Publications by authors named "Christoph H Borchers"

283 Publications

Crystallographic analysis of TarI and TarJ, a cytidylyltransferase and reductase pair for CDP-ribitol synthesis in Staphylococcus aureus wall teichoic acid biogenesis.

J Struct Biol 2021 Apr 2:107733. Epub 2021 Apr 2.

Department of Biochemistry and Molecular Biology and Centre for Blood Research, The University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada. Electronic address:

The cell wall of many pathogenic Gram-positive bacteria contains ribitol-phosphate wall teichoic acid (WTA), a polymer that is linked to virulence and regulation of essential physiological processes including cell division. CDP-ribitol, the activated precursor for ribitol-phosphate polymerization, is synthesized by a cytidylyltransferase and reductase pair known as TarI and TarJ, respectively. In this study, we present crystal structures of Staphylococcus aureus TarI and TarJ in their apo forms and in complex with substrates and products. The TarI structures illustrate the mechanism of CDP-ribitol synthesis from CTP and ribitol-phosphate and reveal structural changes required for substrate binding and catalysis. Insights into the upstream step of ribulose-phosphate reduction to ribitol-phosphate is provided by the structures of TarJ. Furthermore, we propose a general topology of the enzymes in a heterotetrameric form built using restraints from crosslinking mass spectrometry analysis. Together, our data present molecular details of CDP-ribitol production that may aid in the design of inhibitors against WTA biosynthesis.
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http://dx.doi.org/10.1016/j.jsb.2021.107733DOI Listing
April 2021

An Update on MRMAssayDB: A Comprehensive Resource for Targeted Proteomics Assays in the Community.

J Proteome Res 2021 Apr 8;20(4):2105-2115. Epub 2021 Mar 8.

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

Precise multiplexed quantification of proteins in biological samples can be achieved by targeted proteomics using multiple or parallel reaction monitoring (MRM/PRM). Combined with internal standards, the method achieves very good repeatability and reproducibility enabling excellent protein quantification and allowing longitudinal and cohort studies. A laborious part of performing such experiments lies in the preparation steps dedicated to the development and validation of individual protein assays. Several public repositories host information on targeted proteomics assays, including NCI's Clinical Proteomic Tumor Analysis Consortium assay portals, PeptideAtlas SRM Experiment Library, SRMAtlas, PanoramaWeb, and PeptideTracker, with all offering varying levels of details. We introduced MRMAssayDB in 2018 as an integrated resource for targeted proteomics assays. The Web-based application maps and links the assays from the repositories, includes comprehensive up-to-date protein and sequence annotations, and provides multiple visualization options on the peptide and protein level. We have extended MRMAssayDB with more assays and extensive annotations. Currently it contains >828 000 assays covering >51 000 proteins from 94 organisms, of which >17 000 proteins are present in >2400 biological pathways, and >48 000 mapping to >21 000 Gene Ontology terms. This is an increase of about four times the number of assays since introduction. We have expanded annotations of interaction, biological pathways, and disease associations. A newly added visualization module for coupled molecular structural annotation browsing allows the user to interactively examine peptide sequence and any known PTMs and disease mutations, and map all to available protein 3D structures. Because of its integrative approach, MRMAssayDB enables a holistic view of suitable proteotypic peptides and commonly used transitions in empirical data. Availability: http://mrmassaydb.proteincentre.com.
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http://dx.doi.org/10.1021/acs.jproteome.0c00961DOI Listing
April 2021

Profiling of antimicrobial metabolites of plant growth promoting spp. isolated from different plant hosts.

3 Biotech 2021 Feb 11;11(2):48. Epub 2021 Jan 11.

School of Life Sciences, Forman Christian College (A Chartered University), Lahore, 54600 Pakistan.

In this study, nine strains of and , and two isolates of sp: At1RP4 and RS-1, were characterized for the in-vitro production of secondary metabolites in LB, DMB, and King's B media, and of the genes responsible for the production of antagonistic metabolites. Based on 16S rRNA gene sequence, isolates At1RP4 and RS-1 were identified as strains of and . Five phenazine derivatives comprising phenazine, phenazine-1-carboxylic acid (PCA), 2-hydroxyphenazine-1-carboxylic acid (2-OH-Phz-1-COOH), phenazine-1,6-dicarboxylic acid (Phz-1,6-di-COOH), and 2-hydroxyphenazine (2-OH-Phz) were produced by all strains in all three culture media including DMB, King's B and LB. However, 2,8-dihydroxyphenazine, 6-methylphenazine-1-carboxylic acid, pyrrolnitrin, and the -dialkylaromatic acids, were produced by the and strains. In addition, all strains produced 2-acetamidophenol, pyochelin, and diketopiperazine derivatives in variable amounts in all three culture media used. Highest levels of quorum-sensing signal molecules including PQS, 2-Octyl-3-hydroxy-4(1H)-quinolone, and hexahydro-quinoxaline-1,4-dioxide were recorded for At1RP4. Moreover, all strains produced volatile hydrogen cyanide (0.95-6.68 µg/L) and the phytohormone indole-3-acetic acid (0.42-13.9 µM). Production of extracellular lipase and protease was recorded in all pseudomonads, whereas, cellulase production and phosphate solubilization were variable. Genes for hydrogen cyanide and phenazine-1-carboxylic acid were detected in all eleven strains while the gene for pyrrolnitrin biosynthesis was amplified in and strains. Comparative metabolomic analysis provided detailed insights about the strain-specific metabolites in pseudomonads, and their pseudo-relative quantification in different bacterial growth media to be used as single-strain biofertilizer and biocontrol inoculums.

Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-020-02585-8.
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http://dx.doi.org/10.1007/s13205-020-02585-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7801537PMC
February 2021

Mouse Quantitative Proteomics Knowledgebase: reference protein concentration ranges in 20 mouse tissues using 5000 quantitative proteomics assays.

Bioinformatics 2021 Jan 23. Epub 2021 Jan 23.

University of Victoria, Victoria, BC, Canada.

