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Identification of human tear fluid biomarkers in vernal keratoconjunctivitis using iTRAQ quantitative proteomics
Author(s) -
Leonardi A.,
Palmigiano A.,
Mazzola E. A.,
Messina A.,
Milazzo E. M. S.,
Bortolotti M.,
Garozzo D.
Publication year - 2014
Publication title -
allergy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.363
H-Index - 173
eISSN - 1398-9995
pISSN - 0105-4538
DOI - 10.1111/all.12331
Subject(s) - vernal keratoconjunctivitis , hemopexin , tears , transferrin , quantitative proteomics , proteomics , albumin , medicine , proteome , mass spectrometry , tandem mass spectrometry , chemistry , chromatography , immunology , biochemistry , gene , heme , enzyme
Background Understanding and treating vernal keratoconjunctivitis ( VKC ) has been a challenge because the pathogenesis is unclear and antiallergic therapy often unsuccessful. The aim of the study was to analyze peptide profiles in human tears using mass spectrometry to elucidate compositional differences between healthy subjects and patients affected by VKC . Methods Tears were collected from healthy subjects and VKC patients. Digested samples were treated with iTRAQ (isobaric tag for relative and absolute quantitation). Separation of tryptic peptides was realized using a Micro HPLC interfaced with a microfraction collector. MS and MS / MS mass spectra were performed using a MALDI TOF / TOF 4800 A pplied B iosystem spectrometer. Protein P ilot™ software with P aragon™ algorithm v4.1.46 or GPS ™ with M ascot engine was used as search engines with S wiss P rot or IPI human as the databases. Results A significant number of peptides were examined, and 78 proteins were successfully identified. In all VKC samples, levels of serum albumin, transferrin, and hemopexin were found up to 100 times higher than control tear levels and correlated to the severity of disease. Hemopexin, transferrin, mammaglobin B , and secretoglobin 1 D were found significantly over‐expressed in VKC samples compared with the control samples. Tear samples from patients treated with topical cyclosporine or corticosteroids showed a dramatic reduction in these protein levels. Conclusions LC MALDI MS and isobaric tag for relative and absolute quantitation technique may be useful in the quantitative and qualitative characterization of the peptidoma of human tears. These techniques may identify target proteins to be used in the diagnosis and management of VKC and other inflammatory ocular surface conditions.