http://www.researchonline.mq.edu.au/vital/access/services/Feed ${session.getAttribute("locale")} 5 http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:21034 The widespread clinical adoption of protein biomarkers with diagnostic, prognostic and/or predictive value remains a formidable challenge for the biomedical community. From discovery to validation, the path to biomarkers of clinical relevance abounds with many protein candidates, yet so few concrete examples have been substantiated. In this review, we focus on the recent adoption of selected reaction monitoring (SRM) of plasma proteins in the path to clinical use for a broad range of diseases including cancer, cardiovascular disease, genetic disorders and various metabolic disorders. Recent progress reveals a promising outlook for clinical applications using SRM, which now provides the routine analysis of clinically relevant protein markers at low nanogram per millilitre in plasma. 2012-08-24T16:10:17.156Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:14844 In this review we examine techniques, software, and statistical analyses used in label-free quantitative proteomics studies for area under the curve and spectral counting approaches. Recent advances in the field are discussed in an order that reflects a logical workflow design. Examples of studies that follow this design are presented to highlight the requirement for statistical assessment and further experiments to validate results from label-free quantitation. Limitations of label-free approaches are considered, label-free approaches are compared with labelling techniques, and forward-looking applications for label-free quantitative data are presented. We conclude that label-free quantitative proteomics is a reliable, versatile, and cost-effective alternative to labelled quantita tion. 2012-06-14T08:12:29.679Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:15969 Global mean surface temperature has been predicted to increase by 1.8-4°C within this century, accompanied by an increase in the magnitude and frequency of extreme temperature events. Developing rice cultivars better adapted to non-optimal temperatures is essential to increase rice yield in the future and, hence, understanding the molecular response of rice to temperature stress is necessary. In this study, we investigated the proteomic responses of leaves of 24-day-old rice seedlings to sudden temperature changes. Rice seedlings grown at 28/20°C (day/night) were subjected to 3-day exposure to 12/5°C or 20/12°C (day/night) for low-temperature stress, and 36/28°C or 44/36°C (day/night) for high-temperature stress, followed by quantitative label-free shotgun proteomic analysis on biological triplicates of each treatment. Out of over 1100 proteins identified in one or more temperature treatments, more than 400 were found to be responsive to temperature stress. Of these, 43, 126 and 47 proteins were exclusively found at 12/5, 20/12 and 44/36°C (day/night), respectively. Our results showed that a greater change occurs in the rice leaf proteome at 20/12°C (day/night) in comparison to other non-optimal temperature regimes. In addition, our study identified more than 20 novel stress-response proteins. 2011-11-17T16:51:57.388Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:15966 Rice is susceptible to cold stress and with a future of climatic instability we will be unable to produce enough rice to satisfy increasing demand. A thorough understanding of the molecular responses to thermal stress is imperative for engineering cultivars, which have greater resistance to low temperature stress. In this study we investigated the proteomic response of rice seedlings to 48, 72 and 96h of cold stress at 12-14°C. The use of both label-free and iTRAQ approaches in the analysis of global protein expression enabled us to assess the complementarity of the two techniques for use in plant proteomics. The approaches yielded a similar biological response to cold stress despite a disparity in proteins identified. The label-free approach identified 236 cold-responsive proteins compared to 85 in iTRAQ results, with only 24 proteins in common. Functional analysis revealed differential expression of proteins involved in transport, photosynthesis, generation of precursor metabolites and energy; and, more specifically, histones and vitamin B biosynthetic proteins were observed to be affected by cold stress. 2011-11-15T18:10:10.620Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:13604 Global mean temperatures are expected to rise by 2–4.5°C by 2100, accompanied by an increase in frequency and amplitude of extreme temperature events. Greater climatic extremes and an expanded range of cultivation will expose rice to increasing stress in the future. Understanding gene expression in disparate thermal regimes is important for the engineering of cultivars with tolerance to nonoptimal temperatures. Our study investigated the proteomic responses of rice cell suspension cultures to sudden temperature changes. Cell cultures grown at 28°C were subjected to 3-day exposure to 12 or 20°C for low-temperature stress, and 36 or 44°C for high-temperature stress. Quantitative label-free shotgun proteomic analysis was performed on biological triplicates of each treatment. Over 1900 proteins were expressed in one or more temperature treatments, and, of these, more than 850 were found to be responsive to either of the temperature extremes. These temperature-responsive proteins included more than 300 proteins which were uniquely expressed at either 12 or 44°C. Our study also identified 40 novel stress–response proteins and observed that switching between the classical and the alternative pathways of sucrose metabolism occurs in response to extremes of temperature. 