Publications by authors named "Angela M Jackson"

20 Publications

  • Page 1 of 1

Proteomic Portraits Reveal Evolutionarily Conserved and Divergent Responses to Spinal Cord Injury.

Mol Cell Proteomics 2021 Jun 12;20:100096. Epub 2021 Jun 12.

Division of Neurosurgery, University of British Columbia, Vancouver, British Columbia, Canada.

Despite the emergence of promising therapeutic approaches in preclinical studies, the failure of large-scale clinical trials leaves clinicians without effective treatments for acute spinal cord injury (SCI). These trials are hindered by their reliance on detailed neurological examinations to establish outcomes, which inflate the time and resources required for completion. Moreover, therapeutic development takes place in animal models whose relevance to human injury remains unclear. Here, we address these challenges through targeted proteomic analyses of cerebrospinal fluid and serum samples from 111 patients with acute SCI and, in parallel, a large animal (porcine) model of SCI. We develop protein biomarkers of injury severity and recovery, including a prognostic model of neurological improvement at 6 months with an area under the receiver operating characteristic curve of 0.91, and validate these in an independent cohort. Through cross-species proteomic analyses, we dissect evolutionarily conserved and divergent aspects of the SCI response and establish the cerebrospinal fluid abundance of glial fibrillary acidic protein as a biochemical outcome measure in both humans and pigs. Our work opens up new avenues to catalyze translation by facilitating the evaluation of novel SCI therapies, while also providing a resource from which to direct future preclinical efforts.
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http://dx.doi.org/10.1016/j.mcpro.2021.100096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8260874PMC
June 2021

Process and Workflow for Preparation of Disparate Mouse Tissues for Proteomic Analysis.

J Proteome Res 2021 01 5;20(1):305-316. Epub 2020 Nov 5.

University of Victoria-Genome British Columbia Proteomics Centre, Victoria V8Z 7X8, British Columbia, Canada.

We investigated the effect of homogenization strategy and protein precipitation on downstream protein quantitation using multiple reaction monitoring mass spectrometry (MRM-MS). Our objective was to develop a workflow capable of processing disparate tissue types with high throughput, minimal variability, and maximum purity. Similar abundances of endogenous proteins were measured in nine different mouse tissues regardless of the homogenization method used; however, protein precipitation had strong positive effects on several targets. The best throughput was achieved by lyophilizing tissues to dryness, followed by homogenization bead-beating without sample buffer. Finally, the effect of tissue perfusion prior to dissection and collection was explored in 20 mouse tissues. MRM-MS showed decreased abundances of blood-related proteins in perfused tissues; however, complete removal was not achieved. Concentrations of nonblood proteins were largely unchanged, although significantly higher variances were observed for proteins from the perfused lung, indicating that perfusion may not be suitable for this organ. We present a simple yet effective tissue processing workflow consisting of harvest of fresh nonperfused tissue, novel lyophilization and homogenization by bead-beating, and protein precipitation. This workflow can be applied to a range of mouse tissues with the advantages of simplicity, minimal manual manipulation of samples, use of commonly available equipment, and high sample quality.
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http://dx.doi.org/10.1021/acs.jproteome.0c00399DOI Listing
January 2021

Development and validation of protein biomarkers of health in grizzly bears.

Conserv Physiol 2020 24;8(1):coaa056. Epub 2020 Jun 24.

Department of Veterinary Biomedical Sciences, University of Saskatchewan, 44 Campus Drive, Saskatoon, Saskatchewan S7N 5B3, Canada.

