Manitoba Center for Proteomics and Systems Biology

Winnipeg, Canada

Manitoba Center for Proteomics and Systems Biology

Winnipeg, Canada

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Lao Y.W.,University of Manitoba | Mackenzie K.,ShapeFoods Inc | Vincent W.,ShapeFoods Inc | Krokhin O.V.,Manitoba Center for Proteomics and Systems Biology | Krokhin O.V.,University of Manitoba
Journal of Separation Science | Year: 2014

Organoleptic properties of flaxseed oil deteriorate during storage due to methionine oxidation in its major cyclolinopeptides. Cyclolinopeptide E was previously identified as being responsible for the manifestation of bitter taste with flaxseed oil ageing. We developed a chromatographic procedure to monitor the oxidation of major cyclic peptides in flaxseed oil. We also used liquid chromatography with mass spectrometry and high-efficiency core-shell reversed-phase sorbents to study the separation of cyclolinopeptides in detail. The KinetexTM family of stationary phases (C8, C 18, phenyl-hexyl) was tested, along with the standard porous LunaTM C18(2) media. We found that only the phenyl-hexyl stationary phase allows for complete resolution of major cyclolinopeptides, thus permitting direct UV monitoring of degree of conversion for cyclolinopeptide B into C and L into E. We also report, for the first time, a significant effect of peak splitting for some methionine S-oxide (Mso) containing cyclolinopeptides, which most likely appear due to diastereomerization. This results in poor separation efficiency for cyclolinopeptides F, G, and E, and gives baseline resolution of diastereomeric pairs for cyclolinopeptides I and P. Thus, a single oxidation of cyclolinopeptide N yields three distinct chromatographic peaks corresponding to cyclolinopeptide T (cyclo-MsoLMPFFWV, reported for the first time) and pair of cyclolinopeptide I (cyclo-MLMsoPFFWV) diastereomers. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


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.


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.


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.


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.


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.


Reimer J.,Manitoba Center for Proteomics and Systems Biology | Shamshurin D.,Manitoba Center for Proteomics and Systems Biology | Harder M.,University of Manitoba | Yamchuk A.,University of Manitoba | And 3 more authors.
Journal of Chromatography A | Year: 2011

N-terminal loss of ammonia is a typical peptide modification chemical artifact observed in bottom-up proteomics experiments. It occurs both in vivo for N-terminal glutamine and in vitro following enzymatic cleavage for both N-terminal glutamine and cysteine alkylated with iodoacetamide. In addition to a mass change of -17.03. Da, modified peptides exhibit increased chromatographic retention in reversed-phase (RP) HPLC systems. The magnitude of this increase varies significantly depending on the peptide sequence and the chromatographic condition used. We have monitored these changes for extensive sets (more than 200 each) of tryptic Gln and Cys N-terminated species. Peptides were separated on 100. Å pore size C18 phases using identical acetonitrile gradient slopes with 3 different eluent compositions: 0.1% trifluoroacetic acid; 0.1% formic acid and 20. mM ammonium formate at pH 10 as ion-pairing modifiers. The observed effect of this modification on RP retention is the product of increased intrinsic hydrophobicity of the modified N-terminal residue, lowering or removing the effect of ion-pairing formation on the hydrophobicity of adjacent residues at acidic pHs; and possibly the increased formation of amphipathic helical structures when the positive charge is removed. Larger retention shifts were observed for Cys terminated peptides compared to Gln, and for smaller peptides. Also the size of the retention increase depends on the eluent conditions: pH 10 ≤ trifluoroacetic acid < formic acid. Different approaches for incorporation these findings in the peptide retention prediction models are discussed. © 2011 Elsevier B.V.


