Carol E Parker

University of Victoria, Victoria, British Columbia, Canada

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Publications (113)319.67 Total impact

  • Carol E. Parker, Christoph H. Borchers
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    ABSTRACT: In its early years, mass-spectrometry (MS)-based proteomics focused on the cataloging of proteins found in different species or different tissues. By 2005, proteomics was being used for protein quantitation, typically based on "proteotypic" peptides which act as surrogates for the parent proteins. Biomarker discovery is usually done by non-targeted "shotgun" proteomics, using relative quantitation methods to determine protein expression changes that correlate with disease (output given as "up-or-down regulation" or "fold-increases"). MS-based techniques can also perform "absolute" quantitation which is required for clinical applications (output given as protein concentrations). Here we describe the differences between these methods, factors that affect the precision and accuracy of the results, and some examples of recent studies using MS-based proteomics to verify cancer-related biomarkers.
    Molecular Oncology 01/2014; · 6.70 Impact Factor
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    ABSTRACT: Quantitative plasma proteomics, through the use of targeted MRM-MS and isotopically labeled standards, is emerging as a popular technique to address biological- and biomedical-centered queries. High precision and accuracy are essential in such measurements, particularly in protein biomarker research where translation to the clinic is sought. Standardized procedures and routine performance evaluation of all stages of the workflow (both pre-analytical and analytical) are therefore imperative to satisfy these requisites and enable high inter-laboratory reproducibility and transferability. In this review, we first discuss the pre-analytical and analytical variables that can affect the precision and accuracy of 'absolute' quantitative plasma proteomic measurements. Proposed strategies to limit such variability will then be highlighted and unmet needs for future exploration will be noted. Although there is no way to conduct a truly comprehensive review on this broad, rapidly changing topic, we have highlighted key aspects and included references to review articles on various sub-topics.
    Bioanalysis 11/2013; 5(22):2837-2856. · 3.25 Impact Factor
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    Dataset: ja8090752
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    ABSTRACT: The iMALDI (immuno-MALDI) technique involves the affinity capture of target peptides from an enzymatic digest of a sample, followed by the direct analysis of the affinity beads while on a MALDI target. For determination of peptide concentration (and, by inference, protein concentration), stable-isotope-labeled standard peptides (SIS peptides) can be added to the digest and will be captured along with the native peptides. This technique can provide the highest possible specificity by determining two molecular characteristics of the epitope-containing peptides: (1) the molecular weight, typically measured to within 100 ppm or better by MALDI-MS, and (2) the amino acid sequence, by performing MALDI-MS/MS. This technique has been shown to be capable of detecting low-attomole levels of target peptides in environmental samples and in digests of human plasma. This chapter provides a detailed description of how to perform iMALDI analyses, starting with the selection of the target peptides. Examples are shown of the application of iMALDI to the detection of an organism that is a possible bioterrorism threat, and to the detection of two isoforms of human EGFR.
    Methods in molecular biology (Clifton, N.J.) 01/2013; 1023:97-120.
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    ABSTRACT: Multiple reaction monitoring (MRM)-mass spectrometry (MS) with stable isotope-labeled standards (SIS) has proven adept in rapidly, precisely, and accurately quantifying proteins in complex biological samples. The impetus behind the early use of multiplexed MRM in proteomics was to expedite the verification and validation stages of the protein biomarker pipeline for clinical utility, which involves the analysis of hundreds or even thousands of samples. Moreover, once a multiplexed assay has been developed, however, it can be turned around and used for biomarker discovery, as has been demonstrated for cancer biomarkers by our laboratory and by others. Overall, these MRM-based methods compare favorably with antibody-based techniques, such as ELISAs or protein arrays, in that MRM-based methods are less expensive and can be developed more rapidly.There are two MRM-based platforms that are currently being developed: a standard-flow and a nano-flow LC/ESI-MRM-MS (liquid chromatography-electrospray ionization) platform. In this book chapter, we describe a recent study in which we evaluated these two platforms, both interfaced to the same mass spectrometer. This study demonstrated the enhanced performance metrics (in terms of sensitivity, dynamic range, and robustness) of the standard-flow ultra-high performance liquid chromatography (UHPLC) system compared to the nano-flow HPLC-Chip for the absolute quantitation of 48 plasma proteins. Using the standard-flow platform, we also developed two high-throughput assays for the analysis of a panel of 67 cardiovascular disease (CVD) biomarkers in non-depleted and non-enriched human plasma and a panel of 25 putative biomarkers in dried human blood spots (DBS). Since the nanoLC/MRM-MS platform has advantages under sample-limited conditions and for the analysis of certain specific peptides, the protocols for both systems are described here.