Large carnivores play critical roles in the maintenance and function of natural ecosystems; however, the populations of many of these species are in decline across the globe. Therefore, there is an urgent need to develop novel techniques that can be used as sensitive conservation tools to detect new threats to the health of individual animals well in advance of population-level effects. Our study aimed to determine the expression of proteins related to energetics, reproduction and stress in the skin of grizzly bears () using a liquid chromatography and multiple reaction monitoring mass spectrometry assay. We hypothesized that a suite of target proteins could be measured using this technique and that the expression of these proteins would be associated with biological (sex, age, sample location on body) and environmental (geographic area, season, sample year) variables. Small skin biopsies were collected from free-ranging grizzly bears in Alberta, Canada, from 2013 to 2019 (n = 136 samples from 111 individuals). Over 700 proteins were detected in the skin of grizzly bears, 19 of which were chosen as targets because of their established roles in physiological function. Generalized linear mixed model analysis was used for each target protein. Results indicate that sample year influenced the majority of proteins, suggesting that physiological changes may be driven in part by responses to changes in the environment. Season influenced the expression of proteins related to energetics, reproduction and stress, all of which were lower during fall compared to early spring. The expression of proteins related to energetics and stress varied by geographic area, while the majority of proteins that were affected by biological attributes (age class, sex and age class by sex interaction) were related to reproduction and stress. This study provides a novel method by which scientists and managers can further assess and monitor physiological function in wildlife.
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http://dx.doi.org/10.1093/conphys/coaa056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7311831PMC
June 2020

Validation of a proteomic biomarker panel to diagnose minor-stroke and transient ischaemic attack: phase 2 of SpecTRA, a large scale translational study.

Biomarkers 2018 Dec 23;23(8):793-803. Epub 2018 Aug 23.

g Genome British Columbia Proteomics Centre, University of Victoria , Victoria , Canada.

Objective: To validate our previously developed 16 plasma-protein biomarker panel to differentiate between transient ischaemic attack (TIA) and non-cerebrovascular emergency department (ED) patients.

Method: Two consecutive cohorts of ED patients prospectively enrolled at two urban medical centers into the second phase of SpecTRA study (training, cohort 2A, n = 575; test, cohort 2B, n = 528). Plasma samples were analyzed using liquid chromatography/multiple reaction monitoring-mass spectrometry. Logistic regression models which fit cohort 2A were validated on cohort 2B.

Results: Three of the panel proteins failed quality control and were removed from the panel. During validation, panel models did not outperform a simple motor/speech (M/S) deficit variable. Post-hoc analyses suggested the measured behaviour of L-selectin and coagulation factor V contributed to poor model performance. Removal of these proteins increased the external performance of a model containing the panel and the M/S variable.

Conclusions: Univariate analyses suggest insulin-like growth factor-binding protein 3 and serum paraoxonase/lactonase 3 are reliable and reproducible biomarkers for TIA status. Logistic regression models indicated L-selectin, apolipoprotein B-100, coagulation factor IX, and thrombospondin-1 to be significant multivariate predictors of TIA. We discuss multivariate feature subset analyses as an exploratory technique to better understand a panel's full predictive potential.
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http://dx.doi.org/10.1080/1354750X.2018.1499130DOI Listing
December 2018

Verification of a proteomic biomarker panel to diagnose minor stroke and transient ischaemic attack: phase 1 of SpecTRA, a large scale translational study.

Biomarkers 2018 May - Jun;23(4):392-405. Epub 2018 Feb 12.

i Department of Biochemistry and Microbiology , University of Victoria , Victoria , BC , Canada.

Objective: To derive a plasma biomarker protein panel from a list of 141 candidate proteins which can differentiate transient ischaemic attack (TIA)/minor stroke from non-cerebrovascular (mimic) conditions in emergency department (ED) settings.

Design: Prospective clinical study (#NCT03050099) with up to three timed blood draws no more than 36 h following symptom onset. Plasma samples analysed by multiple reaction monitoring-mass spectrometry (MRM-MS).

Participants: Totally 545 participants suspected of TIA enrolled in the EDs of two urban medical centres.

Outcomes: 90-day, neurologist-adjudicated diagnosis of TIA informed by clinical and radiological investigations.

