Proteome analysis of human aqueous humor

Uttio Roy Chowdhury, Benjamin J. Madden, Mary Christine Charlesworth, Michael P Fautsch

Research output: Contribution to journalArticle

86 Citations (Scopus)

Abstract

PURPOSE. Human aqueous humor (hAH) provides nutrition and immunity within the anterior chamber of the eye. Characterization of the protein composition of hAH will identify molecules involved in maintaining a homeostatic environment for anterior segment tissues. The present study was conducted to analyze the proteome of hAH. METHODS. hAH samples obtained during elective cataract surgery were divided into three matched groups and immunodepleted of albumin, IgG, IgA, haploglobin, antitrypsin, and transferrin. Reduced and denatured proteins (20 μg) from each group were separated by gel electrophoresis. Thirty-three gel slices were excised from each of three gel lanes (n = 99), digested with trypsin, and subjected to nanoflow liquid chromatography electrospray ionization tandem mass spectrometry (nano-LC-ESI-MS/MS). The protein component of hAH was also analyzed by antibody-based protein arrays, and selected proteins were quantified. RESULTS. A total of 676 proteins were identified in hAH. Of the 355 proteins identified by nano-LC-ESI-MS/MS, 206 were found in all three groups. Most of the proteins identified by nano-LCESI-MS/MS had catalytic, enzymatic, and structural properties. Using antibody-based protein arrays, 328 cytokines, chemokines, and receptors were identified. Most of the quantified proteins had concentrations that ranged between 0.1 and 2.5 ng/mL. Ten proteins were identified by both nano-LC-ESIMS/ MS and antibody protein arrays. CONCLUSIONS. Proteomic analysis of hAH identified 676 nonredundant proteins. More than 80% of these proteins are novel identifications. The elucidation of the aqueous proteome will establish a foundation for protein function analysis and identification of differentially expressed markers associated with diseases of the anterior segment.

Original languageEnglish (US)
Pages (from-to)4921-4931
Number of pages11
JournalInvestigative Ophthalmology and Visual Science
Volume51
Issue number10
DOIs
StatePublished - Oct 2010
Externally publishedYes

Fingerprint

Aqueous Humor
Proteome
Proteins
Protein Array Analysis
Gels
Antibodies
Cytokine Receptors
Electrospray Ionization Mass Spectrometry
Chemokine Receptors
Anterior Chamber
Transferrin
Tandem Mass Spectrometry
Liquid Chromatography
Proteomics
Trypsin
Cataract
Immunoglobulin A
Electrophoresis
Albumins
Immunity

ASJC Scopus subject areas

  • Medicine(all)
  • Ophthalmology
  • Cellular and Molecular Neuroscience
  • Sensory Systems

Cite this

Proteome analysis of human aqueous humor. / Chowdhury, Uttio Roy; Madden, Benjamin J.; Charlesworth, Mary Christine; Fautsch, Michael P.

In: Investigative Ophthalmology and Visual Science, Vol. 51, No. 10, 10.2010, p. 4921-4931.

Research output: Contribution to journalArticle

Chowdhury, Uttio Roy ; Madden, Benjamin J. ; Charlesworth, Mary Christine ; Fautsch, Michael P. / Proteome analysis of human aqueous humor. In: Investigative Ophthalmology and Visual Science. 2010 ; Vol. 51, No. 10. pp. 4921-4931.
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N2 - PURPOSE. Human aqueous humor (hAH) provides nutrition and immunity within the anterior chamber of the eye. Characterization of the protein composition of hAH will identify molecules involved in maintaining a homeostatic environment for anterior segment tissues. The present study was conducted to analyze the proteome of hAH. METHODS. hAH samples obtained during elective cataract surgery were divided into three matched groups and immunodepleted of albumin, IgG, IgA, haploglobin, antitrypsin, and transferrin. Reduced and denatured proteins (20 μg) from each group were separated by gel electrophoresis. Thirty-three gel slices were excised from each of three gel lanes (n = 99), digested with trypsin, and subjected to nanoflow liquid chromatography electrospray ionization tandem mass spectrometry (nano-LC-ESI-MS/MS). The protein component of hAH was also analyzed by antibody-based protein arrays, and selected proteins were quantified. RESULTS. A total of 676 proteins were identified in hAH. Of the 355 proteins identified by nano-LC-ESI-MS/MS, 206 were found in all three groups. Most of the proteins identified by nano-LCESI-MS/MS had catalytic, enzymatic, and structural properties. Using antibody-based protein arrays, 328 cytokines, chemokines, and receptors were identified. Most of the quantified proteins had concentrations that ranged between 0.1 and 2.5 ng/mL. Ten proteins were identified by both nano-LC-ESIMS/ MS and antibody protein arrays. CONCLUSIONS. Proteomic analysis of hAH identified 676 nonredundant proteins. More than 80% of these proteins are novel identifications. The elucidation of the aqueous proteome will establish a foundation for protein function analysis and identification of differentially expressed markers associated with diseases of the anterior segment.

AB - PURPOSE. Human aqueous humor (hAH) provides nutrition and immunity within the anterior chamber of the eye. Characterization of the protein composition of hAH will identify molecules involved in maintaining a homeostatic environment for anterior segment tissues. The present study was conducted to analyze the proteome of hAH. METHODS. hAH samples obtained during elective cataract surgery were divided into three matched groups and immunodepleted of albumin, IgG, IgA, haploglobin, antitrypsin, and transferrin. Reduced and denatured proteins (20 μg) from each group were separated by gel electrophoresis. Thirty-three gel slices were excised from each of three gel lanes (n = 99), digested with trypsin, and subjected to nanoflow liquid chromatography electrospray ionization tandem mass spectrometry (nano-LC-ESI-MS/MS). The protein component of hAH was also analyzed by antibody-based protein arrays, and selected proteins were quantified. RESULTS. A total of 676 proteins were identified in hAH. Of the 355 proteins identified by nano-LC-ESI-MS/MS, 206 were found in all three groups. Most of the proteins identified by nano-LCESI-MS/MS had catalytic, enzymatic, and structural properties. Using antibody-based protein arrays, 328 cytokines, chemokines, and receptors were identified. Most of the quantified proteins had concentrations that ranged between 0.1 and 2.5 ng/mL. Ten proteins were identified by both nano-LC-ESIMS/ MS and antibody protein arrays. CONCLUSIONS. Proteomic analysis of hAH identified 676 nonredundant proteins. More than 80% of these proteins are novel identifications. The elucidation of the aqueous proteome will establish a foundation for protein function analysis and identification of differentially expressed markers associated with diseases of the anterior segment.

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