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A rapid standardized quantitative microfluidic system approach for evaluating human tear proteins
PURPOSE: To explore the potential of a chip-based miniaturized capillary gel electrophoresis device in a quantitative evaluation of the human tear protein profile and to validate the method. METHODS: A total of 5 μl of tears were collected from 25 patients diagnosed as having mild to moderate dry ey...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Molecular Vision
2012
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3482171/ https://www.ncbi.nlm.nih.gov/pubmed/23112568 |
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author | Versura, Piera Bavelloni, Alberto Blalock, William Fresina, Michela Campos, Emilio C. |
author_facet | Versura, Piera Bavelloni, Alberto Blalock, William Fresina, Michela Campos, Emilio C. |
author_sort | Versura, Piera |
collection | PubMed |
description | PURPOSE: To explore the potential of a chip-based miniaturized capillary gel electrophoresis device in a quantitative evaluation of the human tear protein profile and to validate the method. METHODS: A total of 5 μl of tears were collected from 25 patients diagnosed as having mild to moderate dry eye according to Dry Eye Workshop guidelines and from 20 matched normal volunteers. Protein analysis was performed with the 2100 Bioanalyzer; different protein kit assays were evaluated (Protein 80 kit, Protein 230 kit, High Sensitivity Protein 250 kit) for sizing and quantifying protein samples from 5 to 80 kDa, 14 to 230 kDa, and 5 to 250 kDa, respectively. A standard protein ladder was loaded on each chip to allow an estimation of the appropriate molecular weight of the separated proteins; a sample buffer containing a lower and an upper marker was used to check the correct alignment of each lane. Virtual bands generated by the Bioanalyzer were identified and validated as follows: tear samples were run in parallel and proteins separated by one-dimensional and two-dimensional sodium dodecyl sulfate–PAGE and characterized by immunoblotting, enzymatic digestion, and analysis with liquid chromatography-mass spectrometry followed by a search of the SProt human protein database. RESULTS: Analyses were successfully performed by using as small as a 2 μl tear sample. The Protein 230 kit was selected as the best chip kit, able to differentiate all the proteins of interest. The measurement noise parameters were low, and reproducibility and repeatability exhibited high accuracy (0.998 and 0.995, respectively) and precision (0.974 and 0.977, respectively). The coefficient of variability was slightly higher than that declared by the manufacturer (6.2% versus 5.0%). Total protein content and the following proteins were recognized in all samples: lipophilin A lysozyme C, tear lipocalin-1, zinc-alpha-2-glycoprotein, serotransferrin, lactotransferrin, and exudated serum albumin. CONCLUSIONS: Our data demonstrate that this chip-based tear protein analysis is a reliable method of instrumental diagnosis in daily clinical activity and may provide supporting evaluation parameters for diagnosing and managing tear-based disorders. |
format | Online Article Text |
id | pubmed-3482171 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | Molecular Vision |
record_format | MEDLINE/PubMed |
spelling | pubmed-34821712012-10-30 A rapid standardized quantitative microfluidic system approach for evaluating human tear proteins Versura, Piera Bavelloni, Alberto Blalock, William Fresina, Michela Campos, Emilio C. Mol Vis Research Article PURPOSE: To explore the potential of a chip-based miniaturized capillary gel electrophoresis device in a quantitative evaluation of the human tear protein profile and to validate the method. METHODS: A total of 5 μl of tears were collected from 25 patients diagnosed as having mild to moderate dry eye according to Dry Eye Workshop guidelines and from 20 matched normal volunteers. Protein analysis was performed with the 2100 Bioanalyzer; different protein kit assays were evaluated (Protein 80 kit, Protein 230 kit, High Sensitivity Protein 250 kit) for sizing and quantifying protein samples from 5 to 80 kDa, 14 to 230 kDa, and 5 to 250 kDa, respectively. A standard protein ladder was loaded on each chip to allow an estimation of the appropriate molecular weight of the separated proteins; a sample buffer containing a lower and an upper marker was used to check the correct alignment of each lane. Virtual bands generated by the Bioanalyzer were identified and validated as follows: tear samples were run in parallel and proteins separated by one-dimensional and two-dimensional sodium dodecyl sulfate–PAGE and characterized by immunoblotting, enzymatic digestion, and analysis with liquid chromatography-mass spectrometry followed by a search of the SProt human protein database. RESULTS: Analyses were successfully performed by using as small as a 2 μl tear sample. The Protein 230 kit was selected as the best chip kit, able to differentiate all the proteins of interest. The measurement noise parameters were low, and reproducibility and repeatability exhibited high accuracy (0.998 and 0.995, respectively) and precision (0.974 and 0.977, respectively). The coefficient of variability was slightly higher than that declared by the manufacturer (6.2% versus 5.0%). Total protein content and the following proteins were recognized in all samples: lipophilin A lysozyme C, tear lipocalin-1, zinc-alpha-2-glycoprotein, serotransferrin, lactotransferrin, and exudated serum albumin. CONCLUSIONS: Our data demonstrate that this chip-based tear protein analysis is a reliable method of instrumental diagnosis in daily clinical activity and may provide supporting evaluation parameters for diagnosing and managing tear-based disorders. Molecular Vision 2012-10-12 /pmc/articles/PMC3482171/ /pubmed/23112568 Text en Copyright © 2012 Molecular Vision. http://creativecommons.org/licenses/by/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Article Versura, Piera Bavelloni, Alberto Blalock, William Fresina, Michela Campos, Emilio C. A rapid standardized quantitative microfluidic system approach for evaluating human tear proteins |
title | A rapid standardized quantitative microfluidic system approach for evaluating human tear proteins |
title_full | A rapid standardized quantitative microfluidic system approach for evaluating human tear proteins |
title_fullStr | A rapid standardized quantitative microfluidic system approach for evaluating human tear proteins |
title_full_unstemmed | A rapid standardized quantitative microfluidic system approach for evaluating human tear proteins |
title_short | A rapid standardized quantitative microfluidic system approach for evaluating human tear proteins |
title_sort | rapid standardized quantitative microfluidic system approach for evaluating human tear proteins |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3482171/ https://www.ncbi.nlm.nih.gov/pubmed/23112568 |
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