Results: The final protein panel consists of 16 proteins whose patterns show differential abundance between TIA and mimic patients. Nine of the proteins were significant univariate predictors of TIA [odds ratio (95% confidence interval)]: L-selectin [0.726 (0.596-0.883)]; Insulin-like growth factor-binding protein 3 [0.727 (0.594-0.889)]; Coagulation factor X [0.740 (0.603-0.908)]; Serum paraoxonase/lactonase 3 [0.763 (0.630-0.924)]; Thrombospondin-1 [1.313 (1.081-1.595)]; Hyaluronan-binding protein 2 [0.776 (0.637-0.945)]; Heparin cofactor 2 [0.775 (0.634-0.947)]; Apolipoprotein B-100 [1.249 (1.037-1.503)]; and von Willebrand factor [1.256 (1.034-1.527)]. The scientific plausibility of the panel proteins is discussed.

Conclusions: Our panel has the potential to assist ED physicians in distinguishing TIA from mimic patients.
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http://dx.doi.org/10.1080/1354750X.2018.1434681DOI Listing
September 2018

The Application of Multiple Reaction Monitoring to Assess Apo A-I Methionine Oxidations in Diabetes and Cardiovascular Disease.

Transl Proteom 2014 Dec;4-5:18-24

University of Victoria - Genome British Columbia Proteomics Centre, Victoria, BC ; Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC.

The oxidative modification of apolipoprotein A-I 's methionine148(M148) is associated with defective HDL function in vitro. Multiple Reaction Monitoring (MRM) is a mass spectrometric technique that can be used to quantitate post-translational modifications. In this study, we developed an MRM assay to monitor the abundance ratio of the peptide containing oxidized M148 to the native peptide in Apo A-I. Measurement of the oxidized-to-unoxidized-M148 ratio was reproducible (CV<5%). The extent of methionine M148 oxidation in the HDL of healthy controls, and type 2 diabetic participants with and without prior cardiovascular events (CVD) were then examined. The results suggest a significant increase in the relative ratio of the peptide containing oxidized M148 to the unmodified peptide in the HDL of participants with diabetes and CVD (p<0.001), compared to participants without CVD. Monitoring the abundance ratio of the peptides containing oxidized and unoxidized M148 by MRM provides a means of examining the relationship between M148 oxidation and vascular complications in CVD.
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http://dx.doi.org/10.1016/j.trprot.2014.10.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4331021PMC
December 2014

Identification and validation of potential new biomarkers for prostate cancer diagnosis and prognosis using 2D-DIGE and MS.

Biomed Res Int 2015 15;2015:454256. Epub 2015 Jan 15.

Institute of Pathology, RWTH Aachen University, 52074 Aachen, Germany ; Medizinisches Proteom-Center, Ruhr-University Bochum, 44801 Bochum, Germany ; Leibniz-Institut für Analytische Wissenschaften ISAS e.V., 44139 Dortmund, Germany.

This study was designed to identify and validate potential new biomarkers for prostate cancer and to distinguish patients with and without biochemical relapse. Prostate tissue samples analyzed by 2D-DIGE (two-dimensional difference in gel electrophoresis) and mass spectrometry (MS) revealed downregulation of secernin-1 (P<0.044) in prostate cancer, while vinculin showed significant upregulation (P<0.001). Secernin-1 overexpression in prostate tissue was validated using Western blot and immunohistochemistry while vinculin expression was validated using immunohistochemistry. These findings indicate that secernin-1 and vinculin are potential new tissue biomarkers for prostate cancer diagnosis and prognosis, respectively. For validation, protein levels in urine were also examined by Western blot analysis. Urinary vinculin levels in prostate cancer patients were significantly higher than in urine from nontumor patients (P=0.006). Using multiple reaction monitoring-MS (MRM-MS) analysis, prostatic acid phosphatase (PAP) showed significant higher levels in the urine of prostate cancer patients compared to controls (P=0.012), while galectin-3 showed significant lower levels in the urine of prostate cancer patients with biochemical relapse, compared to those without relapse (P=0.017). Three proteins were successfully differentiated between patients with and without prostate cancer and patients with and without relapse by using MRM. Thus, this technique shows promise for implementation as a noninvasive clinical diagnostic technique.
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http://dx.doi.org/10.1155/2015/454256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4312578PMC
October 2015

PeptidePicker: a scientific workflow with web interface for selecting appropriate peptides for targeted proteomics experiments.