Laboratory mouse is the most used animal model in biological research, largely due to its high conserved synteny with human. Researchers use mice to answer various questions ranging from determining a pathological effect of knocked out/in gene to understanding drug metabolism. Our group developed >5000 quantitative targeted proteomics assays for 20 mouse tissues and determined the concentration ranges of a total of more than 1600 proteins using heavy labelled internal standards. We describe here MouseQuaPro; a knowledgebase that hosts this collection of carefully curated experimental data. The Web-based application includes protein concentrations from >700 mouse tissue samples from three common research strains, corresponding to more than 200k experimentally determined concentrations. The knowledgebase integrates the assay and protein concentration information with their human orthologs, functional and molecular annotations, biological pathways, related human diseases, and known gene expressions. At its core are the protein concentration ranges, which provide insights into (dis)similarities between tissues, strains, and sexes. MouseQuaPro implements advanced search as well as filtering functionalities with a simple interface and interactive visualization. This information-rich resource provides an initial map of protein absolute concentration in mouse tissues and allows guided design of proteomics phenotyping experiments. The knowledgebase is available at mousequapro.proteincentre.com. (Reviewer access username and password: mousequapro_reviewer1234567).
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http://dx.doi.org/10.1093/bioinformatics/btab018DOI Listing
January 2021

Direct and Precise Measurement of Bevacizumab Levels in Human Plasma Based on Controlled Methionine Oxidation and Multiple Reaction Monitoring.

ACS Pharmacol Transl Sci 2020 Dec 13;3(6):1304-1309. Epub 2020 Nov 13.

Gerald Bronfman Department of Oncology, Jewish General Hospital, and Segal Cancer Proteomics Center, Lady Davis Institute, General Hospital, McGill University, Montreal, Quebec H3T1E2, Canada.

Bevacizumab is a monoclonal antibody which targets vascular endothelial growth factor A (VEGF-A) and is used to treat various cancers and recently COVID-19. The dosage recommendations for bevacizumab are determined on the basis of body weight, and the drug is administered after defined time intervals, when it is presumed to still be above its minimum effective serum concentration. Interindividual and disease-stage-related variations in bevacizumab catabolism, however, can affect the proper dosing of patients, resulting in plasma concentrations which may not be within the optimal therapeutic window for the drug. Therapeutic drug monitoring (TDM) enables the assessment of patients' serum concentrations and allows personalized dosing which has the potential to improve efficacy and reduce side effects. While TMD is often performed using ligand-based assays, mass spectrometry (MS)-based TDM offers improved specificity. Here, we present a robust multiple reaction monitoring (MRM)-MS-based TDM method for the precise quantification of bevacizumab plasma concentrations, based on the controlled oxidation of the methionine-containing peptide, STAYLQMNSLR. The assay shows good linearity ( = 0.9951), robustness, and precision (CVs < 20%) for the quantification of bevacizumab, with a lower limit of quantification (/ > 10) of 1.8 μg/mL of plasma, without the need for enrichment and requiring less than 1 μL of plasma and less than 6 h from sampling to result.
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http://dx.doi.org/10.1021/acsptsci.0c00134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7737317PMC
December 2020

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

Depletion of essential isoprenoids and ER stress induction following acute liver-specific deletion of HMG-CoA reductase.

J Lipid Res 2020 12 27;61(12):1675-1686. Epub 2020 Oct 27.

Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX, USA. Electronic address:

HMG-CoA reductase (Hmgcr) is the rate-limiting enzyme in the mevalonate pathway and is inhibited by statins. In addition to cholesterol, Hmgcr activity is also required for synthesizing nonsterol isoprenoids, such as dolichol, ubiquinone, and farnesylated and geranylgeranylated proteins. Here, we investigated the effects of Hmgcr inhibition on nonsterol isoprenoids in the liver. We have generated new genetic models to acutely delete genes in the mevalonate pathway in the liver using AAV-mediated delivery of Cre-recombinase (AAV-) or CRISPR/Cas9 (AAV-CRISPR). The genetic deletion of by AAV- resulted in extensive hepatocyte apoptosis and compensatory liver regeneration. At the biochemical level, we observed decreased levels of sterols and depletion of the nonsterol isoprenoids, dolichol and ubiquinone. At the cellular level, -null hepatocytes showed ER stress and impaired N-glycosylation. We further hypothesized that the depletion of dolichol, essential for N-glycosylation, could be responsible for ER stress. Using AAV-CRISPR, we somatically disrupted dehydrodolichyl diphosphate synthase subunit (), encoding a branch point enzyme required for dolichol biosynthesis. -null livers showed ER stress and impaired N-glycosylation, along with apoptosis and regeneration. Finally, the combined deletion of and synergistically exacerbated hepatocyte ER stress. Our data show a critical role for mevalonate-derived dolichol in the liver and suggest that dolichol depletion is at least partially responsible for ER stress and apoptosis upon potent Hmgcr inhibition.
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http://dx.doi.org/10.1194/jlr.RA120001006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7707164PMC
December 2020

Toward Increased Reliability, Transparency, and Accessibility in Cross-linking Mass Spectrometry.

Structure 2020 11 15;28(11):1259-1268. Epub 2020 Oct 15.

Wellcome Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3BF, Scotland, UK; Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, 13355 Berlin, Germany. Electronic address:

Cross-linking mass spectrometry (MS) has substantially matured as a method over the past 2 decades through parallel development in multiple labs, demonstrating its applicability to protein structure determination, conformation analysis, and mapping protein interactions in complex mixtures. Cross-linking MS has become a much-appreciated and routinely applied tool, especially in structural biology. Therefore, it is timely that the community commits to the development of methodological and reporting standards. This white paper builds on an open process comprising a number of events at community conferences since 2015 and identifies aspects of Cross-linking MS for which guidelines should be developed as part of a Cross-linking MS standards initiative.
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http://dx.doi.org/10.1016/j.str.2020.09.011DOI Listing
November 2020

Semiquantitative Proteomic Research of Protein Plasma Profile of Volunteers in 21-Day Head-Down Bed Rest.

Front Physiol 2020 24;11:678. Epub 2020 Jul 24.

Institute of Biomedical Problems-Russian Federation State Scientific Research Center, Russian Academy of Sciences, Moscow, Russia.

