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Dive into the research topics where Dennis J. Orton is active.

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Featured researches published by Dennis J. Orton.


Journal of Proteome Research | 2014

Resolubilization of precipitated intact membrane proteins with cold formic acid for analysis by mass spectrometry.

Alan A. Doucette; Douglas B. Vieira; Dennis J. Orton; Mark J. Wall

Protein precipitation in organic solvent is an effective strategy to deplete sodium dodecyl sulfate (SDS) ahead of MS analysis. Here we evaluate the recovery of membrane and water-soluble proteins through precipitation with chloroform/methanol/water or with acetone (80%). With each solvent system, membrane protein recovery was greater than 90%, which was generally higher than that of cytosolic proteins. With few exceptions, residual supernatant proteins detected by MS were also detected in the precipitation pellet, having higher MS signal intensity in the pellet fraction. Following precipitation, we present a novel strategy for the quantitative resolubilization of proteins in an MS-compatible solvent system. The pellet is incubated at -20 °C in 80% formic acid/water and then diluted 10-fold with water. Membrane protein recovery matches that of sonication of the pellet in 1% SDS. The resolubilized proteins are stable at room temperature, with no observed formylation as is typical of proteins suspended in formic acid at room temperature. The protocol is applied to the molecular weight determination of membrane proteins from a GELFrEE-fractionated sample of Escherichia coli proteins.


Journal of Proteome Research | 2013

Dual LC-MS platform for high-throughput proteome analysis.

Dennis J. Orton; Mark J. Wall; Alan A. Doucette

We describe a dual-column interface for parallel chromatography to improve throughput during LC-MS experimentation. The system employs a high-voltage switch to operate two capillary column/nanospray emitters fixed at the MS orifice. Sequentially loading one column while operating the second nearly doubles the LC-MS duty cycle. Given the innate run-to-run variation of a nanospray LC-MS (12% RSD peak area; 2% retention time), the intercolumn variability of the platform showed no meaningful difference for proteome analysis, with equal numbers of proteins and peptides identified per column. Applied to GeLC analysis of an E. coli extract, throughput was increased using one of three methods: doubling the number of replicates, increasing the LC gradient length, or sectioning the gel into twice as many fractions. Each method increased the total number of identifications as well as detection throughput (number of peptides/proteins identified per hour). The greatest improvement was achieved by doubling the number of gel slices (10 vs 5). Analysis on the dual column platform provided a 26% increase in peptides identified per hour (24% proteins). This translates into ~50% more total proteins and peptides identified in the experiment using the dual LC-MS platform.


Journal of Proteomics | 2014

Proteomic analysis of rat proximal tubule cells following stretch-induced apoptosis in an in vitro model of kidney obstruction.

Dennis J. Orton; Alan A. Doucette; Geoffrey N. Maksym; Dawn L. MacLellan

UNLABELLED Urinary tract obstruction (UTO) is a commonly noted disorder on prenatal ultrasound that has the potential to lead to permanent loss of renal function. To study the molecular processes of the disease, an in vitro model has been developed which involves mechanical stretch of proximal tubule cells grown on flexible plates which mimics the physiological conditions during UTO. This study employs a one dimensional SDS-PAGE fractionation procedure, followed by in-gel digest and LC-MS/MS analysis in a semi-quantitative experiment using spectral counting to relatively quantify changes in protein expression following the established model of UTO. Quantitative analysis shows 317 of the 1630 identified proteins express altered abundance, with 135 increased and 182 decreased in abundance as a result of stretch. Gene ontology (GO) and KEGG annotations implicate a number of physiological processes that have been previously shown in addition to some potentially novel processes in UTO. The quantitative proteomic analysis we performed here provides a more complete characterization of changes in protein abundance as a result of stretch than previous studies, and provides a number of previously undescribed proteins in proximal tubule cells that may play a role in UTO. BIOLOGICAL SIGNIFICANCE Urinary tract obstruction (UTO) is a commonly noted abnormality on prenatal ultrasound that can either resolve spontaneously or require surgical intervention to prevent permanent renal damage or loss of function. While targeted studies of UTO have shown a number of pathological responses in proximal tubule cells, there are currently no large-scale quantitative studies that aim to elucidate a global cellular response. This study uses a semi-quantitative approach and applies a well characterized model of UTO to determine a number of cellular processes affected by UTO simulation and identifies a number of proteins with altered abundance that have not been noted previously in UTO. This article is part of a Special Issue entitled: Can Proteomics Fill the Gap Between Genomics and Phenotypes?