Shamshurin D.,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: 2011

The two leading RP-HPLC approaches for deriving hydrophobicity values of amino acids utilize either sets of designed synthetic peptides or extended random datasets often extracted from proteomics experiments. We find that the best examples of these two methods provide virtually identical results - with exception of Lys, Arg, and His. The intrinsic hydrophobicity values of the remaining residues as determined by Kovacs et al. (Biopolymers 84 (2006) 283) correlates with an R2-value of 0.995+ against amino acid retention coefficients from our Sequence Specific Retention Calculator model (Anal. Chem. 78 (2006) 7785). This novel finding lays the foundation for establishing consensus amino acids hydrophobicity scales as determined by RP-HPLC. Simultaneously, we find the assignment of hydrophobicity values for charged residues (Lys, Arg and His at pH 2) is ambiguous; their retention contribution is strongly affected by the overall peptide hydrophobicity. The unique behavior of the basic residues is related to the dualistic character of the RP peptide retention mechanism, where both hydrophobic and ion-pairing interactions are involved. We envision the introduction of " sliding" hydrophobicity scales for charged residues as a new element in peptide retention prediction models. We also show that when using a simple additive retention prediction model, the " correct" coefficient value optimization (0.98+ correlation against values determined by synthetic peptide approach) requires a training set of at least 100 randomly selected peptides. © 2011 Elsevier B.V.


Choi K.-Y.G.,Manitoba Center for Proteomics and Systems Biology | Choi K.-Y.G.,University of Manitoba | Lippert D.N.D.,Manitoba Center for Proteomics and Systems Biology | Ezzatti P.,Manitoba Center for Proteomics and Systems Biology | And 2 more authors.
Journal of Immunological Methods | Year: 2012

An impediment in the development of new therapeutic strategies for chronic inflammatory diseases is the limited understanding of underlying molecular mechanisms. The objective of this study was to identify newly synthesized (nascent) proteins induced by critical inflammatory cytokines TNF-α and IL-1β in human monocytic THP-1 cells. We optimized methods to combine two different approaches, bio-orthogonal non-canonical amino acid tagging (BONCAT) along with proteomics using isobaric tags (iTRAQ). BONCAT employed the incorporation of l-azidohomoalanine (AHA), an analog of methionine, into TNF-α or IL-1β induced nascent proteins. The AHA-containing nascent proteins were tagged with alkyne-biotin to allow enrichment using avidin affinity purification. The differential expressions of the enriched proteins were further determined using iTRAQ reagents and mass spectrometry (MS). The combination of BONCAT and proteomics represents a unique approach that has uncovered the nascent proteome induced by inflammatory cytokines TNF-α and IL-1β. © 2012 Elsevier B.V.


Mcqueen P.,Manitoba Center for Proteomics and Systems Biology | Spicer V.,University of Manitoba | Rydzak T.,University of Manitoba | Sparling R.,University of Manitoba | And 5 more authors.
Proteomics | Year: 2012

We have developed a real-time graphic-processor-unit-based search engine capable of high-quality peptide identifications in <500 μs per spectrum. The steps of peptide/protein identification, in-silico prediction of all possible tryptic peptides from these proteins, and the prediction of their expected retention times and m/z values take less than 5 s per cycle over ~3000 MS/MS spectra. This lays the foundation for information-dependent acquisition with exclusion lists generated on-the-fly, as the instrument continues to acquire data. While a complete evaluation of the dynamic exclusion system requires the participation from instrument vendors, we conducted a series of model experiments using a whole cell tryptic digestion of the bacterium Clostridium thermocellum. We ran a series of five iterative LC-MS/MS runs, adding a new exclusion list at each of four chromatographic "tripping points" - the elution times of the four standard peptides spiked into the sample. Retention times of these standard peptides were also used for real-time "chromatographic calibration." The dynamic exclusion approach gave a ~5% increase in confident protein identification (for typical 2 h LC-MS/MS run), and reduced the average number of identified peptides per protein from 4.7 to 2.9. Its application to a two-times shorter gradient gave a ~17% increase in proteins identified. Further improvements are possible for instruments with better mass accuracy, by employing a more accurate retention prediction algorithm and by developing better understanding of the possible chemical modifications and fragmentations produced during electrospray ionization. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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