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http://dx.doi.org/10.3389/fphys.2020.00678DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7396636PMC
July 2020

Editorial.

J Proteomics 2020 09 13;228:103937. Epub 2020 Aug 13.

Segal Cancer Proteomics Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, Quebec H3T 1E2, Canada; Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University, Montreal, Quebec, H3T 1E2, Canada; Center for Computational and Data-Intensive Science and Engineering, Skolkovo Institute of Science and Technology, Moscow 121205, Russia.

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http://dx.doi.org/10.1016/j.jprot.2020.103937DOI Listing
September 2020

An LC-MRM assay for the quantification of metanephrines from dried blood spots for the diagnosis of pheochromocytomas and paragangliomas.

Anal Chim Acta 2020 Sep 9;1128:140-148. Epub 2020 Jul 9.

Segal Cancer Proteomics Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, McGill University, Montreal, QC, Canada; Center for Computational and Data-Intensive Science and Engineering, Skolkovo Institute of Science and Technology, Moscow, Russia; Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University, Montreal, QC, Canada. Electronic address:

The quantitation of metanephrine (MN), normetanephrine (NMN), and 3-methoxytyramine (3-MT) - referred to as metanephrines -- by LC-MS/MS is the gold-standard for screening for pheochromocytoma and paragangliomas (PPGLs), tumours of the adrenal gland and the peripheral nervous system. An assay for metanephrines from dried blood spots (DBSs) would be of high clinical utility as it simplifies sample collection, enables remote sampling, and could increase compliance with the clinical recommendation for supine sampling. Moreover, DBS sampling facilitates the measurement of blood-derived metanephrines in pediatric patients - where DBSs are well-established - in order to diagnose neuroblastomas. Here, we adapted an established derivatization-based LC-MRM-MS assay for plasma catecholamines, and optimized the sample extraction, LC, and MS parameters to produce a fast, sensitive, and robust method for the measurement of metanephrines from DBSs, including 3-methoxytyramine. The DBS samples were excised, derivatized with phenyl isothiocyanate (PITC) on-spot, extracted, and measured by LC-MRM-MS. To validate assay suitability and performance, we assessed the linearity, precision, accuracy, recovery, and matrix effects of the method, and determined the stability of metanephrines in DBSs under different storage conditions. Assay performance for NMN, MN, and 3-MT was sufficient for quantitation from a single DBS within a linear range from 40 to 2000 pg/mL. MN and NMN were stable in DBSs for 2 weeks, whereas 3-MT was stable for one week regardless of storage temperature. Altogether, this work represents the first quantitative LC-MS/MS method for metanephrines from DBSs and provides a novel opportunity for the diagnosis of PPGLs and neuroblastomas in the future.
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http://dx.doi.org/10.1016/j.aca.2020.06.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7346842PMC
September 2020

Mass-Spectrometric Detection of SARS-CoV-2 Virus in Scrapings of the Epithelium of the Nasopharynx of Infected Patients via Nucleocapsid N Protein.

J Proteome Res 2020 11 19;19(11):4393-4397. Epub 2020 Aug 19.

V. I. Kulakov National Medical Research Center for Obstetrics, Gynecology and Perinatology, Ministry of Healthcare of the Russian Federation, Moscow 117997, Russia.

The detection of viral RNA by polymerase chain reaction (PCR) is currently the main diagnostic tool for COVID-19 ( 2019, 25 (3), 1). The PCR-based test, however, shows limited sensitivity, especially in the early and late stages of disease development ( 2020, 581, 465-469; 2020, 119 (6) 1123), and is relatively time-consuming. Fast and reliable complementary methods for detecting the viral infection would be of help in the current pandemic conditions. Mass spectrometry is one of such possibilities. We have developed a mass-spectrometry-based method for the detection of the SARS CoV-2 virus in nasopharynx epithelial swabs based on the detection of the viral nucleocapsid N protein. Our approach shows confident identification of the N protein in patient samples, even those with the lowest viral loads, and a much simpler preparation procedure. Our main protocol consists of virus inactivation by heating and the addition of isopropanol and tryptic digestion of the proteins sedimented from the swabs followed by MS analysis. A set of unique peptides, produced as a result of proteolysis of the nucleocapsid phosphoprotein of SARS-CoV-2, is detected. The obtained results can further be used to create fast parallel mass-spectrometric approaches for the detection of the virus in the nasopharyngeal mucosa, saliva, sputum and other physiological fluids.
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http://dx.doi.org/10.1021/acs.jproteome.0c00412DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7640963PMC
November 2020

Using Two Peptide Isotopologues as Internal Standards for the Streamlined Quantification of Low-Abundance Proteins by Immuno-MRM and Immuno-MALDI.

Anal Chem 2020 09 1;92(18):12407-12414. Epub 2020 Sep 1.

Segal Cancer Proteomics Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montréal, Québec H3T 1E2, Canada.

Mass spectrometry (MS), particularly targeted proteomics, is increasingly being used for quantifying specific proteins and peptides in clinical specimens. The coupling of immuno-enrichment of proteotypic peptides with MS [e.g., immuno-multiple reaction monitoring (MRM) and immuno-matrix-assisted laser desorption ionization (MALDI)] enables the development of highly sensitive and specific assays for low-abundance signaling proteins. By incorporating stable isotope-labeled standards, these workflows allow the determination of endogenous protein concentrations. This is typically achieved through external calibration, often using surrogate matrices, which has inherent limitations for the analysis of clinical specimens as there are often substantial variations in the sample matrix, and sample amounts are typically limited. We have previously introduced the use of two peptide isotopologues for generating external calibration curves in plasma. Here, we present a two-point internal calibration (2-PIC) strategy using two isotopologues for immuno-MS assays and demonstrate its flexibility and robustness. Quantification of the tumor suppressor PTEN in Colo-205 cells by immuno-MRM and immuno-MALDI using 2-PIC and external calibration yielded very similar results (relative standard deviation between 2-PIC and external calibration: 4.9% for immuno-MRM; 1.1% for immuno-MALDI), without the need for a surrogate matrix or additional patient material for calibration, while concurrently reducing the instrument time and cost. Although our PTEN immuno-MRM and immuno-MALDI assays can be considered to be orthogonal as they utilized entirely different sample preparation and MS analysis workflows, targeted different PTEN peptides, and were performed in different laboratories, the endogenous Colo-205 PTEN levels determined with 2-PIC showed a good correlation ( = 0.9966) and good agreement (0.48 ± 0.01 and 0.29 ± 0.02 fmol/μg of total protein) between immuno-MRM and immuno-MALDI.
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http://dx.doi.org/10.1021/acs.analchem.0c02157DOI Listing
September 2020

Systematic Optimization of the iMALDI Workflow for the Robust and Straightforward Quantification of Signaling Proteins in Cancer Cells.