    Methods in molecular biology (Clifton, N.J.) 01/2013; 1000:167-89.
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    ABSTRACT: Multiple reaction monitoring (MRM), sometimes called selected reaction monitoring (SRM), is a directed tandem mass spectrometric technique performed on to triple quadrupole mass spectrometers. MRM assays can be used to sensitively and specifically quantify proteins based on peptides that are specific to the target protein. Stable-isotope-labeled standard peptide analogues (SIS peptides) of target peptides are added to enzymatic digests of samples, and quantified along with the native peptides during MRM analysis. Monitoring of the intact peptide and a collision-induced fragment of this peptide (an ion pair) can be used to provide information on the absolute peptide concentration of the peptide in the sample and, by inference, the concentration of the intact protein. This technique provides high specificity by selecting for biophysical parameters that are unique to the target peptides: (1) the molecular weight of the peptide, (2) the generation of a specific fragment from the peptide, and (3) the HPLC retention time during LC/MRM-MS analysis. MRM is a highly sensitive technique that has been shown to be capable of detecting attomole levels of target peptides in complex samples such as tryptic digests of human plasma. This chapter provides a detailed description of how to develop and use an MRM protein assay. It includes sections on the critical "first step" of selecting the target peptides, as well as optimization of MRM acquisition parameters for maximum sensitivity of the ion pairs that will be used in the final method, and characterization of the final MRM assay.
    Methods in molecular biology (Clifton, N.J.) 01/2013; 1023:53-82.
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    ABSTRACT: Clinical biomarker discovery, verification, and validation are facilitated by the latest technological advances in mass spectrometry. It is now possible to analyze simultaneously group of tens or hundreds of biomarkers in a blood sample using multiple reaction monitoring (MRM), a tandem mass spectrometric method. However, these newly-developed methods face new challenges, including standardization, calibration, and the determination of analytical and biological variation. Here we illustrate the background, pre-analytical sample preparation, and biomarker assay development using an MRM-mass spectrometric method. In addition, special attention is given to future standardization methods to enable widespread use of the technology.
    Topics in current chemistry 08/2012; · 8.46 Impact Factor
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    ABSTRACT: MALDI imaging allows the creation of a "molecular image" of a tissue slice. This image is reconstructed from the ion abundances in spectra obtained while rastering the laser over the tissue. These images can then be correlated with tissue histology to detect potential biomarkers of, for example, aberrant cell types. MALDI, however, is known to have problems with ion suppression, making it difficult to correlate measured ion abundance with concentration. It would be advantageous to have a method which could provide more accurate protein concentration measurements, particularly for screening applications or for precise comparisons between samples. In this paper, we report the development of a novel MALDI imaging method for the localization and accurate quantitation of proteins in tissues. This method involves optimization of in situ tryptic digestion, followed by reproducible and uniform deposition of an isotopically labeled standard peptide from a target protein onto the tissue, using an aerosol-generating device. Data is acquired by MALDI multiple reaction monitoring (MRM) mass spectrometry (MS), and accurate peptide quantitation is determined from the ratio of MRM transitions for the endogenous unlabeled proteolytic peptides to the corresponding transitions from the applied isotopically labeled standard peptides. In a parallel experiment, the quantity of the labeled peptide applied to the tissue was determined using a standard curve generated from MALDI time-of-flight (TOF) MS data. This external calibration curve was then used to determine the quantity of endogenous peptide in a given area. All standard curves generate by this method had coefficients of determination greater than 0.97. These proof-of-concept experiments using MALDI MRM-based imaging show the feasibility for the precise and accurate quantitation of tissue protein concentrations over 2 orders of magnitude, while maintaining the spatial localization information for the proteins.
