Label-Free Quantitative Proteomics Reveals Differentially Regulated Proteins in Experimental Gingivitis

We investigated the sequential protein expression in gingival crevicular fluid samples during the induction (I) and resolution (R) of experimental gingivitis. Periodontally and systemically healthy volunteers (n = 20) participated in a three-week experimental gingivitis protocol, followed by debridement and two weeks of regular plaque control. Gingival crevicular fluid (GCF) samples were collected at baseline, Day 7, 14, and 21 (induction; I-phase), and at Day 21, 25, 30, and 35 (resolution; R-phase). Liquid chromatography–tandem mass spectrometry (LC–MS/MS) for label-free quantitative proteomics was applied. A total of 287 proteins were identified including 254 human, 14 bacterial, 12 fungal, and 7 yeast proteins. Ontology analysis revealed proteins primarily involved in cytoskeletal rearrangements, immune response, antimicrobial function, protein degradation, and DNA binding. There was considerable variation in the number of proteins identified, both among subjects and within subjects across time points. After pooling of samples between subjects at each time point, the levels of 59 proteins in the I-phase and 73 proteins in the R-phase were quantified longitudinally. Our data demonstrate that LC–MS/MS label-free quantitative proteomics is valuable in the assessment of the protein content of the GCF and can facilitate a better understanding of the molecular mechanisms involved in the induction and resolution of plaque-induced gingival inflammation in humans.本研究探究了实验性牙龈炎诱导期（I期）与消退期（R期）龈沟液样本中的时序蛋白质表达情况。招募20名牙周及全身健康的志愿者参与为期三周的实验性牙龈炎造模方案，随后接受牙周清创术并进行为期两周的常规菌斑控制。分别在基线、第7、14、21天（诱导期，即I期）以及第21、25、30、35天（消退期，即R期）采集龈沟液（Gingival Crevicular Fluid, GCF）样本。采用液相色谱-串联质谱（Liquid Chromatography–Tandem Mass Spectrometry, LC-MS/MS）进行无标记定量蛋白质组学分析。共鉴定出287种蛋白质，其中包括254种人类蛋白质、14种细菌蛋白质、12种真菌蛋白质以及7种酵母菌蛋白质。本体分析结果显示，这些蛋白质主要参与细胞骨架重排、免疫应答、抗菌功能、蛋白质降解以及DNA结合等生物学过程。不同受试者之间以及同一受试者在不同时间点的鉴定蛋白质数量均存在显著差异。在将每个时间点的受试者样本合并后，对I期的59种蛋白质以及R期的73种蛋白质进行了纵向定量分析。本研究结果表明，LC-MS/MS无标记定量蛋白质组学技术可有效用于龈沟液蛋白质组分的分析，有助于更深入地理解人类菌斑诱导性牙龈炎的发生与消退相关分子机制。