Proteomics Clin Appl 2020 09 9;14(5):e2000034. Epub 2020 Aug 9.

University of Victoria-Genome BC Proteomics Centre, University of Victoria, Victoria, BC, V8Z 7E8, Canada.

Purpose: Immuno-MALDI (iMALDI) combines immuno-enrichment of biomarkers with MALDI-MS for fast, precise, and specific quantitation, making it a valuable tool for developing clinical assays. iMALDI assays are optimized for the PI3-kinase signaling pathway members phosphatase and tensin homolog (PTEN) and PI3-kinase catalytic subunit alpha (p110α), with regard to sensitivity, robustness, and throughput. A standardized template for developing future iMALDI assays, including automation protocols to streamline assay development and translation, is provided.

Experimental Design: Conditions for tryptic digestion and immuno-enrichment (beads, bead:antibody ratios, incubation times, direct vs. indirect immuno-enrichment) are rigorously tested. Different strategies for calibration and data readout are compared.

Results: Digestion using 1:2 protein:trypsin (wt:wt) for 1 h yielded high and consistent peptide recoveries. Direct immuno-enrichment (antibody-bead coupling prior to antigen-enrichment) yielded 30% higher peptide recovery with a 1 h shorter incubation time than indirect enrichment. Immuno-enrichment incubation overnight yielded 1.5-fold higher sensitivities than 1 h incubation. Quantitation of the endogenous target proteins is not affected by the complexity of the calibration matrix, further simplifying the workflow.

Conclusions And Clinical Relevance: This optimized and automated workflow will facilitate the clinical translation of high-throughput sensitive iMALDI assays for quantifying cell-signaling proteins in individual tumor samples, thereby improving patient stratification for targeted treatment.
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http://dx.doi.org/10.1002/prca.202000034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7539945PMC
September 2020

Changes in plasma bile acid profiles after partial internal biliary diversion in PFIC2 patients.

Ann Transl Med 2020 Mar;8(5):185

Department of Pediatrics, Fudan University Shanghai Medical College, The Center for Pediatric Liver Diseases, Children's Hospital of Fudan University, Shanghai 201102, China.

Background: We ask if plasma bile acid profiles can be used to monitor the effectiveness of partial internal biliary diversion (PIBD) for treating uncontrolled cholestasis in progressive familial intrahepatic cholestasis type 2 (PFIC2) patients.

Methods: Plasma bile acids were profiled in 3 cases of ATP-binding cassette, sub-family B member 11 ()mutated PFIC2 children before and after PIBD compared to healthy controls and 8 PFIC2 patients. The quantitation of bile acids was performed by reversed-phase ultrahigh-performance liquid chromatography/multiple-reaction monitoring-mass spectrometry (UPLC/MRM-MS) with negative ion detection.

Results: Before PIBD, all three patients presented with >50-fold higher levels of total plasma bile acids, 2-7 folds higher ratios of taurine: glycine conjugated primary bile acids, and unchanged secondary bile acids levels compared to healthy controls. After PIBD, only one of the three patients (P3) showed relief of cholestasis. The bile acid profiles of the two nonresponding patients showed little change while that of the responding patient showed a 5-fold reduction in total plasma primary bile acids, a reduced taurine: glycine conjugate ratio, and an unexpected 26- and 12-fold increase in secondary bile acids DCA and LCA respectively. One year later, the responder suffered a recurrence of cholestasis, and the bile acid profile shifted back to a more pre-PIBD-like profile.

Conclusions: Plasma bile acid profiles may potentially be useful as sensitive biomarkers for monitoring the clinical course of PIBD patients. Relief of cholestasis after PIBD appears to be associated with significantly increased circulating toxic secondary bile acids and this may limit the utility of PIBD in PFIC2 patients in the long run.
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http://dx.doi.org/10.21037/atm.2020.01.103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7154393PMC
March 2020

Determination of the concentration range for 267 proteins from 21 lots of commercial human plasma using highly multiplexed multiple reaction monitoring mass spectrometry.

Analyst 2020 May;145(10):3634-3644

University of Victoria - Genome British Columbia Proteomics Centre, University of Victoria, Victoria, British Columbia V8Z 7X8, Canada and Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, V8P 5C2, Canada and Segal Cancer Proteomics Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, Quebec H3 T 1E2, Canada. and Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University, Montreal, Quebec H3 T 1E2, Canada and Department of Data Intensive Science and Engineering, Skolkovo Institute of Science and Technology, Skolkovo Innovation Center, Nobel St., Moscow 143026, Russia.

Multiple reaction monitoring (MRM) is a key tool for biomarker validation and the translation of potential biomarkers into the clinic. To demonstrate the applicability of MRM towards achieving this goal, we set out to determine the concentration ranges of 267 plasma proteins, including 61 FDA-approved/LDT developed biomarkers, in 21 commercial human plasma lots, as well as to assess accuracy and precision. Each target protein was quantified by calculating the area ratio of the endogenous tryptic target peptide to its stable isotope-labelled internal standard equivalent and compared to a standard curve. This highly multiplexed approach utilized a standard-flow UHPLC system linked to a triple quadrupole. All samples were analyzed across three separate days and assessed for robustness and accuracy. The standard curves and quality control samples showed excellent performance, with >93% of standards and QCs meeting the acceptance criteria. A total of 248 proteins were able to be quantified in at least one sample on at least one of the three days, with 111 proteins being quantified in all 21 samples on all three days. The protein concentrations across all proteins covered six orders of magnitude. Furthermore, excellent three-day precision was demonstrated with 86% of CVs falling below 15%. Overall, the protein concentration differences ranged from 1.1-fold for metalloproteinase inhibitor 2, to 69-fold for serum amyloid A-1/A-2.
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http://dx.doi.org/10.1039/c9an01893jDOI Listing
May 2020

Plasma Protein Signatures of a Murine Venous Thrombosis Model and Slc44a2 Knockout Mice Using Quantitative-Targeted Proteomics.

