Ophthalmology Unit, Department of Neuroscience, University of Padua, Padua, Italy.
Allergy. 2014 Feb;69(2):254-60. doi: 10.1111/all.12331. Epub 2013 Dec 13.
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.
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™ software with Paragon™ algorithm v4.1.46 or GPS™ with Mascot engine was used as search engines with SwissProt or IPI human as the databases.
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.
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.
由于发病机制尚不清楚，且抗过敏治疗通常无效，因此理解和治疗春季角结膜炎（VKC）一直具有挑战性。本研究旨在通过质谱分析分析人泪液中的肽谱，以阐明健康受试者和 VKC 患者之间的组成差异。
收集健康受试者和 VKC 患者的泪液。用 iTRAQ（相对和绝对定量的同重同位素标记）对消化后的样本进行处理。采用与微馏分收集器接口的 MicroHPLC 对胰蛋白酶肽进行分离。使用 MALDI TOF/TOF 4800 Applied Biosystem 光谱仪进行 MS 和 MS/MS 质谱分析。使用 Protein Pilot™软件和 Paragon™算法 v4.1.46 或 GPS™与 Mascot 引擎作为搜索引擎，以 SwissProt 或 IPI 人类作为数据库。
检查了大量的肽，成功鉴定了 78 种蛋白质。在所有 VKC 样本中，血清白蛋白、转铁蛋白和血红素结合蛋白的水平均比对照泪液水平高 100 倍，且与疾病的严重程度相关。与对照样本相比，VKC 样本中血红素结合蛋白、转铁蛋白、乳球蛋白 B 和分泌球蛋白 1D 的表达明显上调。与局部环孢素或皮质类固醇治疗的患者的泪液样本相比，这些蛋白水平显著降低。
LC MALDI MS 和同重同位素标记相对和绝对定量技术可用于人泪液肽组的定量和定性特征分析。这些技术可能会确定用于诊断和治疗 VKC 及其他炎症性眼表面疾病的靶蛋白。
