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Zhang H.,Kinexus Bioinformatics Corporation | Pelech S.,Kinexus Bioinformatics Corporation | Pelech S.,University of British Columbia | Pelech S.,The Brain Research Center
Seminars in Cell and Developmental Biology | Year: 2012

Unraveling the complexity of cell regulatory systems and monitoring their operations under normal and pathological circumstances is one of the major outstanding biomedical challenges. The phosphoproteome has emerged as a rich source of biomarkers for tracking cell signaling and disease, and many of the kinases that phosphorylate proteins represent attractive targets for drug development. Over 100,000 phosphorylation sites distributed in most of the 23,000 proteins encoded by the human genome have already been identified in a non-targeted fashion by mass-spectrometry. Antibody microarrays permit ultra-sensitive, semi-quantitative measurements of the levels of hundreds of target proteins and their phosphorylation in parallel with specimens from cells and tissues. Conversely, reverse-phase protein microarrays (RPPMs) that are printed with crude cell/tissue lysates allow tracking of a target protein with a probing antibody in hundreds to thousands of cell and tissue samples simultaneously. While more than half a million commercial antibodies are available, the identification of highly specific and potent antibodies for use in microarrays remains a major impediment. Antibody cross-reactivity is an issue for both antibody microarrays and RPPMs. The low abundance of signal transduction proteins and their substoichiometric levels of phosphorylation are also problematic. Finally, non-denaturing conditions used with standard antibody microarrays permit protein complexes, which can produce false positives and false negatives. Changes in the level of an interacting protein may be misinterpreted as alterations in the amount of a target protein or its phosphorylation state. It is critical that leads from both types of microarrays are validated by complementary approaches such as immunoblotting and ELISA. More than a hundred reports have appeared in the scientific literature that have benefited from utilization of antibody and protein lysate microarrays. We have highlighted some of the pioneering works in this field and provided recent examples of their successful deployment as tools for broad-based, targeted proteomics research. © 2012 Elsevier Ltd.

Carter C.A.,Lorillard Tobacco Company | Misra M.,Lorillard Tobacco Company | Pelech S.,Kinexus Bioinformatics Corporation | Pelech S.,University of British Columbia
Journal of Proteome Research | Year: 2011

A short-term 5 day mainstream cigarette smoke exposure study was conducted in Fischer 344 rats to identify changes in lung proteins. Groups of 10 male and female rats at 5 weeks of age were assigned to one of four exposure groups. Animals received either nose-only filtered air (Air Control) or 75, 200, or 400 mg total particulate matter (TPM)/m 3 of diluted cigarette smoke. Exposures were conducted for 3 h per day, for 5 consecutive days. One lung per animal was frozen in liquid nitrogen and processed for proteomic analyses. Lung lysates from control verses treated animals were screened with 650 antibodies for changes in signaling protein levels and phosphorylation using antibody microarray technology, and then over 100 of the top protein hits were assessed by immunoblotting. The top smoke-altered proteins were further evaluated using reverse lysate microarrays. Major protein changes showed medium to strong bands on Western blots, depended on dose and gender, and included protein-serine kinases (Cot/Tpl2, ERK1/2, GSK3α/β, MEK6, PKCα/γ, RSK1), protein phosphatases (PP4/A′2, PP1Cβ), and other proteins (caspase 5, CRMP2, Hsc70, Hsp60, Rac1 and STAT2). The most pronounced changes occurred with 75 mg TPM/m 3 exposed females and 200 mg TPM/m 3 exposed males. Smoke-altered proteins regulate apoptosis, stress response, cell structure, and inflammation. Changes in identified proteins may serve as early indicators of lung damage. © 2011 American Chemical Society.