Thromb Haemost 2020 Mar 5;120(3):423-436. Epub 2020 Mar 5.

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

The plasma compartment of the blood holds important information on the risk to develop cardiovascular diseases such as venous thrombosis (VT). Mass spectrometry-based targeted proteomics with internal standards quantifies proteins in multiplex allowing generation of signatures associated with a disease or a condition. Here, to demonstrate the method, we investigate the plasma protein signatures in mice following the onset of VT, which was induced by RNA interference targeting the natural anticoagulants antithrombin and protein C. We then study mice lacking , which was recently characterized as a VT-susceptibility gene in human genome-wide association studies. We use a recently developed panel of 375 multiplexed mouse protein assays measured by mass spectrometry. A strong plasma protein siganture was observed when VT was induced. Discriminators included acute phase response proteins, and proteins related to erythrocyte function. In mice lacking , protein signature was primarily overruled by the difference between sexes and not by the absent gene. Upon separate analyses for males and females, we were able to establish a signature for deficiency, in which glycosylation-dependent cell adhesion molecule-1 and thrombospondin-1 were shared by both sexes. The minimal impact of deficiency on the measured plasma proteins suggests that the main effect of on VT does not lay ultimately in the plasma compartment. This suggests further investigation into the role of this VT-susceptibility gene should perhaps also question the possible involvement in cellular mechanisms.
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http://dx.doi.org/10.1055/s-0040-1702229DOI Listing
March 2020

Improving Identification of Protein-Protein Interactions Using an Affinity-enrichable, Isotopically Coded, and Mass Spectrometry-cleavable Chemical Crosslinker.

Mol Cell Proteomics 2020 04 12;19(4):624-639. Epub 2020 Feb 12.

Department of Biochemistry and Microbiology, University of Victoria, 3800 Finnerty Rd., Victoria, BC V8P 5C2, Canada; University of Victoria - Genome British Columbia Proteomics Centre, #3101-4464 Markham Street, Vancouver Island Technology Park, Victoria, BC V8Z7X8, Canada; Segal Cancer Proteomics Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, Quebec, H3T 1E2, Canada; Gerald Bronfman Department of Oncology, Jewish General Hospital, Montreal, Quebec, H3T 1E2, Canada; Department of Data Intensive Science and Engineering, Skolkovo Institute of Science and Technology, Skolkovo Innovation Center, Nobel St., Moscow 143026, Russia. Electronic address:

An experimental and computational approach for identification of protein-protein interactions by chemical crosslinking and mass spectrometry (CLMS) has been developed that takes advantage of the specific characteristics of cyanurbiotindipropionylsuccinimide (CBDPS), an affinity-tagged isotopically coded mass spectrometry (MS)-cleavable crosslinking reagent. Utilizing this reagent in combination with a crosslinker-specific data-dependent acquisition strategy based on MS2 scans, and a software pipeline designed for integrating crosslinker-specific mass spectral information led to demonstrated improvements in the application of the CLMS technique, in terms of the detection, acquisition, and identification of crosslinker-modified peptides. This approach was evaluated on intact yeast mitochondria, and the results showed that hundreds of unique protein-protein interactions could be identified on an organelle proteome-wide scale. Both known and previously unknown protein-protein interactions were identified. These interactions were assessed based on their known sub-compartmental localizations. Additionally, the identified crosslinking distance constraints are in good agreement with existing structural models of protein complexes involved in the mitochondrial electron transport chain.
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http://dx.doi.org/10.1074/mcp.RA119.001839DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7124466PMC
April 2020

Multiplex quantitation of 270 plasma protein markers to identify a signature for early detection of colorectal cancer.

Eur J Cancer 2020 03 21;127:30-40. Epub 2020 Jan 21.

Division of Preventive Oncology, German Cancer Research Center (DKFZ), National Center for Tumour Diseases (NCT), Heidelberg, Germany; Division of Clinical Epidemiology and Aging Research, German Cancer Research Center (DKFZ), Heidelberg, Germany; German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany. Electronic address:

Blood-based protein biomarker signatures might be an alternative or supplement to existing methods for early detection of colorectal cancer (CRC) for population-based screening. The objective of this study was to derive a protein biomarker signature for early detection of CRC and its precursor advanced adenoma (AA). In a two-stage design, 270 protein markers were measured by liquid chromatography/multiple reaction monitoring/mass spectrometry in plasma samples of discovery and validation sets. In the discovery set consisting of 100 newly diagnosed CRC cases and 100 age- and sex-matched controls free of neoplasm at screening colonoscopy, the algorithms predicting the presence of early- or late-stage CRC were derived by Lasso regression and .632 + bootstrap. The prediction algorithms were then externally validated in an independent validation set consisting of participants of screening colonoscopy including 56 participants with CRC, 99 with AA and 99 controls without any colorectal neoplasms. Three different signatures for all-, early- and late-stage CRC consisting of five-, three- and eight-protein markers were obtained in the discovery set with areas under the curves (AUCs) after .632 + bootstrap adjustment of 0.85, 0.83 and 0.96, respectively. External validation in the representative screening population yielded AUCs of 0.79 (95% CI, 0.70-0.86), 0.79 (95% CI, 0.66-0.89) and 0.80 (95% CI, 0.70-0.89) for all-, early- and late-stage CRCs, respectively. The three-marker early-stage algorithm yielded an AUC of 0.65 (95% CI, 0.56-0.73) for detection of AA in the validation set. Although not yet competitive with available stool-based tests for CRC early detection, the identified proteins may contribute to the development of powerful blood-based tests for early detection of CRC and its precursors AAs.
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http://dx.doi.org/10.1016/j.ejca.2019.11.021DOI Listing
March 2020

pH Biosensing by PI4P Regulates Cargo Sorting at the TGN.

