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Dive into the research topics where Darryl B. Hardie is active.

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Featured researches published by Darryl B. Hardie.


Molecular & Cellular Proteomics | 2009

Multiple Reaction Monitoring-based, Multiplexed, Absolute Quantitation of 45 Proteins in Human Plasma

Michael A. Kuzyk; Derek Smith; Juncong Yang; Tyra J. Cross; Angela M. Jackson; Darryl B. Hardie; N. Leigh Anderson; Christoph H. Borchers

Mass spectrometry-based multiple reaction monitoring (MRM) quantitation of proteins can dramatically impact the discovery and quantitation of biomarkers via rapid, targeted, multiplexed protein expression profiling of clinical samples. A mixture of 45 peptide standards, easily adaptable to common plasma proteomics work flows, was created to permit absolute quantitation of 45 endogenous proteins in human plasma trypsin digests. All experiments were performed on simple tryptic digests of human EDTA-plasma without prior affinity depletion or enrichment. Stable isotope-labeled standard peptides were added immediately following tryptic digestion because addition of stable isotope-labeled standard peptides prior to trypsin digestion was found to generate elevated and unpredictable results. Proteotypic tryptic peptides containing isotopically coded amino acids ([13C6]Arg or [13C6]Lys) were synthesized for all 45 proteins. Peptide purity was assessed by capillary zone electrophoresis, and the peptide quantity was determined by amino acid analysis. For maximum sensitivity and specificity, instrumental parameters were empirically determined to generate the most abundant precursor ions and y ion fragments. Concentrations of individual peptide standards in the mixture were optimized to approximate endogenous concentrations of analytes and to ensure the maximum linear dynamic range of the MRM assays. Excellent linear responses (r > 0.99) were obtained for 43 of the 45 proteins with attomole level limits of quantitation (<20% coefficient of variation) for 27 of the 45 proteins. Analytical precision for 44 of the 45 assays varied by <10%. LC-MRM/MS analyses performed on 3 different days on different batches of plasma trypsin digests resulted in coefficients of variation of <20% for 42 of the 45 assays. Concentrations for 39 of the 45 proteins are within a factor of 2 of reported literature values. This mixture of internal standards has many uses and can be applied to the characterization of trypsin digestion kinetics and plasma protein expression profiling because 31 of the 45 proteins are putative biomarkers of cardiovascular disease.


Journal of Proteome Research | 2010

A Quantitative Study of the Effects of Chaotropic Agents, Surfactants, and Solvents on the Digestion Efficiency of Human Plasma Proteins by Trypsin

Jennifer L. Proc; Michael A. Kuzyk; Darryl B. Hardie; Juncong Yang; Derek Smith; Angela M. Jackson; Carol E. Parker; Christoph H. Borchers

Plasma biomarkers studies are based on the differential expression of proteins between different treatment groups or between diseased and control populations. Most mass spectrometry-based methods of protein quantitation, however, are based on the detection and quantitation of peptides, not intact proteins. For peptide-based protein quantitation to be accurate, the digestion protocols used in proteomic analyses must be both efficient and reproducible. There have been very few studies, however, where plasma denaturation/digestion protocols have been compared using absolute quantitation methods. In this paper, 14 combinations of heat, solvent [acetonitrile, methanol, trifluoroethanol], chaotropic agents [guanidine hydrochloride, urea], and surfactants [sodium dodecyl sulfate (SDS) and sodium deoxycholate (DOC)] were compared with respect to their effectiveness in improving subsequent tryptic digestion. These digestion protocols were evaluated by quantitating the production of proteotypic tryptic peptides from 45 moderate- to high-abundance plasma proteins, using tandem mass spectrometry in multiple reaction monitoring mode, with a mixture of stable-isotope labeled analogues of these proteotypic peptides as internal standards. When the digestion efficiencies of these 14 methods were compared, we found that both of the surfactants (SDS and DOC) produced an increase in the overall yield of tryptic peptides from these 45 proteins, when compared to the more commonly used urea protocol. SDS, however, can be a serious interference for subsequent mass spectrometry. DOC, on the other hand, can be easily removed from the samples by acid precipitation. Examining the results of a reproducibility study, done with 5 replicate digestions, DOC and SDS with a 9 h digestion time produced the highest average digestion efficiencies (∼80%), with the highest average reproducibility (<5% error, defined as the relative deviation from the mean value). However, because of potential interferences resulting from the use of SDS, we recommend DOC with a 9 h digestion procedure as the optimum protocol.


