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You J.,University of Sydney | Willcox M.D.,University of New South Wales | Madigan M.C.,University of Sydney | Madigan M.C.,University of New South Wales | And 10 more authors.
Advances in Clinical Chemistry | Year: 2013

The tear film covers and protects the ocular surface. It contains various molecules including a large variety of proteins. The protein composition of the tear fluid can change with respect to various local and systemic diseases. Prior to the advent of the proteomic era, tear protein analysis was limited to a few analytical techniques, the most common of which was immunoelectrophoresis, an approach dependent on antibody availability. Using proteomics, hundreds of tear proteins could potentially be identified and subsequently studied. Although detection of low-abundance proteins in the complex tear proteome remains a challenge, advances in sample fractionation and mass spectrometry have greatly enhanced our ability to detect these proteins. With increasing proteomic applications, tears show great potential as biomarkers in the development of clinical assays for various human diseases.In this chapter, we discuss the structure and functions of the tear film and methods for its collection. We also summarize potential tear protein biomarkers identified using proteomic techniques for both ocular and systemic diseases. Finally, modern proteomic techniques for tear biomarker research and future challenges are explored. © 2013 Elsevier Inc.


Fitzgerald A.,Minomic International Ltd | Walsh B.J.,Minomic International Ltd
Electrophoresis | Year: 2010

The depth of proteome analysis is severely limited in complex samples with a wide dynamic range of protein abundance such as plasma. Removal of high-abundance proteins should reveal the signal of lower abundance plasma proteins. However, smaller proteins may be part of larger protein complexes and hence the removal of proteins involved in complexes with high-abundance proteins such as albumin may inhibit the search for disease biomarkers. Prefractionation of a sample divides it into fractions of reduced complexity, allowing improved detection of lower abundance proteins. Using a prefractionation device, which employs Gradiflow™ technology, we were able to separate small volume plasma samples into multiple fractions based on the molecular weight and/or charge. The resulting samples of reduced complexity were directly compatible with 2-DE. The use of this prefractionation machine may therefore be useful in the hunt for disease biomarkers. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


You J.,Minomic International Ltd | You J.,University of New South Wales | Fitzgerald A.,Minomic International Ltd | Cozzi P.J.,University of New South Wales | And 13 more authors.
Electrophoresis | Year: 2010

This is the first 2-DE study using sequential dyes to analyse phospho-, glyco- and total tear protein profiles (Pro-Q Diamond for phosphoprotein, Pro-Q Emerald for glycoprotein and Sypro Ruby for total protein). This method minimised the gel-gel variations, allowing better comparisons among the three profiles and generated a whole map of PTM profiles of tear protein. A novel tear protein, dermcidin, was identified for the first time in this study. The identification of this antimicrobial protein suggests a new model of defence in tears. In addition, we are able to present the first experimental evidence of the presence of glycosylated lipocalin 1 and cystatin S. Nucleobindin 2 was only detected using phospho staining, suggesting it is only phosphorylated in tears. This study provides the groundwork for understanding the PTM of tear proteins and consequently these methods could be useful in the search for biomarkers in tears. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.


You J.,University of New South Wales | You J.,Minomic International Ltd | Cozzi P.,University of New South Wales | Walsh B.,Minomic International Ltd | And 8 more authors.
Critical Reviews in Oncology/Hematology | Year: 2010

The marker currently used for prostate cancer (CaP) detection is an increase in serum prostate-specific antigen (PSA). However, the PSA test which may give false positive or negative information, is not reliable and does not allow the differentiation of benign prostate hyperplasia.(BPH), non-aggressive CaP and aggressive CaP. There is thus an urgent need to search for novel CaP biomarkers to improve the early detection and accuracy of diagnosis, determine the aggressiveness of CaP and to monitor the efficacy of treatment. Proteomic techniques allow for a high-throughput analysis of bio-fluids with the visualization and quantification of thousands of potential protein markers and represent very promising tools in the search for new, improved molecular markers of CaP. In this review, we will summarize conventional CaP biomarkers and focus on novel identified biomarkers for CaP early diagnosis and progression that might be used in the future. © 2009 Elsevier Ireland Ltd.


You J.,University of New South Wales | You J.,Minomic International Ltd | You J.,In.Sight | Willcox M.,University of New South Wales | And 10 more authors.
Analytical Biochemistry | Year: 2016

The mass spectrometry technique of multiple reaction monitoring (MRM) was used to quantify and compare the expression level of lactoferrin in tear films among control, prostate cancer (CaP), and benign prostate hyperplasia (BPH) groups. Tear samples from 14 men with CaP, 15 men with BPH, and 14 controls were analyzed in the study. Collected tears (2 μl) of each sample were digested with trypsin overnight at 37°C without any pretreatment, and tear lactoferrin was quantified using a lactoferrin-specific peptide, VPSHAVVAR, both using natural/light and isotopic-labeled/heavy peptides with MRM. The average tear lactoferrin concentration was 1.01 ± 0.07 μg/μl in control samples, 0.96 ± 0.07 μg/μl in the BPH group, and 0.98 ± 0.07 μg/μl in the CaP group. Our study is the first to quantify tear proteins using a total of 43 individual (non-pooled) tear samples and showed that direct digestion of tear samples is suitable for MRM studies. The calculated average lactoferrin concentration in the control group matched that in the published range of human tear lactoferrin concentration measured by enzyme-linked immunosorbent assay (ELISA). Moreover, the lactoferrin was stably expressed across all of the samples, with no significant differences being observed among the control, BPH, and CaP groups. © 2015 Elsevier Inc. All rights reserved.

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