Dev Cell 2020 02 9;52(4):461-476.e4. Epub 2020 Jan 9.

Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada. Electronic address:

Phosphoinositides, diacylglycerolpyrophosphate, ceramide-1-phosphate, and phosphatidic acid belong to a unique class of membrane signaling lipids that contain phosphomonoesters in their headgroups having pK values in the physiological range. The phosphomonoester headgroup of phosphatidic acid enables this lipid to act as a pH biosensor as changes in its protonation state with intracellular pH regulate binding to effector proteins. Here, we demonstrate that binding of pleckstrin homology (PH) domains to phosphatidylinositol 4-phosphate (PI4P) in the yeast trans-Golgi network (TGN) is dependent on intracellular pH, indicating PI4P is a pH biosensor. pH biosensing by TGN PI4P in response to nutrient availability governs protein sorting at the TGN, likely by regulating sterol transfer to the TGN by Osh1, a member of the conserved oxysterol-binding protein (OSBP) family of lipid transfer proteins. Thus, pH biosensing by TGN PI4P allows for direct metabolic regulation of protein trafficking and cell growth.
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http://dx.doi.org/10.1016/j.devcel.2019.12.010DOI Listing
February 2020

Concentration Determination of >200 Proteins in Dried Blood Spots for Biomarker Discovery and Validation.

Mol Cell Proteomics 2020 03 2;19(3):540-553. Epub 2020 Jan 2.

University of Victoria - Genome BC Proteomics Centre, University of Victoria, Victoria, British Columbia V8Z 7X8, Canada; Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8P 5C2, Canada; Segal Cancer Proteomics Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, Quebec H3T 1E2, Canada; Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University, Montreal, Quebec H3T 1E2, Canada; Department of Data Intensive Science and Engineering, Skolkovo Institute of Science and Technology, Skolkovo Innovation Center, Nobel St., Moscow143026, Russia. Electronic address:

The use of protein biomarkers as surrogates for clinical endpoints requires extensive multilevel validation including development of robust and sensitive assays for precise measurement of protein concentration. Multiple reaction monitoring (MRM) is a well-established mass-spectrometric method that can be used for reproducible protein-concentration measurements in biological specimens collected via microsampling. The dried blood spot (DBS) microsampling technique can be performed non-invasively without the expertise of a phlebotomist, and can enhance analyte stability which facilitate the application of this technique in retrospective studies while providing lower storage and shipping costs, because cold-chain logistics can be eliminated. Thus, precise, sensitive, and multiplexed methods for measuring protein concentrations in DBSs can be used for biomarker discovery and for biomarker quantification or verification experiments. To achieve this goal, MRM assays were developed for multiplexed concentration measurement of proteins in DBSs.The lower limit of quantification (LLOQ) was found to have a median total coefficient of variation (CV) of 18% for 245 proteins, whereas the median LLOQ was 5 fmol of peptide injected on column, and the median inter-day CV over 4 days for measuring endogenous protein concentration was 8%. The majority (88%) of the assays displayed parallelism, whereas the peptide standards remained stable throughout the assay workflow and after exposure to multiple freeze-thaw cycles. For 190 proteins, the measured protein concentrations remained stable in DBS stored at ambient laboratory temperature for up to 2 months. Finally, the developed assays were used to measure the concentration ranges for 200 proteins in twenty same sex, same race and age matched individuals.
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http://dx.doi.org/10.1074/mcp.TIR119.001820DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7050112PMC
March 2020

LC-MS/MS based comparative proteomics of floral nectars reveal different mechanisms involved in floral defense of Nicotiana spp., Petunia hybrida and Datura stramonium.

J Proteomics 2020 02 14;213:103618. Epub 2019 Dec 14.

Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA. Electronic address:

Tobacco floral nectar (FN) is a biological fluid produced by nectaries composed of sugars, amino acids and proteins called nectarins, involved in the floral defense. FN provides an ideal source of nutrients for microorganisms. Understanding the role of nectar proteins is essential to predict impacts in microbial growth, composition and plants-pollinators interactions. Using LC-MS/MS-based comparative proteomic analysis we identified 22 proteins from P. hybrida, 35 proteins from D. stramonium, and 144 proteins from 23 species of Nicotiana. The data are available at ProteomeXchance (PXD014760). GO analysis and secretory signal prediction demonstrated that defense/stress was the largest group of proteins in the genus Nicotiana. The Nicotiana spp. proteome consisted of 105 exclusive proteins such as lipid transfer proteins (LTPs), Nectar Redox Cycle proteins, proteases inhibitors, and PR-proteins. Analysis by taxonomic sections demonstrated that LTPs were most abundant in Undulatae and Noctiflora, while nectarins were more abundant in Rusticae, Suaveolens, Polydicliae, and Alata sections. Peroxidases (Pox) and chitinases (Chit) were exclusive to P. hybrida, while D. stramonium had only seven unique proteins. Biochemical analysis confirmed these differences. These findings support the hypothesis that, although conserved, there is differential abundance of proteins related to defense/stress which may impact the mechanisms of floral defense. SIGNIFICANCE: This study represents a comparative proteomic analysis of floral nectars of the Nicotiana spp. with two correlated Solanaceous species. Significant differences were identified between the proteome of taxonomic sections providing relevant insights into the group of proteins related to defense/stress associated with Nectar Redox Cycle, antimicrobial proteins and signaling pathways. The activity of FNs proteins is suggested impact the microbial growth. The knowledge about these proteomes provides significant insights into the diversity of proteins secreted in the nectars and the array of mechanisms used by Nicotiana spp. in its floral defense.
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http://dx.doi.org/10.1016/j.jprot.2019.103618DOI Listing
February 2020

The intestinal microbiome potentially affects thrombin generation in human subjects.

J Thromb Haemost 2020 03 27;18(3):642-650. Epub 2019 Dec 27.

Department of Vascular Medicine, Amsterdam Cardiovascular Sciences, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands.

Background: The intestinal microbiome plays a versatile role in the etiology of arterial thrombosis. In venous thrombosis, driven chiefly by plasma coagulation, no such role has yet been established. We hypothesized that the intestinal microbiome composition affects coagulation in humans.

