Harman Jarrod C, Guidry Jessie J, Gidday Jeffrey M
Department of Ophthalmology, Louisiana State University Health Science Center (LSUHSC), New Orleans, LA.
Department of Physiology, LSUHSC, New Orleans, LA.
Mol Vis. 2018 Dec 31;24:875-889. eCollection 2018.
Diverse groups of proteins play integral roles in both the physiology and pathophysiology of the retina. However, thorough proteomic analyses of retinas of experimental species are currently unavailable. The purpose of the present paper is providing the field with a comprehensive proteomic characterization of the retina of a commonly used laboratory mouse using a discovery-based mass spectrometry (MS) approach.
Retinas from eight male and eight female 30-week-old outbred ND4 Swiss Webster mice were harvested and immediately processed for MS analysis on a Thermo Fisher (TF) Fusion Orbitrap MS. The retinal proteome and phosphoproteome were identified and subsequently analyzed using Proteome Discoverer 2.2 and Panther-GeneGo. SEQUEST-HT scoring was used for analysis, and the reference protein FASTA database was from . Specifically, three technical repeats were performed for each biological sample. For characterization, only high-scoring peptides were considered, with a false discovery rate (FDR) of <1%. Downstream bioinformatic analysis used Ingenuity Pathway Analysis (IPA; Qiagen).
Using Proteome Discoverer 2.2, 4,767 different proteins were identified and segregated into 26 major protein classes, 9 functional molecular classes, and 12 categories of biological processes. The five largest protein classes included the following: nucleic acid binding (17%), hydrolases (13%), enzyme modulators (10%), transferases (9%), and oxidoreductases (6%). "Binding" and "catalytic" proteins contributed to 81% of the molecular function class at 37% and 42%, respectively. "Cellular processing" and "metabolic processes" contributed the most to biologic activity, at 31% and 26%, respectively. Phosphopeptide enrichment yielded the identification of 610 additional unique proteins that were not originally identified. The two datasets combined produced an adult mouse retinal proteome consisting of 5,377 unique proteins. Overall, 41% of the retinal proteome was phosphorylated. The overwhelming diversity of retinal protein functionality was reflected through further analyses revealing 2,086 unique pathway hits across 241 different pathways (TF). A core analysis summary report was performed in IPA (Qiagen) to analyze the top signaling networks, protein-protein interaction (PPI) enrichments, and canonical pathways.
Using this high-throughput technique, we have further deciphered and updated the diverse proteome of the mouse retina, including the phosphoproteome, thereby providing the most comprehensive proteomic profile for this tissue known to date. These findings, and the bioinformatic analyses we also provided, establish a platform for future studies, facilitating the elucidation of the relevance of these proteins to the molecular and cellular pathologies that underlie retinal function and disease.
多种蛋白质组在视网膜的生理和病理生理过程中发挥着不可或缺的作用。然而，目前尚无对实验物种视网膜进行全面蛋白质组分析的研究。本文旨在通过基于发现的质谱（MS）方法，为该领域提供常用实验小鼠视网膜的全面蛋白质组特征描述。
采集8只30周龄雄性和8只雌性远交系ND4瑞士韦伯斯特小鼠的视网膜，并立即在赛默飞世尔（TF）Fusion Orbitrap质谱仪上进行MS分析。利用Proteome Discoverer 2.2和Panther-GeneGo对视网膜蛋白质组和磷酸化蛋白质组进行鉴定和后续分析。采用SEQUEST-HT评分进行分析，参考蛋白质FASTA数据库来自……。具体而言，每个生物样本进行三次技术重复。为进行特征描述，仅考虑高分肽段，错误发现率（FDR）<1%。下游生物信息学分析使用 Ingenuity Pathway Analysis（IPA；Qiagen）。
使用Proteome Discoverer 2.2，共鉴定出4767种不同蛋白质，并将其分为26个主要蛋白质类别、9个功能分子类别和12类生物过程。五大蛋白质类别如下：核酸结合（17%）水解酶（13%）、酶调节剂（10%）、转移酶（9%）和氧化还原酶（6%）。“结合”和“催化”蛋白分别占分子功能类别的37%和42%，共占81%。“细胞加工”和“代谢过程”对生物活性的贡献最大，分别为31%和26%。磷酸肽富集鉴定出另外610种最初未鉴定的独特蛋白质。两个数据集合并产生了一个由5377种独特蛋白质组成的成年小鼠视网膜蛋白质组。总体而言，视网膜蛋白质组中有41%发生了磷酸化。通过进一步分析揭示了241条不同途径（TF）中的2086个独特途径命中，反映了视网膜蛋白质功能的极度多样性。在IPA（Qiagen）中进行了核心分析总结报告，以分析顶级信号网络、蛋白质-蛋白质相互作用（PPI）富集和经典途径。
使用这种高通量技术，我们进一步解读并更新了小鼠视网膜的多样化蛋白质组，包括磷酸化蛋白质组，从而提供了迄今为止该组织最全面的蛋白质组图谱。我们提供的这些发现以及生物信息学分析，为未来的研究建立了一个平台，有助于阐明这些蛋白质与视网膜功能和疾病背后的分子和细胞病理学的相关性。