Molecular & Cellular Proteomics | 2009

SISCAPA Peptide Enrichment on Magnetic Beads Using an In-line Bead Trap Device

N. Leigh Anderson; Angela M. Jackson; Derek Smith; Darryl B. Hardie; Christoph H. Borchers; Terry W. Pearson

A SISCAPA (stable isotope standards and capture by anti-peptide antibodies) method for specific antibody-based capture of individual tryptic peptides from a digest of whole human plasma was developed using a simplified magnetic bead protocol and a novel rotary magnetic bead trap device. Following off-line equilibrium binding of peptides by antibodies and subsequent capture of the antibodies on magnetic beads, the bead trap permitted washing of the beads and elution of bound peptides inside a 150-μm-inner diameter capillary that forms part of a nanoflow LC-MS/MS system. The bead trap sweeps beads against the direction of liquid flow using a continuous succession of moving high magnetic field-gradient trap regions while mixing the beads with the flowing liquid. This approach prevents loss of low abundance captured peptides and allows automated processing of a series of SISCAPA reactions. Selected tryptic peptides of α1-antichymotrypsin and lipopolysaccharide-binding protein were enriched relative to a high abundance serum albumin peptide by 1,800 and 18,000-fold, respectively, as measured by multiple reaction monitoring. A large majority of the peptides that are bound nonspecifically in SISCAPA reactions were shown to bind to components other than the antibody (e.g. the magnetic beads), suggesting that substantial improvement in enrichment could be achieved by development of improved inert bead surfaces.


Biochimica et Biophysica Acta | 2014

Advances in multiplexed MRM-based protein biomarker quantitation toward clinical utility

Andrew J. Percy; Andrew G. Chambers; Juncong Yang; Darryl B. Hardie; Christoph H. Borchers

Accurate and rapid protein quantitation is essential for screening biomarkers for disease stratification and monitoring, and to validate the hundreds of putative markers in human biofluids, including blood plasma. An analytical method that utilizes stable isotope-labeled standard (SIS) peptides and selected/multiple reaction monitoring-mass spectrometry (SRM/MRM-MS) has emerged as a promising technique for determining protein concentrations. This targeted approach has analytical merit, but its true potential (in terms of sensitivity and multiplexing) has yet to be realized. Described herein is a method that extends the multiplexing ability of the MRM method to enable the quantitation 142 high-to-moderate abundance proteins (from 31mg/mL to 44ng/mL) in undepleted and non-enriched human plasma in a single run. The proteins have been reported to be associated to a wide variety of non-communicable diseases (NCDs), from cardiovascular disease (CVD) to diabetes. The concentrations of these proteins in human plasma are inferred from interference-free peptides functioning as molecular surrogates (2 peptides per protein, on average). A revised data analysis strategy, involving the linear regression equation of normal control plasma, has been instituted to enable the facile application to patient samples, as demonstrated in separate nutrigenomics and CVD studies. The exceptional robustness of the LC/MS platform and the quantitative method, as well as its high throughput, makes the assay suitable for application to patient samples for the verification of a condensed or complete protein panel. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.


Sexual Plant Reproduction | 2007

Proteomic evaluation of gymnosperm pollination drop proteins indicates highly conserved and complex biological functions

Rebecca Wagner; Serena Mugnaini; Richard A. Sniezko; Darryl B. Hardie; Brett A. D. Poulis; Massimo Nepi; Ettore Pacini; Patrick von Aderkas