J Proteomics 2014 Jun 22;106:151-61. Epub 2014 Apr 22.

University of Victoria - Genome British Columbia Proteomics Centre, University of Victoria, Victoria, BC V8Z7X8, Canada; Department of Biochemistry & Microbiology, University of Victoria, Victoria, BC V8P 5C2, Canada. Electronic address:

Unlabelled: One challenge in Multiple Reaction Monitoring (MRM)-based proteomics is to select the most appropriate surrogate peptides to represent a target protein. We present here a software package to automatically generate these most appropriate surrogate peptides for an LC/MRM-MS analysis. Our method integrates information about the proteins, their tryptic peptides, and the suitability of these peptides for MRM which is available online in UniProtKB, NCBI's dbSNP, ExPASy, PeptideAtlas, PRIDE, and GPMDB. The scoring algorithm reflects our knowledge in choosing the best candidate peptides for MRM, based on the uniqueness of the peptide in the targeted proteome, its physiochemical properties, and whether it previously has been observed. The modularity of the workflow allows further extension and additional selection criteria to be incorporated. We have developed a simple Web interface where the researcher provides the protein accession number, the subject organism, and peptide-specific options. Currently, the software is designed for human and mouse proteomes, but additional species can be easily be added. Our software improved the peptide selection by eliminating human error, considering multiple data sources and all of the isoforms of the protein, and resulted in faster peptide selection - approximately 50 proteins per hour compared to 8 per day.

Biological Significance: Compiling a list of optimal surrogate peptides for target proteins to be analyzed by LC/MRM-MS has been a cumbersome process, in which expert researchers retrieved information from different online repositories and used their own reasoning to find the most appropriate peptides. Our scientific workflow automates this process by integrating information from different data sources including UniProt, Global Proteome Machine, NCBI's dbSNP, and PeptideAtlas, simulating the researchers' reasoning, and incorporating their knowledge of how to select the best proteotypic peptides for an MRM analysis. The developed software can help to standardize the selection of peptides, eliminate human error, and increase productivity.
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http://dx.doi.org/10.1016/j.jprot.2014.04.018DOI Listing
June 2014

The application of multiple reaction monitoring and multi-analyte profiling to HDL proteins.

Lipids Health Dis 2014 Jan 8;13. Epub 2014 Jan 8.

Department of Medicine, University of Southern California, Los Angeles, CA, USA.

Background: HDL carries a rich protein cargo and examining HDL protein composition promises to improve our understanding of its functions. Conventional mass spectrometry methods can be lengthy and difficult to extend to large populations. In addition, without prior enrichment of the sample, the ability of these methods to detect low abundance proteins is limited. Our objective was to develop a high-throughput approach to examine HDL protein composition applicable to diabetes and cardiovascular disease (CVD).

Methods: We optimized two multiplexed assays to examine HDL proteins using a quantitative immunoassay (Multi-Analyte Profiling- MAP) and mass spectrometric-based quantitative proteomics (Multiple Reaction Monitoring-MRM). We screened HDL proteins using human xMAP (90 protein panel) and MRM (56 protein panel). We extended the application of these two methods to HDL isolated from a group of participants with diabetes and prior cardiovascular events and a group of non-diabetic controls.

Results: We were able to quantitate 69 HDL proteins using MAP and 32 proteins using MRM. For several common proteins, the use of MRM and MAP was highly correlated (p < 0.01). Using MAP, several low abundance proteins implicated in atherosclerosis and inflammation were found on HDL. On the other hand, MRM allowed the examination of several HDL proteins not available by MAP.

