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 MicroHPLC interfaced with a microfraction collector. MS and MS/MS mass spectra were performed using a MALDI TOF/TOF 4800 Applied Biosystem spectrometer. Protein Pilot(TM) software with Paragon(TM) algorithm v4.1.46 or GPS(TM) with Mascot engine was used as search engines with SwissProt 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 1D 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. The tear film is a highly complex entity that contains hundreds proteins many of which are bioactive even in trace amounts [1, 2]. Several studies focused on quantification of certain target proteins and investigated the relationship between normal subjects and patients with different ocular diseases [3]. Proteomic analysis of tear fluids revealed that the composition of tear proteins is correlated with certain conditions such as dry eye [4, 5]. Isobaric tag for relative and absolute quantitation (iTRAQ) quantitative proteomics has been successfully used for the identification of tear fluid biomarkers in dry eye syndrome [5, 6], in climatic droplet keratopathy [7] and in glaucoma [8]. Vernal keratoconjunctivitis (VKC) is a severe ocular allergic disease that occurs predominately in children and young adults [9]. It is characterized by intense ocular itching, photophobia, foreign body sensation, conjunctival hyperemia, and mucous secretion, typically accompanied by giant papillae formation on the upper tarsal conjunctiva and/or by limbal infiltrates and nodules. Corneal involvement is common, characterized by punctate keratitis or corneal ulcers, as a result of the epitheliotoxic effects of proteins and enzymes released by activated eosinophils. The disease is accompanied by tissue remodeling, neovascularization, and scarring [9]. To date, no specific laboratory test is suitable for VKC diagnosis and monitoring. Tear levels of inflammatory mediators have been extensively used in ocular allergy to find either a 'disease biomarker', to better understand the immune mechanisms involved, or to identify potential therapeutic targets [10, 11]. In this study, we analyzed quantitative and qualitative proteomic profiles in human tears of healthy subjects and VKC patients, using the 4-plex iTRAQ labeling [12] analysis to compare qualitative and quantitative peptide profiles of normal subjects to those of VKC patients and to identify potential biomarkers for disease diagnosis. Subsequently, using the 8-plex iTRAQ labeling, we monitored the effect of topical treatments on quantitative peptide profiles. Differently from previous studies, our analyses were performed on single and not pooled tear samples.
Identification of human tear fluid biomarkers in vernal keratoconjunctivitis using iTRAQ quantitative proteomics
A Palmigiano;A Messina;D Garozzo
2014
Abstract
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 MicroHPLC interfaced with a microfraction collector. MS and MS/MS mass spectra were performed using a MALDI TOF/TOF 4800 Applied Biosystem spectrometer. Protein Pilot(TM) software with Paragon(TM) algorithm v4.1.46 or GPS(TM) with Mascot engine was used as search engines with SwissProt 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 1D 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. The tear film is a highly complex entity that contains hundreds proteins many of which are bioactive even in trace amounts [1, 2]. Several studies focused on quantification of certain target proteins and investigated the relationship between normal subjects and patients with different ocular diseases [3]. Proteomic analysis of tear fluids revealed that the composition of tear proteins is correlated with certain conditions such as dry eye [4, 5]. Isobaric tag for relative and absolute quantitation (iTRAQ) quantitative proteomics has been successfully used for the identification of tear fluid biomarkers in dry eye syndrome [5, 6], in climatic droplet keratopathy [7] and in glaucoma [8]. Vernal keratoconjunctivitis (VKC) is a severe ocular allergic disease that occurs predominately in children and young adults [9]. It is characterized by intense ocular itching, photophobia, foreign body sensation, conjunctival hyperemia, and mucous secretion, typically accompanied by giant papillae formation on the upper tarsal conjunctiva and/or by limbal infiltrates and nodules. Corneal involvement is common, characterized by punctate keratitis or corneal ulcers, as a result of the epitheliotoxic effects of proteins and enzymes released by activated eosinophils. The disease is accompanied by tissue remodeling, neovascularization, and scarring [9]. To date, no specific laboratory test is suitable for VKC diagnosis and monitoring. Tear levels of inflammatory mediators have been extensively used in ocular allergy to find either a 'disease biomarker', to better understand the immune mechanisms involved, or to identify potential therapeutic targets [10, 11]. In this study, we analyzed quantitative and qualitative proteomic profiles in human tears of healthy subjects and VKC patients, using the 4-plex iTRAQ labeling [12] analysis to compare qualitative and quantitative peptide profiles of normal subjects to those of VKC patients and to identify potential biomarkers for disease diagnosis. Subsequently, using the 8-plex iTRAQ labeling, we monitored the effect of topical treatments on quantitative peptide profiles. Differently from previous studies, our analyses were performed on single and not pooled tear samples.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