The pollination droplet is a highly conservative pollination mechanism that is observed in all major gymnosperm taxa. Proteomics analysis of the pollination drops was carried out on four gymnosperm species: Juniperus communis (common juniper), Juniperus oxycedrus (prickly juniper), Chamaecyparis lawsoniana (Port Orford cedar), and Welwitschia mirabilis. Pollination drop proteins were purified by SDS-PAGE, and the most abundant proteins were analyzed by mass spectrometry and sequenced. Based on BLAST searching of combined amino acid sequences, the following proteins were identified in the following species: an 83-kDa subtilisin-like proteinase, a 62-kDa glycosyl hydrolase, a 47.5-kDa glucan 1,3-β-glucosidase precursor, a 30-kDa chitinase, and a 25-kDa thaumatin-like protein were identified in J. communis; a 30-kDa chitinase, a 25-kDa thaumatin-like protein, and a 32.5-kDa glucanase-like protein were identified in J. oxycedrus; an 83-kDa subtilisin-like proteinase, a 62-kDa β-d-glucan exohydrolase, a 47.5-kDa glucan 1,3-β-glucosidase, and two 25-kDa thaumatin-like proteins were identified in C. lawsoniana, and a 25-kDa chitinase was identified in W. mirabilis. Based on protein identifications, there is strong evidence that the pollination drop functions in both pathogen defense and pollen development. The discovery of similarities in terms of peptide sequence and protein identifications indicates that ovular secretions are functionally conservative, and that they are essential to reproductive success.


Journal of Proteome Research | 2014

Multiplexed MRM with Internal Standards for Cerebrospinal Fluid Candidate Protein Biomarker Quantitation.

Andrew J. Percy; Juncong Yang; Andrew G. Chambers; Romain Simon; Darryl B. Hardie; Christoph H. Borchers

Multiplexed quantitation is essential for discovering, verifying, and validating biomarkers for risk stratification, disease prognostication, and therapeutic monitoring. The most promising strategy for quantifying unverified protein biomarkers in biofluids relies on selected/multiple reaction monitoring (SRM or MRM) technology with isotopically labeled standards employed within a bottom-up proteomic workflow. Since cerebrospinal fluid (CSF) is an important fluid for studying central nervous system (CNS) related diseases, we sought to develop a rapid, antibody- and fractionation-free MRM-based approach with a complex mixture of peptide standards to quantify a highly multiplexed panel of candidate protein biomarkers in human CSF. Development involved peptide transition optimization, denaturation/digestion protocol evaluation, transition interference screening, and protein quantitation via peptide standard curves. The final method exhibited excellent reproducibility (average coefficient of variation of <1% for retention time and <6% for signal) and breadth of quantitation (130 proteins from 311 interference-free peptides) in a single 43-min run. These proteins are of high-to-low abundance with determined concentrations from 118 μg/mL (serum albumin) to 550 pg/mL (apolipoprotein C-I). Overall, the method consists of the most highly multiplexed and broadest panel of candidate protein biomarkers in human CSF reported thus far and is well suited for subsequent verification studies on patient samples.


Molecular & Cellular Proteomics | 2012

Use of Proteinase K Nonspecific Digestion for Selective and Comprehensive Identification of Interpeptide Cross-links: Application to Prion Proteins

Evgeniy V. Petrotchenko; Jason J. Serpa; Darryl B. Hardie; Mark V. Berjanskii; Bow P. Suriyamongkol; David S. Wishart; Christoph H. Borchers

Chemical cross-linking combined with mass spectrometry is a rapidly developing technique for structural proteomics. Cross-linked proteins are usually digested with trypsin to generate cross-linked peptides, which are then analyzed by mass spectrometry. The most informative cross-links, the interpeptide cross-links, are often large in size, because they consist of two peptides that are connected by a cross-linker. In addition, trypsin targets the same residues as amino-reactive cross-linkers, and cleavage will not occur at these cross-linker-modified residues. This produces high molecular weight cross-linked peptides, which complicates their mass spectrometric analysis and identification. In this paper, we examine a nonspecific protease, proteinase K, as an alternative to trypsin for cross-linking studies. Initial tests on a model peptide that was digested by proteinase K resulted in a “family” of related cross-linked peptides, all of which contained the same cross-linking sites, thus providing additional verification of the cross-linking results, as was previously noted for other post-translational modification studies. The procedure was next applied to the native (PrPC) and oligomeric form of prion protein (PrPβ). Using proteinase K, the affinity-purifiable CID-cleavable and isotopically coded cross-linker cyanurbiotindipropionylsuccinimide and MALDI-MS cross-links were found for all of the possible cross-linking sites. After digestion with proteinase K, we obtained a mass distribution of the cross-linked peptides that is very suitable for MALDI-MS analysis. Using this new method, we were able to detect over 60 interpeptide cross-links in the native PrPC and PrPβ prion protein. The set of cross-links for the native form was used as distance constraints in developing a model of the native prion protein structure, which includes the 90–124-amino acid N-terminal portion of the protein. Several cross-links were unique to each form of the prion protein, including a Lys185–Lys220 cross-link, which is unique to the PrPβ and thus may be indicative of the conformational change involved in the formation of prion protein oligomers.