Conclusions: MAP and MRM offer a sensitive and high-throughput approach to examine changes in HDL proteins in diabetes and CVD. This approach can be used to measure the presented HDL proteins in large clinical studies.
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http://dx.doi.org/10.1186/1476-511X-13-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3900256PMC
January 2014

Method and platform standardization in MRM-based quantitative plasma proteomics.

J Proteomics 2013 Dec 7;95:66-76. Epub 2013 Aug 7.

University of Victoria-Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101-4464 Markham St., Victoria, BC V8Z 7X8, Canada.

Unlabelled: There exists a growing demand in the proteomics community to standardize experimental methods and liquid chromatography-mass spectrometry (LC/MS) platforms in order to enable the acquisition of more precise and accurate quantitative data. This necessity is heightened by the evolving trend of verifying and validating candidate disease biomarkers in complex biofluids, such as blood plasma, through targeted multiple reaction monitoring (MRM)-based approaches with stable isotope-labeled standards (SIS). Considering the lack of performance standards for quantitative plasma proteomics, we previously developed two reference kits to evaluate the MRM with SIS peptide approach using undepleted and non-enriched human plasma. The first kit tests the effectiveness of the LC/MRM-MS platform (kit #1), while the second evaluates the performance of an entire analytical workflow (kit #2). Here, these kits have been refined for practical use and then evaluated through intra- and inter-laboratory testing on 6 common LC/MS platforms. For an identical panel of 22 plasma proteins, similar concentrations were determined, regardless of the kit, instrument platform, and laboratory of analysis. These results demonstrate the value of the kit and reinforce the utility of standardized methods and protocols.

Biological Significance: The proteomics community needs standardized experimental protocols and quality control methods in order to improve the reproducibility of MS-based quantitative data. This need is heightened by the evolving trend for MRM-based validation of proposed disease biomarkers in complex biofluids such as blood plasma. We have developed two kits to assist in the inter- and intra-laboratory quality control of MRM experiments: the first kit tests the effectiveness of the LC/MRM-MS platform (kit #1), while the second evaluates the performance of an entire analytical workflow (kit #2). In this paper, we report the use of these kits in intra- and inter-laboratory testing on 6 common LC/MS platforms. This article is part of a Special Issue entitled: Standardization and Quality Control in Proteomics.
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http://dx.doi.org/10.1016/j.jprot.2013.07.026DOI Listing
December 2013

Interlaboratory evaluation of automated, multiplexed peptide immunoaffinity enrichment coupled to multiple reaction monitoring mass spectrometry for quantifying proteins in plasma.

Mol Cell Proteomics 2012 Jun 22;11(6):M111.013854. Epub 2011 Dec 22.

Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.

The inability to quantify large numbers of proteins in tissues and biofluids with high precision, sensitivity, and throughput is a major bottleneck in biomarker studies. We previously demonstrated that coupling immunoaffinity enrichment using anti-peptide antibodies (SISCAPA) to multiple reaction monitoring mass spectrometry (MRM-MS) produces Immunoprecipitation MRM-MS (immuno-MRM-MS) assays that can be multiplexed to quantify proteins in plasma with high sensitivity, specificity, and precision. Here we report the first systematic evaluation of the interlaboratory performance of multiplexed (8-plex) immuno-MRM-MS in three independent labs. A staged study was carried out in which the effect of each processing and analysis step on assay coefficient of variance, limit of detection, limit of quantification, and recovery was evaluated. Limits of detection were at or below 1 ng/ml for the assayed proteins in 30 μl of plasma. Assay reproducibility was acceptable for verification studies, with median intra- and interlaboratory coefficients of variance above the limit of quantification of 11% and <14%, respectively, for the entire immuno-MRM-MS assay process, including enzymatic digestion of plasma. Trypsin digestion and its requisite sample handling contributed the most to assay variability and reduced the recovery of target peptides from digested proteins. Using a stable isotope-labeled protein as an internal standard instead of stable isotope-labeled peptides to account for losses in the digestion process nearly doubled assay accuracy for this while improving assay precision 5%. Our results demonstrate that multiplexed immuno-MRM-MS can be made reproducible across independent laboratories and has the potential to be adopted widely for assaying proteins in matrices as complex as plasma.
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http://dx.doi.org/10.1074/mcp.M111.013854DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3433918PMC
June 2012