Proteome | 2013

Proteomic Workflows for Biomarker Identification Using Mass Spectrometry — Technical and Statistical Considerations during Initial Discovery

Dennis J. Orton; Alan A. Doucette

Identification of biomarkers capable of differentiating between pathophysiological states of an individual is a laudable goal in the field of proteomics. Protein biomarker discovery generally employs high throughput sample characterization by mass spectrometry (MS), being capable of identifying and quantifying thousands of proteins per sample. While MS-based technologies have rapidly matured, the identification of truly informative biomarkers remains elusive, with only a handful of clinically applicable tests stemming from proteomic workflows. This underlying lack of progress is attributed in large part to erroneous experimental design, biased sample handling, as well as improper statistical analysis of the resulting data. This review will discuss in detail the importance of experimental design and provide some insight into the overall workflow required for biomarker identification experiments. Proper balance between the degree of biological vs. technical replication is required for confident biomarker identification.


Journal of Chromatography B | 2013

A universal, high recovery assay for protein quantitation through temperature programmed liquid chromatography (TPLC)

Dennis J. Orton; Alan A. Doucette

As an alternative to direct UV absorbance measurements, estimation of total protein concentration is typically conducted through colorimetric reagent assays. However, for protein-limited applications, the proportion of the sample sacrificed to the assay becomes increasingly significant. This work demonstrates a method for quantitation of protein samples with high recovery. Temperature programmed liquid chromatography (TPLC) with absorbance detection at 214nm permits accurate estimation of total protein concentration from samples containing as little as 0.75μg. The method incorporates a temperature gradient from 25 to 80°C to facilitate elution of total protein into a single fraction. Analyte recovery, as measured from 1 and 10μg protein extracts of Escherichia coli, is shown to exceed 93%. Extinction coefficients at 214nm were calculated across the human proteome, providing a relative standard deviation of 21% (versus 42% at 280nm), suggesting absorbance values at 214nm provide a more consistent measure of protein concentration. These results translate to a universal protein detection strategy exhibiting a coefficient of variation below 10%. Together with the sensitivity and tolerance to contaminants, TPLC with UV detection is a favorable alternative to colorimetric assay for total protein quantitation, particularly in sample-limited applications.


Canadian Journal of Microbiology | 2011

The htpAB operon of Legionella pneumophila cannot be deleted in the presence of the groE chaperonin operon of Escherichia coli

Gheyath K. Nasrallah; Elizabeth Gagnon; Dennis J. Orton; Rafael A. Garduño

HtpB, the chaperonin of the intracellular bacterial pathogen Legionella pneumophila , displays several virulence-related functions in vitro. To confirm HtpBs role in vivo, host infections with an htpB deletion mutant would be required. However, we previously reported that the htpAB operon (encoding co-chaperonin and chaperonin) is essential. We attempted here to delete htpAB in a L. pneumophila strain carrying the groE operon (encoding the Escherichia coli co-chaperonin and chaperonin). The groE operon was inserted into the chromosome of L. pneumophila Lp02, and then allelic replacement of htpAB with a gentamicin resistance cassette was attempted. Although numerous potential postallelic replacement transformants showed a correct selection phenotype, we still detected htpAB by PCR and full-size HtpB by immunoblot. Southern blot and PCR analysis indicated that the gentamicin resistance cassette had apparently integrated in a duplicated htpAB region. However, we showed by Southern blot that strain Lp02, and the Lp02 derivative carrying the groE operon, have only one copy of htpAB. These results confirmed that the htpAB operon cannot be deleted, not even in the presence of the groE operon, and suggested that attempts to delete htpAB under strong phenotypic selection result in aberrant genetic recombinations that could involve duplication of the htpAB locus.


Clinical Biochemistry | 2016

One-step extraction and quantitation of toxic alcohols and ethylene glycol in plasma by capillary gas chromatography (GC) with flame ionization detection (FID).

Dennis J. Orton; Jessica Boyd; Darlene Affleck; Donna Duce; Warren Walsh; Isolde Seiden-Long

