Tear Protein Biomarkers for Ocular Mucous Membrane Pemphigoid Uncovered Using Targeted LC-MS/MS

Mucous membrane pemphigoid (MMP) is a multisystemic rare autoimmune disease affecting the skin and mucous membranes. Ocular involvement is characterized by chronic conjunctival inflammation causing scar formation, leading to corneal opacification and vision loss. Conjunctival biopsies are currently used to confirm diagnosis, and the associated immunosuppression treatments prescribed can have serious consequences on patients. To address these challenges, a noninvasive approach to collect patient tears using untargeted and targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses has been developed to identify a list of potential biomarkers of ocular MMP. Samples were collected on Schirmer strips and subjected to tryptic digestion and LC-MS/MS analysis. Three cohorts of patients were studied, and targeted LC-scheduled multiple reaction monitoring (LC-sMRM) methods were developed for the verification of putative biomarkers. The comparison of three groups of patients, those diagnosed with ocular MMP, non-ocular MMP, and lichen planus, another ocular cicatricial conjunctivitis disorder, yielded 56 biomarkers of interest. Proteins distinguishing MMP patients with and without ocular involvement were linked to the extracellular matrix, metabolism, and neutrophil degranulation. The comparison between MMP and lichen planus patients highlighted lower levels of metabolic enzymes in the latter. This study highlights the use of multiple patient cohorts and tailored targeted quantitative proteomics methods for the discovery of biomarkers for diagnostic and prognostic purposes.黏膜类天疱疮（Mucous membrane pemphigoid, MMP）是一种累及皮肤与黏膜的多系统罕见自身免疫疾病。眼部受累以慢性结膜炎性反应伴瘢痕形成为主要特征，可进一步引发角膜混浊与视力丧失。当前临床主要通过结膜活检确诊该病，而所采用的免疫抑制治疗方案可能对患者造成严重不良反应。为解决上述临床痛点，本研究开发了一种非侵入性泪液采集方法，结合非靶向与靶向液相色谱-串联质谱（LC-MS/MS）分析，以筛选眼部MMP的潜在生物标志物。研究使用施墨试纸条（Schirmer strips）采集泪液样本，经胰蛋白酶消化后开展LC-MS/MS分析。本研究共纳入三队列患者群体，并开发了靶向液相色谱- scheduled多反应监测（LC-sMRM）方法用于候选生物标志物的验证。通过对比眼部MMP患者、非眼部MMP患者以及另一种眼部瘢痕性结膜炎疾病——扁平苔藓（lichen planus）患者三组人群，最终筛选得到56个具有研究价值的生物标志物。区分伴与不伴眼部受累的MMP患者的差异蛋白主要与细胞外基质、代谢过程及中性粒细胞脱颗粒相关。对比MMP与扁平苔藓患者的蛋白表达谱发现，后者的代谢酶水平显著更低。本研究表明，多队列患者联合定制化靶向定量蛋白质组学方法，可有效挖掘用于疾病诊断与预后评估的生物标志物。