Methods: We used healthy donor fecal microbiota transplant (FMT) to experimentally change the microbiome composition in metabolic syndrome patients. Thirty-five subjects were randomized in a blinded fashion to healthy donor FMT or autologous FMT as a control in a 2:1 ratio. We measured thrombin generation at baseline and after 6 weeks using automated calibrated thrombinography, and we determined plasma abundance of 32 coagulation related proteins using a targeted mass spectrometry-based quantitative proteomics assay with heavy labeled internal standards.

Results: Healthy donor FMT prolonged the thrombinography lag time (median delta 0.0 versus 0.25 minutes, P = .039). The other thrombinography parameters showed no significant difference. Unsupervised cluster analysis suggested overall downregulation of coagulation related plasma proteins in subject clusters containing predominantly subjects that had a metabolic response to healthy donor FMT. FMT treatment status itself showed no clear clustering pattern with up- or downregulation, however, and proteins did not cluster according to an apparent biological grouping.

Discussion: A single healthy donor FMT tends to modestly suppress the onset thrombin generation in metabolic syndrome patients, representing initial proof-of-principle that the intestinal microbiota composition might affect the coagulation system in humans. The findings merit external validation as a role for intestinal microbiota in coagulation can have clinically important implications.
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http://dx.doi.org/10.1111/jth.14699DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7065057PMC
March 2020

Proteogenomics of Colorectal Cancer Liver Metastases: Complementing Precision Oncology with Phenotypic Data.

Cancers (Basel) 2019 12 1;11(12). Epub 2019 Dec 1.

Segal Cancer Proteomics Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, QC H3T1E2, Canada.

Hotspot testing for activating mutations is used in precision oncology to select colorectal cancer (CRC) patients who are eligible for anti-EGFR treatment. However, even for tumors anti-EGFR response rates are <30%, while mutated- does not entirely rule out response, indicating the need for improved patient stratification. We performed proteogenomic phenotyping of and CRC liver metastases (mCRC). Among >9000 proteins we detected considerable expression changes including numerous proteins involved in progression and resistance in CRC. We identified peptides representing a number of predicted somatic mutations, including KRAS. For eight of these, we developed a multiplexed parallel reaction monitoring (PRM) mass spectrometry assay to precisely quantify the mutated and canonical protein variants. This allowed phenotyping of eight mCRC tumors and six paired healthy tissues, by determining mutation rates on the protein level. Total KRAS expression varied between tumors (0.47-1.01 fmol/µg total protein) and healthy tissues (0.13-0.64 fmol/µg). In -mCRC, G12V-mutation levels were 42-100%, while one patient had only 10% KRAS but 90% KRAS. This might represent a missed therapeutic opportunity: based on hotspot sequencing, the patient was excluded from anti-EGFR treatment and instead received chemotherapy, while PRM-based tumor-phenotyping indicates the patient might have benefitted from anti-EGFR therapy.
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http://dx.doi.org/10.3390/cancers11121907DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6966481PMC
December 2019

Targeted and Untargeted Proteomics Approaches in Biomarker Development.

Proteomics 2020 05 30;20(9):e1900029. Epub 2020 Jan 30.

Segal Cancer Proteomics Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, Quebec, H3T 1E2, Canada.

An enormous amount of research effort has been devoted to biomarker discovery and validation. With the completion of the human genome, proteomics is now playing an increasing role in this search for new and better biomarkers. Here, what leads to successful biomarker development is reviewed and how these features may be applied in the context of proteomic biomarker research is considered. The "fit-for-purpose" approach to biomarker development suggests that untargeted proteomic approaches may be better suited for early stages of biomarker discovery, while targeted approaches are preferred for validation and implementation. A systematic screening of published biomarker articles using MS-based proteomics reveals that while both targeted and untargeted technologies are used in proteomic biomarker development, most researchers do not combine these approaches. i) The reasons for this discrepancy, (ii) how proteomic technologies can overcome technical challenges that seem to limit their translation into the clinic, and (iii) how MS can improve, complement, or replace existing clinically important assays in the future are discussed.
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http://dx.doi.org/10.1002/pmic.201900029DOI Listing
May 2020

Ligand-induced disorder-to-order transitions characterized by structural proteomics and molecular dynamics simulations.

J Proteomics 2020 01 1;211:103544. Epub 2019 Nov 1.

University of Victoria -Genome British Columbia Proteomics Centre, #3101-4464 Markham Street, Vancouver Island Technology Park, Victoria, BC V8Z7X8, Canada; Department of Biochemistry and Microbiology, University of Victoria, Petch Building, Room 270d, 3800 Finnerty Rd, Victoria, BC V8P 5C2, Canada; Segal Cancer Proteomics Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, Quebec, H3T 1E2, Canada; Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University, Montreal, Quebec, H3T 1E2, Canada. Electronic address:

For disordered proteins, ligand binding can be a critical event that changes their structural dynamics. The ability to characterize such changes would facilitate the development of drugs designed to stabilize disordered proteins, whose mis-folding is important for a number of pathologies, including neurodegenerative diseases such as Parkinson's and Alzheimer's diseases. In this study, we used hydrogen/deuterium exchange, differential crosslinking, differential surface modification, and molecular dynamics (MD) simulations to characterize the structural changes in disordered proteins that result from ligand binding. We show here that both an ATP-independent protein chaperone, Spy L32P, and the FK506 binding domain of a prolyl isomerase, FKBP-25 F145A/I223P, are disordered, yet exhibit structures that are distinct from chemically denatured unfolded states in solution, and that they undergo transitions to a more structured state upon ligand binding. These systems may serve as models for the characterization of ligand-induced disorder-to-order transitions in proteins using structural proteomics approaches. SIGNIFICANCE: In this study, we used hydrogen/deuterium exchange, differential crosslinking, differential surface modification, and molecular-dynamics simulations to characterize the structural changes in disordered proteins that result from ligand binding. The protein-ligand systems studied here (the ATP-independent protein chaperone, Spy L32P, and the FK506 binding domain of a prolyl isomerase, FKBP-25 F145A/I223P) may serve as models for understanding ligand-induced disorder-to-order transitions in proteins. Additionally, the structural proteomic techniques demonstrated here are shown to be effective tools for the characterization of disorder-to-order transitions and can be used to facilitate study of other systems in which this class of structural transition can be used for modulating major pathological features of disease, such as the abnormal protein aggregation that occurs with Parkinson's disease and Alzheimer's disease.
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http://dx.doi.org/10.1016/j.jprot.2019.103544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6878985PMC
January 2020

Insight into the Structure of the "Unstructured" Tau Protein.

