular Biology, §Department of Chemistry,

The Mass Spec Studio package was designed to support the extraction of hydrogen-deuterium exchange and covalent labeling data for a range of mass spectrometry (MS)-based workflows, to integrate with restraint-driven protein modeling activities. In this report, we present an extension of the underlying Studio framework and provide a plug-in for crosslink (XL) detection. To accommodate flexibility in XL methods and applications, while maintaining efficient data processing, the plug-in employs a peptide library reduction strategy via a presearch of the tandem-MS data. We demonstrate that prescoring linear unmodified peptide tags using a probabilistic approach substantially reduces search space by requiring both crosslinked peptides to generate sparse data attributable to their linear forms. The method demonstrates highly sensitive crosslink peptide identification with a low false positive rate. Integration with a Haddock plug-in provides a resource that can combine multiple sources of data for protein modeling activities. We generated a structural model of porcine transferrin bound to TbpB, a membrane-bound receptor essential for iron acquisition in Actinobacillus pleuropneumoniae Using mutational data and crosslinking restraints, we confirm the mechanism by which TbpB recognizes the iron-loaded form of transferrin, and note the requirement for disparate sources of restraint data for accurate model construction. The software plugin is freely available at www.msstudio.ca.
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http://dx.doi.org/10.1074/mcp.O116.058685DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5013318PMC
September 2016

A robust capillary liquid chromatography/tandem mass spectrometry method for quantitation of neuromodulatory endocannabinoids.

Rapid Commun Mass Spectrom 2015 Oct;29(20):1889-97

Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada, T2N 4N1.

Rationale: Methods for quantifying anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) are needed to support programs investigating molecular mechanisms of the central nervous system. Existing methods, while useful, are not well adapted to efficiently process large numbers of very small tissue samples. A unique challenge involves the disparity in endogenous levels of AEA (pmol/g tissue) and 2-AG (nmol/g tissue).

Methods: A simplified one-step solvent extraction procedure was developed for recovering endocannabinoids from rat brain tissues, and combined with capillary liquid chromatography/tandem mass spectrometry (LC/MS/MS). Various multiple reaction monitoring (MRM)-based methods were evaluated for limit of detection (LOD) and robustness.

Results: The optimized simultaneous quantitation method achieves an LOQ of 50 amol for AEA and 25 fmol for 2-AG, both with a linearity over 3 orders of magnitude, and elution times under 3 min. Accuracy, expressed as relative error (RE), is less than 12% for AEA and less than 6% for 2-AG. Precision, expressed as relative standard deviation (RSD), is less than 6% for AEA and less than 3% for 2-AG. Sample handling routines are sufficiently robust to support the automated analysis of thousands of samples from a range of tissue types.

Conclusions: The microscale method is a sensitive, economical and robust alternative to the larger scale LC/MS methods currently implemented for quantitation of AEA and 2-AG.
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http://dx.doi.org/10.1002/rcm.7277DOI Listing
October 2015

A hypothesis-directed approach to the targeted development of a multiplexed proteomic biomarker assay for cancer.

Cancer Inform 2015 17;14:65-70. Epub 2015 May 17.

Department of Surgery, University of Calgary, Calgary, AB, Canada. ; Department of Oncology, University of Calgary, Calgary, AB, Canada.

In recent years, hundreds of candidate protein biomarkers have been identified using discovery-based proteomics. Despite the large number of candidate biomarkers, few proteins advance to clinical validation. We propose a hypothesis-driven approach to identify candidate biomarkers, previously characterized in the literature, with the highest probability of clinical applicability. A ranking method, called the "hypothesis-directed biomarker ranking" (HDBR) system, was developed to score candidate biomarkers based on seven criteria deemed important in the selection of clinically useful biomarkers. To demonstrate its application, we applied the HDBR system to identify candidate biomarkers for the development of a diagnostic test for the early detection of colorectal cancer. One-hundred and fifty-one candidate biomarkers were identified from the literature and ranked based on the specified criteria. The top-ranked candidates represent a group of biomarkers whose further study and validation would be justified in order to expedite the development of biomarkers that could be used in a clinical setting.
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http://dx.doi.org/10.4137/CIN.S24388DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4438726PMC
May 2015

HX-MS2 for high performance conformational analysis of complex protein states.

Protein Sci 2015 Aug 29;24(8):1313-24. Epub 2015 May 29.

Department of Biochemistry and Molecular Biology and the Southern Alberta Cancer Research Institute, University of Calgary, Calgary, Alberta, Canada, T2N 4N1.

Water-mediated hydrogen exchange (HX) processes involving the protein main chain are sensitive to structural dynamics and molecular interactions. Measuring deuterium uptake in amide bonds provides information on conformational states, structural transitions and binding events. Increasingly, deuterium levels are measured by mass spectrometry (MS) from proteolytically generated peptide fragments of large molecular systems. However, this bottom-up method has limited spectral capacity and requires a burdensome manual validation exercise, both of which restrict analysis of protein systems to generally less than 150 kDa. In this study, we present a bottom-up HX-MS(2) method that improves peptide identification rates, localizes high-quality HX data and simplifies validation. The method combines a new peptide scoring algorithm (WUF, weighted unique fragment) with data-independent acquisition of peptide fragmentation data. Scoring incorporates the validation process and emphasizes identification accuracy. The HX-MS(2) method is illustrated using data from a conformational analysis of microtubules treated with dimeric kinesin MCAK. When compared to a conventional Mascot-driven HX-MS method, HX-MS(2) produces two-fold higher α/β-tubulin sequence depth at a peptide utilization rate of 74%. A Mascot approach delivers a utilization rate of 44%. The WUF score can be constrained by false utilization rate (FUR) calculations to return utilization values exceeding 90% without serious data loss, indicating that automated validation should be possible. The HX-MS(2) data confirm that N-terminal MCAK domains anchor kinesin force generation in kinesin-mediated depolymerization, while the C-terminal tails regulate MCAK-tubulin interactions.
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http://dx.doi.org/10.1002/pro.2707DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4534182PMC
August 2015