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Coombs K.M.,University of Manitoba | Coombs K.M.,Manitoba Center for Proteomics and Systems Biology | Coombs K.M.,Manitoba Institute of Child Health
Expert Review of Proteomics | Year: 2011

Measurement of biologically important effector protein molecules has been a long-standing essential component of biological research. Advances in biotechnology, in the form of high-resolution mass spectrometers, and in bioinformatics, now allow the simultaneous quantitative analysis of thousands of proteins. While these techniques still do not allow definitive identification of the entire proteome of complex mixtures, such as cells, quantitative analyses of hundreds to thousands of proteins in such complex mixtures provides a means to elucidate molecular alterations that occur during perturbation of cellular systems. This article will outline considerations of reducing sample complexity, by strategies such as multidimensional separations (gel-based and chromatography-based, including multidimensional protein identification technology). In addition, some of the most common methods used to quantitatively measure proteins in complex mixtures (2D difference in-gel electrophoresis, isotope-coded affinity tags, isotope-coded protein labeling, tandem mass tags, isobaric tags for relative and absolute quantitation, stable isotope labeling of amino acids in cell culture and label-free), as well as recent examples of each strategy, are described. © 2011 Expert Reviews Ltd.

Kanwar N.,Manitoba Center for Proteomics and Systems Biology | Wilkins J.A.,Manitoba Center for Proteomics and Systems Biology | Wilkins J.A.,University of Manitoba
European Journal of Immunology | Year: 2011

Natural killer (NK) cells form a region of tight contact called the NK immunological synapse (NKIS) with their target cells. This is a dynamic region serving as a platform for targeted signaling and exocytotic events. We previously identified IQGAP1 as a cytoskeletal component of the NK-like cell line YTS. The present study was undertaken to determine the role of IQGAP1 in the function of NK cells. Silencing of IQGAP1 expression resulted in almost complete loss of the cytotoxic activity of YTS cells. Loss of IQGAP1 did not prevent conjugate formation with target cells but it did result in a failure to reorient the microtubule organizing centre to the immune synapse. Significantly, IQGAP1 expression was required for the perigranular accumulation of an F-actin network. IQGAP1 was shown to undergo marked rearrangements during synapse maturation in effector target conjugates of YTS or primary NK cells. These results suggest previously undescribed role(s) for IQGAP1 in regulating multiple aspects of cytoskeletal organization and granule polarization in NK cells. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Blydt-Hansen T.D.,University of Manitoba | Gibson I.W.,University of Manitoba | Gao A.,Manitoba Center for Proteomics and Systems Biology | Dufault B.,University of Manitoba | Ho J.,University of Manitoba
Transplantation | Year: 2015

Subclinical and clinical Tcell-mediated rejection (TCMR) has significant prognostic implications in pediatric renal transplantation. The goal of this study was to independently validate urinary CXCL10 as a noninvasive biomarker for detecting acute rejection in children and to extend these findings to subclinical rejection. Methods. Urines (n = 140) from 51 patients with surveillance or indication biopsies were assayed for urinary CXCL10 using enzyme-linked immunosorbent assay and corrected with urinary creatinine. Results.Median urinary CXCL10-to-creatinine (Cr) ratio (ng/mmol)was significantly elevated in subclinical TCMR(4.4 [2.6, 25.4], P<0.001, n = 17); clinical TCMR(24.3 [11.2, 44.8], P<0.001, n = 9); and antibody-mediated rejection (6.0 [3.3, 13.7], P = 0.002, n = 9) compared to noninflamed histology (1.4 [0.4, 4.2], normal and interstitial fibrosis and tubular atrophy, n = 52), and borderline tubulitis (3.3, [1.3, 4.9], n = 36). Elevated urinary CXCL10:Cr was independently associated with t scores (P < 0.001) and g scores (P = 0.006) on multivariate analysis. The area under receiver operating curve for subclinical and clinical TCMR was 0.81 (P = 0.045) and 0.88 (P = 0.019), respectively. This corresponded to a sensitivity-specificity of 0.59-0.67 and 0.77-0.60 for subclinical and clinical TCMR at cutoffs of 4.82 and 4.72 ng/mmol, respectively. Conclusion. This study demonstrates that urinary CXCL10:Cr corresponds with microvascular inflammation and is a sensitive and specific biomarker for subclinical and clinical TCMR in children. This may provide a noninvasive monitoring tool for posttransplant immune surveillance for pediatric renal transplant recipients. © Wolters Kluwer Health, Inc.

Reimer J.,Manitoba Center for Proteomics and Systems Biology | Spicer V.,University of Manitoba | Krokhin O.V.,Manitoba Center for Proteomics and Systems Biology | Krokhin O.V.,University of Manitoba
Journal of Chromatography A | Year: 2012

Twenty five years ago Houghten and DeGraw published a groundbreaking study of reversed-phase (RP)-HPLC retention of 298 peptide analogs, including 260 peptides coding the positional substitution in a 13-mer molecule with all 20 naturally occurring amino acids [1]. The authors challenged the state-of-the-art assumption that peptide retention can be represented as a sum of individual hydrophobicities of the constituent amino acids, and suggested an additional dependence on the ordering (sequence) of the residues. Here we explore the accuracy of modern peptide retention prediction models when applied to this retention dataset. We find that all of them perform below their claimed prediction accuracies. Clearly, the question raised 25 years ago remains unanswered, despite significant progress in the field over the past few years. Analysis of the prediction errors shows that the vast majority of outliers occur due to the amphipathic character of the framework Ac-YPYDVPDYASLRS-Amide peptide. This indicates that the understanding and quantitative description of stabilization of helical structures upon interaction with C18 phase is underdeveloped and should be a priority moving forward. In this report we also show that the presence of N-cap stabilizing residues increases peptide RP retention and should be taken into account. Capping effects have not been considered in peptide RP-HPLC studies, despite the clear evidence hidden in the quarter-century old Houghten and DeGraw's experimental results. © 2012 Elsevier B.V.

Shahiduzzaman M.,University of Manitoba | Shahiduzzaman M.,Manitoba Center for Proteomics and Systems Biology | Shahiduzzaman M.,Bangladesh Agricultural University | Coombs K.M.,University of Manitoba | And 2 more authors.
Frontiers in Microbiology | Year: 2012

Activity-based protein profiling (ABPP) is a newly emerging technique that uses active sitedirected probes to monitor the functional status of enzymes. Serine hydrolases are one of the largest families of enzymes in mammals. More than 200 serine hydrolases have been identified, but little is known about their specific roles. Serine hydrolases are involved in a variety of physiological functions, including digestion, immune response, blood coagulation, and reproduction. ABPP has been used recently to investigate host-virus interactions and to understand the molecular pathogenesis of virus infections. Monitoring the altered serine hydrolases during viral infection gives insight into the catalytic activity of these enzymes that will help to identify novel targets for diagnostic and therapeutic application. This review presents the usefulness of ABPP in detecting and analyzing functional annotation of host cell serine hydrolases as a result of host-virus interaction. © 2012 Shahiduzzaman and Coombs.

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