Proteomics | 2009

Quantitative iTRAQ proteome and comparative transcriptome analysis of elicitor-induced Norway spruce (Picea abies) cells reveals elements of calcium signaling in the early conifer defense response

Dustin Lippert; Steven Ralph; Michael A. Phillips; Rick White; Derek Smith; Darryl B. Hardie; Jonathan Gershenzon; Kermit Ritland; Christoph H. Borchers; Jörg Bohlmann

Long‐lived conifer trees depend on both constitutive and induced defenses for resistance against a myriad of potential pathogens and herbivores. In species of spruce (Picea spp.), several of the late events of pathogen‐, insect‐, or elicitor‐induced defense responses have previously been characterized at the anatomical, biochemical, transcriptome, and proteome levels in stems and needles. However, accurately measuring the early events of induced cellular responses in a conifer is technically challenging due to limitations in the precise timing of induction and tissue sampling from intact trees following insect or fungal treatment. In the present study, we used the advantages of Norway spruce (Picea abies) cell suspensions combined with chitosan elicitation to investigate the early proteome response in a conifer. A combination of iTRAQ labeling and a new design of iterative sample analysis employing data‐dependent exclusion lists were used for proteome analysis. This approach improved the coverage of the spruce proteome beyond that achieved in any prior study in a conifer system. Comparison of elicitor‐induced proteome and transcriptome responses in Norway spruce cells consistently identified features associated with calcium‐mediated signaling and response to oxidative stress that have not previously been observed in the response of intact trees to fungal attack.


Methods | 2015

Precise quantitation of 136 urinary proteins by LC/MRM-MS using stable isotope labeled peptides as internal standards for biomarker discovery and/or verification studies.

Andrew J. Percy; Juncong Yang; Darryl B. Hardie; Andrew G. Chambers; Jessica Tamura-Wells; Christoph H. Borchers

Spurred on by the growing demand for panels of validated disease biomarkers, increasing efforts have focused on advancing qualitative and quantitative tools for more highly multiplexed and sensitive analyses of a multitude of analytes in various human biofluids. In quantitative proteomics, evolving strategies involve the use of the targeted multiple reaction monitoring (MRM) mode of mass spectrometry (MS) with stable isotope-labeled standards (SIS) used for internal normalization. Using that preferred approach with non-invasive urine samples, we have systematically advanced and rigorously assessed the methodology toward the precise quantitation of the largest, multiplexed panel of candidate protein biomarkers in human urine to date. The concentrations of the 136 proteins span >5 orders of magnitude (from 8.6 μg/mL to 25 pg/mL), with average CVs of 8.6% over process triplicate. Detailed here is our quantitative method, the analysis strategy, a feasibility application to prostate cancer samples, and a discussion of the utility of this method in translational studies.


Analytical Chemistry | 2015

Fast Comparative Structural Characterization of Intact Therapeutic Antibodies Using Hydrogen–Deuterium Exchange and Electron Transfer Dissociation

Jingxi Pan; Suping Zhang; Albert Chou; Darryl B. Hardie; Christoph H. Borchers

Higher-order structural characterization plays an important role in many stages of therapeutic antibody production. Herein, we report a new top-down mass spectrometry approach for characterizing the higher-order structure of intact antibodies, by combining hydrogen/deuterium exchange (HDX), subzero temperature chromatography, and electron transfer dissociation on the Orbitrap mass spectrometer. Individual IgG domain-level deuteration information was obtained for 6 IgG domains on Herceptin (HER), which included the antigen binding sites. This is the first time that top-down HDX has been applied to an intact protein as large as 150 kDa, which has never been done before on any instrument. Ligand-binding induced structural differences in HER were determined to be located only on the variable region of the light chain. Global glycosylation profile of antibodies and HDX property of the glycoforms were also determined by accurate intact mass measurements. Although the presence of disulfide bonds prevent the current approach from being able to obtain amino acid level structural information within the disulfide-linked regions, the advantages such as minimal sample manipulation, fast workflow, very low level of back exchange, and simple data analysis, make it well-suited for fast comparative structural evaluation of intact antibodies.

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Derek Smith

University of Victoria

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Yassene Mohammed

Leiden University Medical Center

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