Characterization of the Human Submandibular/Sublingual Saliva Glycoproteome Using Lectin Affinity Chromatography Coupled to Multidimensional Protein Identification Technology

In-depth analysis of the salivary proteome is fundamental to understanding the functions of salivary proteins in the oral cavity and to reveal disease biomarkers involved in different pathophysiological conditions, with the ultimate goal of improving patient diagnosis and prognosis. Submandibular and sublingual glands contribute saliva rich in glycoproteins to the total saliva output, making them valuable sources for glycoproteomic analysis. Lectin-affinity chromatography coupled to mass spectrometry-based shotgun proteomics was used to explore the submandibular/sublingual (SM/SL) saliva glycoproteome. A total of 262 N- and O-linked glycoproteins were identified by multidimensional protein identification technology (MudPIT). Only 38 were previously described in SM and SL salivas from the human salivary N-linked glycoproteome, while 224 were unique. Further comparison analysis with SM/SL saliva of the human saliva proteome, revealed 125 glycoproteins not formerly reported in this secretion. KEGG pathway analyses demonstrated that many of these glycoproteins are involved in processes such as complement and coagulation cascades, cell communication, glycosphingolipid biosynthesis neo-lactoseries, O-glycan biosynthesis, glycan structures-biosynthesis 2, starch and sucrose metabolism, peptidoglycan biosynthesis or others pathways. In summary, lectin-affinity chromatography coupled to MudPIT mass spectrometry identified many novel glycoproteins in SM/SL saliva. These new additions to the salivary proteome may prove to be a critical step for providing reliable biomarkers in the diagnosis of a myriad of oral and systemic diseases.

对唾液蛋白组的深入分析对于理解口腔中唾液蛋白的功能、揭示涉及不同病理生理条件的相关疾病生物标志物至关重要，其最终目标在于提升患者的诊断和预后水平。下颌下腺和舌下腺向总唾液输出贡献了富含糖蛋白的唾液，使其成为糖蛋白组分析中的宝贵来源。通过结合质谱分析技术的糖蛋白亲和层析法（Lectin-affinity chromatography coupled to shotgun proteomics based on mass spectrometry）对下颌下腺/舌下腺（SM/SL）唾液糖蛋白组进行了探索。利用多维度蛋白质鉴定技术（MudPIT）共鉴定出262个N型和O型链接的糖蛋白，其中仅有38个在人类唾液N型链接糖蛋白组中已有描述，而224个为独特发现。进一步与人类唾液蛋白组的SM/SL唾液进行比较分析，揭示了125个在此分泌物中未曾报道的糖蛋白。KEGG通路分析表明，许多这些糖蛋白参与如补体和凝血级联反应、细胞通讯、神经节苷脂生物合成新乳糖系列、O-聚糖生物合成、聚糖结构-生物合成2、淀粉和蔗糖代谢、肽聚糖生物合成或其他途径等过程。总之，结合糖蛋白亲和层析法和MudPIT质谱分析识别了SM/SL唾液中的许多新型糖蛋白。这些新增的唾液蛋白组成分可能成为提供多种口腔和系统性疾病可靠生物标志物诊断的关键步骤。