High-flow multiplexed MRM-based analysis of proteins in human plasma without depletion or enrichment.

Clin Lab Med 2011 Sep;31(3):371-84

University of Victoria-Genome BC Proteomics Centre, Vancouver Island Technology Park, #3101-4464 Markham Street, Victoria, BC V8Z 7X8, Canada.

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http://dx.doi.org/10.1016/j.cll.2011.07.005DOI Listing
September 2011

MALDI immunoscreening (MiSCREEN): a method for selection of anti-peptide monoclonal antibodies for use in immunoproteomics.

J Immunol Methods 2011 Feb 13;364(1-2):50-64. Epub 2010 Nov 13.

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

A scalable method for screening and selection of peptide-specific monoclonal antibodies (mAbs) is described. To identify high affinity anti-peptide mAbs in hybridoma supernatants, antibodies were captured by magnetic affinity beads followed by binding of specific peptides from solution. After timed washing steps, the remaining bound peptides were eluted from the beads and detected by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). This allowed measurement of monovalent interactions of peptides with single antigen binding sites on the antibodies, thus reflecting antibody affinity rather than avidity. Antibodies that were able to bind target peptides from solution phase and retain them during washing for a minimum of 10 min were identified by the strength of the appropriate m/z peptide MS signals obtained. This wash time reflects the minimum peptide dissociation time required for use of these antibodies in several current immuno-mass spectrometry assays. Kinetic analysis of antibody-peptide binding by surface plasmon resonance (SPR) showed that the selected antibodies were of high affinity and, most importantly, had low dissociation constants. This method, called MALDI immunoscreening (MiSCREEN), thus enables rapid screening and selection of high affinity anti-peptide antibodies that are useful for a variety of immunoproteomics applications. To demonstrate their functional utility in immuno-mass spectrometry assays, we used the selected, purified RabMAbs to enrich natural (tryptic) peptides from digested human plasma.
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http://dx.doi.org/10.1016/j.jim.2010.11.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3018550PMC
February 2011

A quantitative study of the effects of chaotropic agents, surfactants, and solvents on the digestion efficiency of human plasma proteins by trypsin.

J Proteome Res 2010 Oct;9(10):5422-37

University of Victoria-Genome BC Proteomics Centre, Victoria, BC, Canada.

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.
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http://dx.doi.org/10.1021/pr100656uDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2996461PMC
October 2010

Self-help groups for patients with coronary heart disease as a resource for rehabilitation and secondary prevention-what is the evidence?

Heart Lung 2009 May-Jun;38(3):192-200. Epub 2009 Apr 3.

Research Unit on Health Behavior and Change, The University of Edinburgh, Medical School, Teviot Place, Edinburgh.

