Abstract：
Assessment of craniofacial skeletal maturity is of great importance in orthodontic diagnosis and treatment planning. Traditional radiographic methods suffer from clinician subjectivity and low reproducibility. Recent biochemical methods, such as the use of gingival crevicular fluid (GCF) protein biomarkers involved in bone metabolism, have provided new opportunities to assess skeletal maturity. However, mass spectrometry (MS)-based GCF proteomic analysis still faces significant challenges, including the interference of high abundance proteins, laborious sample prefractionation and relatively limited coverage of GCF proteome. To improve GCF sample processing and further discover novel biomarkers, we herein developed a single-pot, solid-phase-enhanced sample-preparation (SP3)-based high-field asymmetric waveform ion mobility spectrometry (FAIMS)-MS protocol for deep quantitative analysis of the GCF proteome for skeletal maturity indicators. SP3 combined with FAIMS could minimize sample loss and eliminate tedious and time-consuming offline fractionation, thereby simplifying GCF sample preparation and improving analytical coverage and reproducibility of the GCF proteome. A total of 5407 proteins were identified in GCF samples from prepubertal and circumpubertal groups, representing the largest dataset of human GCF proteome to date. Compared to the prepubertal group, 61 proteins were differentially expressed (31 up-regulated, 30 down-regulated) in the circumpubertal group. The six-protein marker panel, including ATP5D, CLTA, CLTB, DNM2, HSPA8 and NCK1, showed great potential to predict the circumpubertal stage (ROC-AUC 0.937), which provided new insights into skeletal maturity assessment.
摘要：
颅面骨骼成熟度评估在正畸诊断和治疗计划中非常重要。传统的射线照相方法存在临床医生主观性和低再现性的问题。最近的生化方法，例如使用参与骨代谢的龈沟液（GCF）蛋白生物标志物，为评估骨骼成熟度提供了新的机会。然而,基于质谱（MS）的GCF蛋白质组学分析仍然面临重大挑战，包括高丰度蛋白质的干扰，费力的样品分割和GCF蛋白质组的相对有限的覆盖范围。为了改进GCF样品处理并进一步发现新的生物标志物，我们在这里开发了一个单罐，基于固相增强样品制备（SP3）的高场非对称波形离子迁移谱（FAIMS）-MS协议，用于对GCF蛋白质组进行骨骼成熟度指标的深度定量分析。SP3结合FAIMS可以最大限度地减少样品损失，消除繁琐和耗时的离线分馏，从而简化GCF样品制备并提高GCF蛋白质组的分析覆盖率和再现性。在青春期前和青春期周围组的GCF样本中总共鉴定出5407种蛋白质，代表迄今为止最大的人类GCF蛋白质组数据集。与青春期前组相比，61种蛋白质差异表达（31种上调，30下调）在青春期周游组中。六蛋白标记组，包括ATP5D,CLTA,CLTB,DNM2，HSPA8和NCK1，显示出预测生殖青春期阶段的巨大潜力（ROC-AUC0.937），这为骨骼成熟度评估提供了新的见解。