Structure 2019 11 15;27(11):1710-1715.e4. Epub 2019 Oct 15.

University of Victoria-Genome British Columbia Proteomics Centre, 3101-4464 Markham Street, Vancouver Island Technology Park, Victoria, BC V8Z7X8, Canada; Segal Cancer Proteomics Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, QC, H3T 1E2, Canada; Department of Biochemistry and Microbiology, University of Victoria, Petch Building, Room 270d, 3800 Finnerty Road, Victoria, BC V8P 5C2, Canada; Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University, Montreal, QC, H3T 1E2, Canada. Electronic address:

Combining structural proteomics experimental data with computational methods is a powerful tool for protein structure prediction. Here, we apply a recently developed approach for de novo protein structure determination based on the incorporation of short-distance crosslinking data as constraints in discrete molecular dynamics simulations (CL-DMD), for the determination of the conformational ensemble of tau protein in solution. The predicted structures were in agreement with surface modification and long-distance crosslinking data. Tau in solution was found as an ensemble of rather compact globular conformations with distinct topology, inter-residue contacts, and a number of transient secondary-structure elements. Regions important for pathological aggregation consistently were found to contain β strands. The determined structures are compatible with the tau protein in solution being a molten globule at near-ground state with persistent residual structural features which we were able to capture by CL-DMD. The predicted structure may facilitate an understanding of the misfolding and oligomerization pathways of the tau protein.
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http://dx.doi.org/10.1016/j.str.2019.09.003DOI Listing
November 2019

MeCP2-E1 isoform is a dynamically expressed, weakly DNA-bound protein with different protein and DNA interactions compared to MeCP2-E2.

Epigenetics Chromatin 2019 10 10;12(1):63. Epub 2019 Oct 10.

Department of Biochemistry and Microbiology, University of Victoria, Petch Building 260, Victoria, BC, V8W 3P6, Canada.

Background: MeCP2-a chromatin-binding protein associated with Rett syndrome-has two main isoforms, MeCP2-E1 and MeCP2-E2, differing in a few N-terminal amino acid residues. Previous studies have shown brain region-specific expression of these isoforms which, in addition to their different cellular localization and differential expression during brain development, suggest that they may also have non-overlapping molecular mechanisms. However, differential functions of MeCP2-E1 and E2 remain largely unexplored.

Results: Here, we show that the N-terminal domains (NTD) of MeCP2-E1 and E2 modulate the ability of the methyl-binding domain (MBD) to interact with DNA as well as influencing the turn-over rates, binding dynamics, response to neuronal depolarization, and circadian oscillations of the two isoforms. Our proteomics data indicate that both isoforms exhibit unique interacting protein partners. Moreover, genome-wide analysis using ChIP-seq provide evidence for a shared as well as a specific regulation of different sets of genes.

Conclusions: Our study supports the idea that Rett syndrome might arise from simultaneous impairment of cellular processes involving non-overlapping functions of MECP2 isoforms. For instance, MeCP2-E1 mutations might impact stimuli-dependent chromatin regulation, while MeCP2-E2 mutations could result in aberrant ribosomal expression. Overall, our findings provide insight into the functional complexity of MeCP2 by dissecting differential aspects of its two isoforms.
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http://dx.doi.org/10.1186/s13072-019-0298-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6786283PMC
October 2019

Evaluation and Validation of Plasma Proteins Using Two Different Protein Detection Methods for Early Detection of Colorectal Cancer.

Cancers (Basel) 2019 Sep 25;11(10). Epub 2019 Sep 25.

Division of Preventive Oncology, German Cancer Research Center (DKFZ) and National Center for Tumor Diseases (NCT), 69120 Heidelberg, Germany.

Objective: Plasma protein biomarkers could be an efficient alternative for population-based screening for early detection of colorectal cancer (CRC). The objective of this study was to evaluate and validate plasma proteins individually and as a signature for early detection of CRC.

Methods: In a three-stage design, proteins were measured firstly by liquid chromatography/multiple reaction monitoring-mass spectrometry (LC/MRM-MS) and later by proximity extension assay (PEA) in a discovery set consisting of 96 newly diagnosed CRC cases and 94 controls free of neoplasms at screening colonoscopy. Two algorithms (one for each measurement method) were derived by Lasso regression and .632+ bootstrap based on 11 proteins that were included in both the LC/MRM-MS and PEA measurements. Additionally, another algorithm was constructed from the same eleven biomarkers plus amphireglin, the most promising protein marker in the PEA measurements that had not been available from the LC/MRM-MS measurements. Lastly the three prediction signatures were validated with PEA in independent samples of participants of screening colonoscopy (CRC ( = 56), advanced adenoma ( = 101), and participants free of neoplasm ( = 102)).

Results: The same four proteins were included in all three prediction signatures; mannan binding lectin serine protease 1, osteopontin, serum paraoxonase lactonase 3 and transferrin receptor protein 1, and the third prediction signature additionally included amphiregulin. In the independent validation set from a true screening setting, the five-marker blood-based signature including AREG presented areas under the curves of 0.82 (95% CI, 0.74-0.89), 0.86 (95% CI, 0.77-0.92) and 0.76 (95% CI, 0.64-0.86) for all, early and late stages CRC, respectively.

Conclusion: Two different measurement methods consistently identified four protein markers and an algorithm additionally including amphiregulin, a marker measured by PEA only, showed promising performance for detecting early stage CRC in an independent validation in a true screening setting. These proteins may be potential candidates for blood-based tests for early detection of CRC.
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http://dx.doi.org/10.3390/cancers11101426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6826652PMC
September 2019