Cardiovascular heart disease (CHD) is a major health care concern worldwide. Maintaining regular cardiac rehabilitation attendance and secondary-prevention strategies are significant health care challenges. Although self-help groups provide benefit for many chronic health conditions, it is not clear if they address the challenges of CHD rehabilitation and self-management. This literature review was guided by the following question: Can self-help groups address the challenges of CHD rehabilitation and self-management? This article reviews the traditional published and "grey" literature on CHD-related self-help groups identified from a database search (Cochrane Library, PubMed, PsychINFO, Medline, Cumulative Index to Nursing and Allied Health Literature, Applied Social Sciences Index and Abstracts, and Social Sciences Citation Index). Identified articles were screened based on the type of initiative: Community-based non-health service-organized groups were included, but hospital-based group treatment and therapy interventions or programs were excluded. Published research and analysis of CHD-related self-help groups is scarce. Sixteen articles focusing on self-help groups were identified. The review results indicate that the limited quantity, limited range, and variable quality of studies prevents reliable conclusions being made regarding effects and outcomes as well as the extent and profile of participation. Strengthening the evidence base regarding the impact of CHD-related self-help groups, the reasons for participation versus nonparticipation in such groups, and determining nonparticipants support needs must be done to establish if and for which patients such groups constitute an effective resource for rehabilitation and secondary prevention.
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http://dx.doi.org/10.1016/j.hrtlng.2009.01.009DOI Listing
September 2010

Multiple reaction monitoring-based, multiplexed, absolute quantitation of 45 proteins in human plasma.

Mol Cell Proteomics 2009 Aug 1;8(8):1860-77. Epub 2009 May 1.

University of Victoria-Genome British Columbia Proteomics Centre, University of Victoria, Victoria, British Columbia V8Z 7X8, Canada.

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 ([(13)C(6)]Arg or [(13)C(6)]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.
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http://dx.doi.org/10.1074/mcp.M800540-MCP200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2722777PMC
August 2009

Killing of trypanosomatid parasites by a modified bovine host defense peptide, BMAP-18.

PLoS Negl Trop Dis 2009 3;3(2):e373. Epub 2009 Feb 3.

Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada.

Background: Tropical diseases caused by parasites continue to cause socioeconomic devastation that reverberates worldwide. There is a growing need for new control measures for many of these diseases due to increasing drug resistance exhibited by the parasites and problems with drug toxicity. One new approach is to apply host defense peptides (HDP; formerly called antimicrobial peptides) to disease control, either to treat infected hosts, or to prevent disease transmission by interfering with parasites in their insect vectors. A potent anti-parasite effector is bovine myeloid antimicrobial peptide-27 (BMAP-27), a member of the cathelicidin family. Although BMAP-27 is a potent inhibitor of microbial growth, at higher concentrations it also exhibits cytotoxicity to mammalian cells. We tested the anti-parasite activity of BMAP-18, a truncated peptide that lacks the hydrophobic C-terminal sequence of the BMAP-27 parent molecule, an alteration that confers reduced toxicity to mammalian cells.

Methodology/principal Findings: BMAP-18 showed strong growth inhibitory activity against several species and life cycle stages of African trypanosomes, fish trypanosomes and Leishmania parasites in vitro. When compared to native BMAP-27, the truncated BMAP-18 peptide showed reduced cytotoxicity on a wide variety of mammalian and insect cells and on Sodalis glossindius, a bacterial symbiont of the tsetse vector. The fluorescent stain rhodamine 123 was used in immunofluorescence microscopy and flow cytometry experiments to show that BMAP-18 at low concentrations rapidly disrupted mitochondrial potential without obvious alteration of parasite plasma membranes, thus inducing death by apoptosis. Scanning electron microscopy revealed that higher concentrations of BMAP-18 induced membrane lesions in the parasites as early as 15 minutes after exposure, thus killing them by necrosis. In addition to direct killing of parasites, BMAP-18 was shown to inhibit LPS-induced secretion of tumour necrosis factor alpha (TNF-alpha), a cytokine that is associated with inflammation and cachexia (wasting) in sleeping sickness patients. As a prelude to in vivo applications, high affinity antibodies to BMAP-18 were produced in rabbits and used in immuno-mass spectrometry assays to detect the intact peptide in human blood and plasma.