OBJECTIVES Clinical analysis of volatile alcohols (i.e. methanol, ethanol, isopropanol, and metabolite acetone) and ethylene glycol (EG) generally employs separate gas chromatography (GC) methods for analysis. Here, a method for combined analysis of volatile alcohols and EG is described. DESIGN AND METHODS Volatile alcohols and EG were extracted with 2:1 (v:v) acetonitrile containing internal standards (IS) 1,2 butanediol (for EG) and n-propanol (for alcohols). Samples were analyzed on an Agilent 6890 GC FID. The method was evaluated for precision, accuracy, reproducibility, linearity, selectivity and limit of quantitation (LOQ), followed by correlation to existing GC methods using patient samples, Bio-Rad QC, and in-house prepared QC material. RESULTS Inter-day precision was from 6.5-11.3% CV, and linearity was verified from down to 0.6mmol/L up to 150mmol/L for each analyte. The method showed good recovery (~100%) and the LOQ was calculated to be between 0.25 and 0.44mmol/L. Patient correlation against current GC methods showed good agreement (slopes from 1.03-1.12, and y-intercepts from 0 to 0.85mmol/L; R(2)>0.98; N=35). Carryover was negligible for volatile alcohols in the measuring range, and of the potential interferences tested, only toluene and 1,3 propanediol interfered. The method was able to resolve 2,3 butanediol, diethylene glycol, and propylene glycol in addition to the peaks quantified. CONCLUSIONS Here we describe a simple procedure for simultaneous analysis of EG and volatile alcohols that comes at low cost and with a simple liquid-liquid extraction requiring no derivitization to obtain adequate sensitivity for clinical specimens.


Molecular and Cellular Probes | 2013

Reprint of “GELFrEE fractionation combined with mass spectrometry for proteome analysis of secreted toxins from Enteropathogenic Escherichia coli (EPEC)”☆

Dennis J. Orton; Dan J. Arsenault; Nikhil A. Thomas; Alan A. Doucette

Enteropathogenic Escherichia coli, or EPEC, is a human pathogen associated with gastroenteritis and diarrheal disease whose pathogenicity is related to the secretion of effector proteins (exotoxins). Determining exotoxin expression level is of considerable interest to those studying toxin function and pathological phenotypes. Mass spectrometry (MS) provides an ideal platform for detection and quantification of proteins from complex mixtures. Here, we apply a solution-phase electrophoretic platform (GELFrEE) followed by MS to characterize the secreted proteome of a wild type and mutant strain of EPEC (ΔsepD), exhibiting enhanced secretion of effector proteins. Through peptide-level analysis, a total of 363 and 155 proteins were identified from the wild type and mutant strains, respectively. Semi-quantitative analysis of the MS data reveals the effector proteins EspB, EspC, and EspD appear in a relatively greater abundance from wild type EPEC, while two major virulence factors in EPEC, Tir and NleA appear in greater abundance from the secreted proteome of the mutant strain. Additionally, intact proteins may further be characterized following GELFrEE with MS to improve throughput of analysis. This study demonstrates the application of GELFrEE-MS to differentiate wild type and mutant strains of EPEC. This platform is therefore a powerful tool to study exotoxin secretion from pathogenic bacteria.


Clinica Chimica Acta | 2016

Fasting time and vitamin B12 levels in a community-based population.

Dennis J. Orton; Christopher Naugler; S.M. Hossein Sadrzadeh

OBJECTIVES Vitamin B12, also known as cobalamin (Cbl), is an essential vitamin that manifests with numerous severe but non-specific symptoms in cases of deficiency. Assessing Cbl status often requires fasting, although this requirement is not standard between institutions. This study evaluated the impact of fasting on Cbl levels in a large community-based cohort in an effort to promote standardization of Cbl testing between sites. DESIGN AND METHODS Laboratory data for Cbl, fasting time, patient age and sex were obtained from laboratory information service from Calgary Laboratory Services (CLS) for the period of April 2011 to June 2015. CLS is the sole supplier of laboratory services in the Southern Alberta region in Canada (population, approximately 1.4 million). To investigate potential sex-specific effects of fasting on Cbl levels, males and females were analyzed separately using linear regression models. RESULTS A total of 346,957 individual patient results (196,849 females, 146,085 males) were obtained. The mean plasma Cbl level was 386.5 (±195.6) pmol/L and 412.0 (±220.8) pmol/L for males and females, respectively. Linear regression analysis showed fasting had no significant association with Cbl levels in females; however a statistically significant decrease of 0.9pmol/L/hour fasting (p<0.001) was noted in males. CONCLUSIONS The broad population variance in Cbl suggests the slight gender-specific differences noted in this study are insignificant. Despite this, fasting has the potential to contribute to higher rates of Cbl deficiency in men. Together, these data suggest fasting should be excluded as a requirement for evaluating plasma Cbl.


Clinical Chemistry | 2016

Critically High Plasma Ammonia in an Adolescent Girl

Dennis J. Orton; Jessica L. Gifford; Isolde Seiden-Long; Aneal Khan; Lawrence de Koning

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Donna Duce

Foothills Medical Centre

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