    Analytical Chemistry 02/2012; 84(8):3514-22. · 5.70 Impact Factor
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    ABSTRACT: Three common urological diseases are bladder cancer, urinary tract infection, and hematuria. Seventeen bladder cancer biomarkers were previously discovered using iTRAQ - these findings were verified by MRM-MS in this current study. Urine samples from 156 patients with hernia (n=57, control), bladder cancer (n=76), or urinary tract infection/hematuria (n=23) were collected and subjected to multiplexed LC-MRM/MS to determine the concentrations of 63 proteins that are normally considered to be plasma proteins, but which include proteins found in our earlier iTRAQ study. Sixty-five stable isotope-labeled standard proteotypic peptides were used as internal standards for 63 targeted proteins. Twelve proteins showed higher concentrations in the bladder cancer group than in the hernia and the urinary tract infection/hematuria groups, and thus represent potential urinary biomarkers for detection of bladder cancer. Prothrombin had the highest AUC (0.796), with 71.1% sensitivity and 75.0% specificity for differentiating bladder cancer (n=76) from non-cancerous (n=80) patients. The multiplexed MRM-MS data was used to generate a six-peptide marker panel. This six-peptide panel (afamin, adiponectin, complement C4 gamma chain, apolipoprotein A-II precursor, ceruloplasmin, and prothrombin) can discriminate bladder cancer subjects from non-cancerous subjects with an AUC of 0.814, with a 76.3% positive predictive value, and a 77.5% negative predictive value. This article is part of a Special Section entitled: Understanding genome regulation and genetic diversity by mass spectrometry.
    Journal of proteomics 01/2012; 75(12):3529-45. · 5.07 Impact Factor
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    ABSTRACT: Structural proteomics is the application of protein chemistry and modern mass spectrometric techniques to problems such as the characterization of protein structures and assemblies and the detailed determination of protein-protein interactions. The techniques used in structural proteomics include crosslinking, photoaffinity labeling, limited proteolysis, chemical protein modification and hydrogen/deuterium exchange, all followed by mass spectrometric analysis. None of these methods alone can provide complete structural information, but a "combination" of these complementary approaches can be used to provide enough information for answering important biological questions. Structural proteomics can help to determine, for example, the detailed structure of the interfaces between proteins that may be important drug targets and the interactions between proteins and ligands. In this review, we have tried to provide a brief overview of structural proteomics methodologies, illustrated with examples from our laboratory and from the literature.
    European Journal of Mass Spectrometry 01/2012; 18(2):251-67. · 1.26 Impact Factor
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    ABSTRACT: In this study we demonstrate the use of a multiplexed MRM-based assay to distinguish among normal (NL) and iron-metabolism disorder mouse models, particularly, iron-deficiency anemia (IDA), inflammation (INFL), and inflammation and anemia (INFL+IDA). Our initial panel of potential biomarkers was based on the analysis of 14 proteins expressed by candidate genes involved in iron transport and metabolism. Based on this study, we were able to identify a panel of 8 biomarker proteins: apolipoprotein A4 (APO4), transferrin, transferrin receptor 1, ceruloplasmin, haptoglobin, lactoferrin, hemopexin, and matrix metalloproteinase-8 (MMP8) that clearly distinguish among the normal and disease models. Within this set of proteins, transferrin showed the best individual classification accuracy over all samples (72%) and within the NL group (94%). Compared to the best single-protein biomarker, transferrin, the use of the composite 8-protein biomarker panel improved the classification accuracy from 94% to 100% in the NL group, from 50% to 72% in the INFL group, from 66% to 96% in the IDA group, and from 79% to 83% in the INFL+IDA group. Based on these findings, validation of the utility of this potentially important biomarker panel in human samples in an effort to differentiate IDA, inflammation, and combinations thereof, is now warranted. This article is part of a Special Section entitled: Understanding genome regulation and genetic diversity by mass spectrometry.
    Journal of proteomics 11/2011; 75(12):3514-28. · 5.07 Impact Factor
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    Clinics in laboratory medicine 09/2011; 31(3):371-84. · 1.17 Impact Factor
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    ABSTRACT: Lysyl oxidase (LOX) is an amine oxidase that is critical for the stability of connective tissues. The secreted proLOX is enzymatically quiescent and is activated through proteolytic cleavage between residues Gly(162) and Asp(163) (residue numbers according to the mouse LOX) by bone morphogenetic protein (BMP)-1 gene products. Here we report a novel processing of proLOX identified in vitro and in vivo. Two forms of mature LOX were identified and characterized by their immunoreactivity to specific antibodies, amine oxidase activity, and mass spectrometry. One form was identified as a well-characterized BMP-1 processed LOX protein. Another was found to be a truncated form of LOX resulting from the cleavage at the carboxy terminus of Arg(192). The truncated form of LOX still appeared to retain amine oxidase activity. The results from the proLOX gene deletion and mutation experiments indicated that the processing occurs independent of the cleavage of proLOX by BMP-1 gene products and likely requires the presence of LOX propeptide. These results indicate that proLOX could be processed by two different mechanisms producing two forms of active LOX.