2011-06-14T07:51:18.797Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:13605 An emphasis of current proteomic research is the validation of plasma protein biomarkers. The process of blood collection itself is critical to the accuracy and reproducibility of quantitative biomarker assays. We have developed selected reaction monitoring (SRM) assays to analyse thirteen abundant plasma proteins and evaluated the impact of three different blood collection tubes on the levels of these proteins. We also assessed the implications of the time taken to analyse plasma samples by evaluating the recovery of these proteins. We showed that SRM detects minor differences in the levels of some proteins which can be attributed to collection tube type. The average recovery for 12 of 18 assays was higher for proteins that were collected in tubes containing protease inhibitors compared to conventional collection tubes. For five of the assays, the differential recovery was statistically significant. Delaying MS analysis of a freeze-thawed sample for 1 hour showed greatly reduced recovery of these analytes; however differences attributed to tube type were only evident at the baseline timepoint. Finally, we assessed the natural variation of circulating levels of these proteins in a cohort of seven healthy individuals. This study provides useful information for researchers contemplating blood collection for undertaking protein biomarker studies. 2011-06-14T07:51:16.915Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:4451 The development of a sensitive fluorescence-based assay for the quantitative determination of protein concentration is described. The assay is based on the natural product epicocconone, which produces a large increase in fluorescence quantum yield upon binding to detergent-coated proteins in solution. There is a concomitant shift in the emission maximum from 520 to 605 nm after binding, which results in low background signal allowing a linear dynamic range of 40 ng/mL to 200 μg/mL for most proteins. There is little protein-to-protein variation except for iron-containing proteins and the assay can be used so that it is tolerant of chemicals commonly used in 2-D sample buffers. The assay is more sensitive than standard absorption assays such as the Bradford and Lowry assays, and has a greater dynamic range and sensitivity than other fluorescent assays. 2010-01-27T22:41:30.215Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:5913 There is wide interpatient variability in toxicity to chemotherapeutic drugs and a lack of routine clinical tests for prospectively identifying patients at risk of developing toxicity from chemotherapy. An empirically driven MS strategy has been developed to monitor liver-derived plasma proteins as potential biomarkers of early toxicity. Multiple reaction monitoring (MRM) has been used to assess 46 candidate peptides from 18 liver-derived proteins. Following an iterative process of assay design, optimisation and assessment we selected 29 MRM assays (median CV 4.6%, range 1.2-11.6%) and monitored changes in levels of plasma proteins from a small number of colorectal cancer (CRC) patients undergoing chemotherapy. We demonstrated MRM assay robustness, and show that patients undergo minor elevation in plasma proteins when profiled on Day 3 of the chemotherapeutic regime. The MRM assays were in general agreement with 2-D DIGE-based quantitation from the same patient samples. The data supports the application of MRM-based methods as facile, highly reproducible, medium-throughput techniques that warrant expanded investigation for clinical utility in identifying patients at risk of developing chemotoxicity. 2010-01-27T22:24:43.739Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:5967 Two-dimensional gel electrophoresis (2-DE) image analysis is conventionally used for comparative proteomics. However, there are a number of technical difficulties associated with 2-DE protein separation that limit the depth of proteome coverage, and the image analysis steps are typically labor-intensive and low-throughput. Recently, mass spectrometry-based quantitation strategies have been described as alternative differential proteome analysis techniques. In this study, we investigated changes in protein expression using an ovarian cancer cell line, OVMZ6, 24 h post-stimulation with the relatively weak agonist, urokinase-type plasminogen activator (uPA). Quantitative protein profiles were obtained by MALDI−TOF/TOF from stable isotope-labeled cells in culture (SILAC), and these results were compared to the quantitative ratios obtained using 2-DE gel image analysis. MALDI-TOF/TOF mass spectrometry showed that differential quantitation using SILAC was highly reproducible (~8% coefficient of variation (CV)), and this variance was considerably lower than that achieved using automated 2-DE image analysis strategies (CV~25%). Both techniques revealed subtle alterations in cellular protein expression following uPA stimulation. However, due to the lower variances associated with the SILAC technique, smaller changes in expression of uPA-inducible proteins could be found with greater certainty. 2010-01-27T22:24:01.943Z ]]>