Conclusions/significance: BMAP-18, a truncated form of the potent antimicrobial BMAP-27, showed low toxicity to mammalian cells, insect cells and the tsetse bacterial symbiont Sodalis glossinidius while retaining an ability to kill a variety of species and life cycle stages of pathogenic kinetoplastid parasites in vitro. BMAP-18 also inhibited secretion of TNF-alpha, an inflammatory cytokine that plays a role in the cachexia associated with African sleeping sickness. These findings support the idea that BMAP-18 should be explored as a candidate for therapy of economically important trypanosome-infected hosts, such as cattle, fish and humans, and for paratransgenic expression in Sodalis glossinidius, a bacterial symbiont in the tsetse vector, as a strategy for interference with trypanosome transmission.
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http://dx.doi.org/10.1371/journal.pntd.0000373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2628741PMC
March 2010

On the mechanism of mitochondrial uncoupling protein 1 function.

J Biol Chem 2006 Jan 16;281(4):2114-9. Epub 2005 Nov 16.

School of Biochemistry and Immunology, Trinity College Dublin, College Green, Dublin 2, Ireland.

Native uncoupling protein 1 (UCP 1) was purified from rat mitochondria by hydroxyapatite chromatography and identified by peptide mass mapping and tandem mass spectrometry. Native and expressed UCP 1 were reconstituted into liposomes, and proton flux through UCP 1 was shown to be fatty acid-dependent and GDP-sensitive. To investigate the mechanism of action of UCP 1, we determined whether hydrophilic modification of the omega-carbon of palmitate effected its transport function. We show that proton flux was greater through native UCP 1-containing proteoliposomes when facilitated by less hydrophilically modified palmitate (palmitate > omega-methoxypalmitate > omega-hydroxypalmitate with little or no proton flux due to glucose-O-omega-palmitate or undecanesulfonate). We show that non-proton-dependent charge transfer was greater when facilitated by less hydrophilically modified palmitate (palmitate/undecanesulfonate > omega-methoxypalmitate > omega-hydroxypalmitate, with no non-proton-dependent charge transfer flux due to glucose-O-omega-palmitate). We show that the GDP-inhibitable oxygen consumption rate in brown adipose tissue mitochondria was reversed by palmitate (as expected) but not by glucose-O-omega-palmitate. Our data are consistent with the model that UCP 1 flips long-chain fatty acid anions and contradict the "cofactor" model of UCP 1 function.
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http://dx.doi.org/10.1074/jbc.M511575200DOI Listing
January 2006

'Follow the Fish': involving young people in primary care in Midlothian.

Authors:
Angela M Jackson

Health Expect 2003 Dec;6(4):342-51

Patient Involvement Worker, Midlothian Local Health Care Co-operative, Dalkeith Medical Centre, Dalkeith, Midlothian, UK.

Objectives: The project aims were to enable young people to contribute their views about health services, to encourage professionals and policy makers to listen to the young people and to stimulate action to address the issues raised.

Design: Peer interviews were undertaken by a team of five young people to identify the experience and views of young people of various ages about health services. Drama workshop sessions were conducted with 10-15 young people, encompassing initial issue-identifying activities and group discussion about their own experience of, and views about health services, followed by role-play and improvisation to construct drama scenarios about the issues gathered from the interviews and discussions.

Setting And Participants: Twenty young people aged 12-16 years from the Mayfield and Gorebridge areas of Midlothian were recruited from Newbattle Community High school. The project was conducted as a voluntary after-school activity for 12 weeks.

Results: A drama was constructed from research conducted by young people into the experiences and views of their peer group about health services. A cast of young people performed the drama to an invited audience of 30 health and education professionals and held a post-performance question and answer session with the audience to explore the issues raised. The drama engendered a number of practical outcomes related to improving the usage and experience of health services of young people.

Conclusions: Drama can offer a means to encourage participation, facilitate participants' self-expression and explore health/health service themes and issues. In conjunction with conventional techniques such as interviews and group discussions, a drama project can also be used to communicate the experience, views and needs of the wider client group to service providers and planners. Such initiatives can generate outcomes to improve service users' experience of health services.
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http://dx.doi.org/10.1046/j.1369-7625.2003.00233.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5060202PMC
December 2003
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