    Connective tissue research 05/2011; 52(6):479-86. · 1.55 Impact Factor
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    ABSTRACT: Due to the lack of precise markers indicative of its occurrence and progression, coronary artery disease (CAD), the most common type of heart diseases, is currently associated with high mortality in the United States. To systemically identify novel protein biomarkers associated with CAD progression for early diagnosis and possible therapeutic intervention, we employed an iTRAQ-based quantitative proteomic approach to analyze the proteome changes in the plasma collected from a pair of wild-type versus apolipoprotein E knockout (APOE(-/-) ) mice which were fed with a high fat diet. In a multiplex manner, iTRAQ serves as the quantitative 'in-spectra' marker for 'cross-sample' comparisons to determine the differentially expressed/secreted proteins caused by APOE knock-out. To obtain the most comprehensive proteomic data sets from this CAD-associated mouse model, we applied both MALDI and ESI-based mass spectrometric (MS) platforms coupled with two different schemes of multidimensional liquid chromatography (2-D LC) separation. We then comparatively analyzed a series of the plasma samples collected at 6 and 12 wk of age after the mice were fed with fat diets, where the 6- or 12-wk time point represents the early or intermediate phase of the fat-induced CAD, respectively. We then categorized those proteins showing abundance changes in accordance with APOE depletion. Several proteins such as the γ and β chains of fibrinogen, apolipoprotein B, apolipoprotein C-I, and thrombospondin-4 were among the previously known CAD markers identified by other methods. Our results suggested that these unbiased proteomic methods are both feasible and a practical means of discovering potential biomarkers associated with CAD progression.
    Proteomics 05/2011; 11(14):2763-76. · 4.43 Impact Factor
  • Christoph H Borchers, Carol E Parker
    Clinical Chemistry 10/2010; 56(12):1786-8. · 7.15 Impact Factor
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    ABSTRACT: 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.
    Journal of Proteome Research 10/2010; 9(10):5422-37. · 5.06 Impact Factor
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    ABSTRACT: This review reports on the current and emerging technologies for the use of mass-spectrometry-based proteomics in clinical applications.
    The Analyst 08/2010; 135(8):1830-8. · 4.23 Impact Factor
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    ABSTRACT: It was inevitable that as soon as mass spectrometrists were able to tell biologists which proteins were in their samples, the next question would be how much of these proteins were present. This has turned out to be a much more challenging question. In this review, we describe the multiple ways that mass spectrometry has attempted to address this issue, both for relative quantitation and for absolute quantitation of proteins. There is no single method that will work for every problem or for every sample. What we present here is a variety of techniques, with guidelines that we hope will assist the researcher in selecting the most appropriate technique for the particular biological problem that needs to be addressed. We need to emphasize that this is a very active area of proteomics research—new quantitative methods are continuously being introduced and some ‘pitfalls’ of older methods are just being discovered. However, even though there is no perfect technique—and a better technique may be developed tomorrow—valuable information on biomarkers and pathways can be obtained using these currently available methods Copyright © 2009 John Wiley & Sons, Ltd.
