Investigation of the human tear film proteome using multiple proteomic approaches

Mass Spectrometry and Proteomics Facility, The Ohio State University, Columbus, OH 43210, USA.
Molecular vision (Impact Factor: 2.25). 02/2008; 14:456-70.
Source: PubMed

ABSTRACT The purpose of this work was to examine the tear film proteome using a combination of one-dimensional (1D) and two dimensional (2D) gel electrophoresis and mass spectrometry-based techniques and to explore the effect of the tear collection methods on the tear proteome.
Tear samples from eight normal non-contact lens wearing human subjects collected by Drummond glass microcapillary and Schirmer strips were subjected to 1D-sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 2D-SDS-PAGE, and 2D LC-MS/MS (Multidimensional protein identification technology - MudPIT). Bands or cores from the 1D- and 2D-SDS-PAGE were cut, digested with trypsin, and analyzed by tandem mass spectrometry for identification by the generation of sequence tags.
In total (across sampling and proteomic methods), 97 unique proteins were observed, and a significant number of the spots/bands in the PAGE were from posttranslational modifications. Fifty-four unique proteins were identified from proteins extracted from the Schirmer strips in comparison to 13 unique proteins identified from capillary tubes, and 30 unique proteins were identified by both collection methods. Secreted (serum) proteins were predominantly observed from tears collected by capillary whereas a combination of cellular and serum proteins were identified from tear film collected by Schirmer strips.
Overall, these results suggest that the tear film collection and the proteomic method impacts the proteins present in the tear film and that care should be exercised in choosing a tear collection method to best correlate to the experiment being conducted or the hypothesis that is being tested.

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    • "Ammonium bicarbonate has been previously used for protein extraction from Schirmer strips [15] [17]. For instance, Green-Church and coworkers used ammonium bicarbonate to extract proteins from Schirmer strips and then precipitated the extract with acetone [17]. Because tear fluid contains NaCl, adding NaCl to the extraction buffer may increase the solubility of tear proteins and promote their extraction from the strips [18]. "
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    ABSTRACT: Human tear fluid is a complex mixture containing high concentrations of proteins and is increasingly becoming an important source for studying protein biomarkers of eye-related diseases, such as Graves' ophthalmopathy. Today, the Schirmer tear test is the most widely used technique for tear collection. However, sample handling and protein extraction from these strips has been highly challenging. Cutting and removal of the Schirmer strips after extraction, which may lead to sample loss prior to downstream analyses, are some of the challenges to consider. To address some of these limitations, we have developed a single unit filter-aided method for both sample handling and protein extraction. Additionally, we systematically investigated the most suitable conditions for protein extraction from these strips. Among the different extraction conditions applied, extraction with 100 mM ammonium bicarbonate containing 50 mM NaCl resulted in the highest number of identified proteins using 1D-LC MS/MS. Moreover, 1526 proteins were identified when the optimized extraction method was combined with 2D LC MS/MS analyses, which demonstrates the applicability of this novel approach towards the study of the tear proteome. This dataset of identified proteins represent a comprehensive catalogue of the tear proteome and may serve as a list for future biomarker research. Copyright © 2015. Published by Elsevier Inc.
    Analytical Biochemistry 04/2015; 480. DOI:10.1016/j.ab.2015.04.002 · 2.31 Impact Factor
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    • "(Abs = absorbance; BSA = bovine serum albumin) and pathological environment if the analysis of the ocular surface is desirable (Li et al. 2008). However, we cannot determine if these proteins came from ocular surface or from tears (Li et al. 2008), since STT strips can interact with the epithelium of the ocular surface (Van Haeringen et al. 1981, Green-Church et al. 2008). On the other side, samples collected without touching the cornea or conjunctiva, like with the use of microcapillary cannot carry ocular surface proteins (Li et al. 2008). "
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    ABSTRACT: RESUMO.-[Comparação de dois métodos de coleta de lágrima para quantificação proteica pelo método de Bradford.] Compararam-se dois métodos de coleta de lá-grima para quantificação proteica, utilizando-se 58 olhos de 29 cães hígidos. As amostras foram coletadas utilizando--se fitas de teste da lágrima de Schirmer (Schirmer tear test -STT) e tubos microcapilares. Após obtidas, as amostras foram congeladas a -80 o C e posteriormente analisadas pelo método de Bradford. Os resultados foram analisados pelo teste T de Student. A média da concentração proteica e des-vio padrão das amostras obtidas com microcapilar foi de 4,45mg/mL ±0,35 e 4,52mg/mL ±0,29 para olhos direito e. 2013. Comparison of two methods of tear sampling for protein quantification by Bradford method. Pesquisa Veterinária Brasileira The aim of this study was to compare two methods of tear sampling for protein quanti-fication. Tear samples were collected from 29 healthy dogs (58 eyes) using Schirmer tear test (STT) strip and microcapillary tubes. The samples were frozen at -80 o C and analyzed by the Bradford method. Results were analyzed by Student's t test. The average protein concentration and standard deviation from tears collected with microcapillary tube were 4.45mg/mL ±0.35 and 4,52mg/mL ±0.29 for right and left eyes respectively. The average protein concentration and standard deviation from tears collected with Schirmer Tear Test (STT) strip were and 54.5mg/mL ±0.63 and 54.15mg/mL ±0.65 to right and left eyes res-pectively. Statistically significant differences (p<0.001) were found between the methods. In the conditions in which this study was conducted, the average protein concentration obtained with the Bradford test from tear samples obtained by Schirmer Tear Test (STT) strip showed values higher than those obtained with microcapillary tube. It is important that concentration of tear protein pattern values should be analyzed according the method used to collect tear samples. STT os resultados foram 4,45mg/mL ±0,35 and 4,52mg/ mL ±0,29 para olhos direito e esquerdo, respectivamente. Diferenças estatisticamente significativas (p<0,001) foram encontradas entre os dois métodos. Nas condições em que o trabalho foi conduzido, a média da concentração proteica pelo método de Bradford das amostras obtidas através das tiras do STT foi superior à obtida com o tubo microcapilar. Os valores padrão da concentração protéica da lágrima ob-tida pelo método de Bradford devem ser analisados con-siderando-se o método de coleta da lágrima, uma vez que este interfere significativamente nos resultados obtidos.
    Pesquisa Veterinária Brasileira 02/2013; 33(2):261-264. DOI:10.1590/S0100-736X2013000200021 · 0.44 Impact Factor
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    • "This difference could be due to the fact that in validation 1 we employed the same tear collection methodology used in the discovery phase (sponge), whereas in validation 2 we used capillary. Similarly in previous reports, two tear collection methods (Schirmer test and capillary), and their possible effect on proteome information have been addressed [12] [16]. These studies also concluded that both collection methods yielded similar results with slight differences. "
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    Journal of proteomics 11/2012; 78. DOI:10.1016/j.jprot.2012.11.017 · 3.93 Impact Factor
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