Davies A.H.,University of British Columbia | Barrett I.,University of British Columbia | Pambid M.R.,University of British Columbia | Hu K.,University of British Columbia | And 8 more authors.
Oncogene | Year: 2011

Y-box binding protein-1 (YB-1) expression in the mammary gland promotes breast carcinoma that demonstrates a high degree of genomic instability. In the present study, we developed a model of pre-malignancy to characterize the role of this gene during breast cancer initiation and early progression. Antibody microarray technology was used to ascertain global changes in signal transduction following the conditional expression of YB-1 in human mammary epithelial cells (HMEC). Cell cycle-associated proteins were frequently altered with the most dramatic being LIM kinase 1/2 (LIMK1/2). Consequently, the misexpression of LIMK1/2 was associated with cytokinesis failure that acted as a precursor to centrosome amplification. Detailed investigation revealed that YB-1 localized to the centrosome in a phosphorylation-dependent manner, where it complexed with pericentrin and γ-tubulin. This was found to be essential in maintaining the structural integrity and microtubule nucleation capacity of the organelle. Prolonged exposure to YB-1 led to rampant acceleration toward tumorigenesis, with the majority of cells acquiring numerical and structural chromosomal abnormalities. Slippage through the G1/S checkpoint due to overexpression of cyclin E promoted continued proliferation of these genomically compromised cells. As malignancy further progressed, we identified a subset of cells harboring HER2 amplification. Our results recognize YB-1 as a cancer susceptibility gene, with the capacity to prime cells for tumorigenesis. © 2011 Macmillan Publishers Limited All rights reserved.

Silva J.V.,University of Aveiro | Freitas M.J.,University of Aveiro | Correia B.R.,University of Aveiro | Korrodi-Gregorio L.,University of Aveiro | And 4 more authors.
Fertility and Sterility | Year: 2015

Objective To determine the correlation between semen basic parameters and the expression and activity of signaling proteins. Design In vitro studies with human spermatozoa. Setting Academic research institute. Patient(s) Thirty-seven men provided semen samples for routine analysis. Intervention(s) None. Main Outcome Measure(s) Basic semen parameters tracked included sperm DNA fragmentation (SDF), the expression levels of 75 protein kinases, and the phosphorylation/cleavage patterns of 18 signaling proteins in human spermatozoa. Result(s) The results indicated that the phosphorylated levels of several proteins (Bad, GSK-3β, HSP27, JNK/SAPK, mTOR, p38 MAPK, and p53), as well as cleavage of PARP (at D214) and Caspase-3 (at D175), were significantly correlated with motility parameters. Additionally, the percentage of morphologically normal spermatozoa demonstrated a significant positive correlation with the phosphorylated levels of p70 S6 kinase and, in turn, head defects and the teratozoospermia index (TZI) showed a significant negative correlation with the phosphorylated levels of Stat3. There was a significant positive correlation between SDF and the teratozoospermia index, as well as the presence of head defects. In contrast, SDF negatively correlated with the percentage of morphologically normal spermatozoa and the phosphorylation of Akt and p70 S6 kinase. Subjects with varicocele demonstrated a significant negative correlation between head morphological defects and the phosphorylated levels of Akt, GSK3β, p38 MAPK, and Stat1. Additionally, 34 protein kinases were identified as expressed in their total protein levels in normozoospermic samples. Conclusion(s) This study contributed toward establishing a biomarker "fingerprint" to assess sperm quality on the basis of molecular parameters. © 2015 American Society for Reproductive Medicine.

Winkler D.F.H.,Kinexus Bioinformatics Corporation
Mini-Reviews in Organic Chemistry | Year: 2011

The SPOT technique is one of the most frequently used methods for synthesis and screening of peptides on arrays. Materials such as polypropylene and glass are used for the preparation of peptide arrays, however the most commonly used material for SPOT membranes is cellulose. This paper focuses on materials and procedures used in the SPOT synthesis on cellulose membranes as a special type of solid phase peptide synthesis. In particular, different strategies for the modification of cellulose are described which make it more suitable for solid-phase peptide synthesis. This review also provides a short overview of the synthesis procedures including some important types of peptide modification. © 2011 Bentham Science Publishers Ltd.

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