Hexapeptide Libraries for Enhanced Protein PTM Identification and Relative Abundance Profiling in Whole Human Saliva

Dynamic range compression (DRC) by hexapeptide libraries increases MS/MS-based identification of lower-abundance proteins in complex mixtures. However, two unanswered questions impede fully realizing DRC’s potential in shotgun proteomics. First, does DRC enhance identification of post-translationally modified proteins? Second, can DRC be incorporated into a workflow enabling relative protein abundance profiling? We sought to answer both questions analyzing human whole saliva. Addressing question one, we coupled DRC with covalent glycopeptide enrichment and MS/MS. With DRC we identified ∼2 times more N-linked glycoproteins and their glycosylation sites than without DRC, dramatically increasing the known salivary glycoprotein catalog. Addressing question two, we compared differentially stable isotope-labeled saliva samples pooled from healthy and metastatic breast cancer women using a multidimensional peptide fractionation-based workflow, analyzing in parallel one sample portion with DRC and one portion without. Our workflow categorizes proteins with higher absolute abundance, whose relative abundance ratios are altered by DRC, from proteins of lower absolute abundance detected only after DRC. Within each of these salivary protein categories, we identified novel abundance changes putatively associated with breast cancer, demonstrating feasibility and benefits of DRC for relative abundance profiling. Collectively, our results bring us closer to realizing the full potential of DRC for proteomic studies.基于六肽文库（hexapeptide libraries）的动态范围压缩（Dynamic range compression, DRC）可提升复杂混合物中低丰度蛋白质的串联质谱（MS/MS）鉴定效率。然而，仍有两个悬而未决的问题阻碍了鸟枪蛋白质组学（shotgun proteomics）中DRC应用潜力的充分释放。其一，DRC是否能够提升翻译后修饰蛋白质（post-translationally modified proteins）的鉴定效率？其二，能否将DRC整合至可实现蛋白质相对丰度定量的实验流程中？本研究以人类全唾液为研究对象，旨在解答上述两个问题。针对第一个问题，我们将DRC与共价糖肽富集（covalent glycopeptide enrichment）及MS/MS联用。相较于未使用DRC的对照组，使用DRC时我们鉴定到的N-连接糖蛋白（N-linked glycoproteins）及其糖基化位点数量提升约一倍，极大扩充了已知唾液糖蛋白数据库。针对第二个问题，我们采用基于多维肽段分级分离（multidimensional peptide fractionation）的实验流程，对比了来自健康女性与转移性乳腺癌女性的差异性稳定同位素标记（differentially stable isotope-labeled）唾液混合样本，并并行分析了其中添加DRC与未添加DRC的样本分样。本实验流程可将蛋白质划分为两类：一类为绝对丰度较高、其相对丰度比值会受DRC影响的蛋白质，另一类为仅在DRC处理后方可被检测到的低绝对丰度蛋白质。在上述两类唾液蛋白质中，我们均鉴定到了疑似与乳腺癌相关的新型丰度变化，证实了DRC用于蛋白质相对丰度定量的可行性与应用优势。综上，本研究结果进一步推动了DRC在蛋白质组学研究中应用潜力的充分发挥。