    Biological Mass Spectrometry 11/2009; 44(12):1637 - 1660. · 3.41 Impact Factor
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    ABSTRACT: Salvinorin A, the most potent naturally occurring hallucinogen, has attracted an increasing amount of attention since the kappa-opioid receptor (KOR) was identified as its principal molecular target by us [Roth, B. L., et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 11934-11939]. Here we report the design, synthesis, and biochemical characterization of novel, irreversible, salvinorin A-derived ligands suitable as active state probes of the KOR. On the basis of prior substituted cysteine accessibility and molecular modeling studies, C315(7.38) was chosen as a potential anchoring point for covalent labeling of salvinorin A-derived ligands. Automated docking of a series of potential covalently bound ligands suggested that either a haloacetate moiety or other similar electrophilic groups could irreversibly bind with C315(7.38). 22-Thiocyanatosalvinorin A (RB-64) and 22-chlorosalvinorin A (RB-48) were both found to be extraordinarily potent and selective KOR agonists in vitro and in vivo. As predicted on the basis of molecular modeling studies, RB-64 induced wash-resistant inhibition of binding with a strict requirement for a free cysteine in or near the binding pocket. Mass spectrometry (MS) studies utilizing synthetic KOR peptides and RB-64 supported the hypothesis that the anchoring residue was C315(7.38) and suggested one biochemical mechanism for covalent binding. These studies provide direct evidence of the presence of a free cysteine in the agonist-bound state of the KOR and provide novel insights into the mechanism by which salvinorin A binds to and activates the KOR.
    Biochemistry 07/2009; 48(29):6898-908. · 3.38 Impact Factor
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    ABSTRACT: The oxidation of guanine to 5-carboxamido-5-formamido-2-iminohydantoin (2-Ih) is shown to be a major transformation in the oxidation of the single-stranded DNA 5-mer d(TTGTT) by m-chloroperbenzoic acid (m-CPBA) and dimethyldioxirane (DMDO) as a model for peracid oxidants and in the oxidation of the 5-base pair duplex d[(TTGTT).(AACAA)] with DMDO. 2-Ih has not been reported as an oxidative lesion at the level of single/double-stranded DNA or at the nucleoside/nucleotide level. The lesion is stable to DNA digestion and chromatographic purification, suggesting that 2-Ih may be a stable biomarker in vivo. The oxidation products have been structurally characterized and the reaction mechanism has been probed by oxidation of the monomeric species dGuo, dGMP, and dGTP. DMDO selectively oxidizes the guanine moiety of dGuo, dGMP, and dGTP to 2-Ih, and both peracetic and m-chloroperbenzoic acids exhibit the same selectivity. The presence of the glycosidic bond results in the stereoselective induction of an asymmetric center at the spiro carbon to give a mixture of diastereomers, with each diastereomer in equilibrium with a minor conformer through rotation about the formamido C-N bond. Labeling studies with [(18)O(2)]-m-CPBA and H(2)(18)O to determine the source of the added oxygen atoms have established initial epoxidation of the guanine 4-5 bond with pyrimidine ring contraction by an acyl 1,2-migration of guanine carbonyl C6 to form a transient dehydrodeoxyspiroiminodihydantoin followed by hydrolytic ring-opening of the imidazolone ring. Consistent with the proposed mechanism, no 8-oxoguanine was detected as a product of the oxidations of the oligonucleotides or monomeric species mediated by DMDO or the peracids. The 2-Ih base thus appears to be a pathway-specific lesion generated by peracids and possibly other epoxidizing agents and holds promise as a potential biomarker.
    Journal of the American Chemical Society 05/2009; 131(17):6114-23. · 10.68 Impact Factor

Publication Stats

1k Citations
319.67 Total Impact Points

Institutions

  • 2010–2013
    • University of Victoria
      • Department of Biochemistry and Microbiology
      Victoria, British Columbia, Canada
    • Genome British Columbia
      Vancouver, British Columbia, Canada
  • 2007
    • Institute of Biochemistry and Biophysics
      Teheran, Tehrān, Iran
  • 2002–2005
    • University of North Carolina at Chapel Hill
      • Department of Biochemistry and Biophysics
      Chapel Hill, NC, United States
  • 1979–2005
    • National Institute of Environmental Health Sciences
      • Laboratory of Structural Biology (LSB)
      Durham, North Carolina, United States
  • 2003
    • University of British Columbia - Vancouver
      • Biomedical Research Centre (BRC)
      Vancouver, British Columbia, Canada
  • 2001
    • Weill Cornell Medical College
      • Department of Microbiology and Immunology
      New York City, New York, United States
  • 1995–2001
    • National Institutes of Health
      • Structural Biophysics Laboratory
      Bethesda, MD, United States
  • 1994
    • Universität Konstanz
      Constance, Baden-Württemberg, Germany
  • 1990
    • North Carolina State University
      • Department of Crop Science
      Raleigh, NC, United States
  • 1982–1990
    • Research Triangle Park Laboratories, Inc.
      Raleigh, North Carolina, United States