Niobium(V) Oxide (Nb₂O₅): Application to Phosphoproteomics

Proteomics-based analysis of signaling cascades relies on a growing suite of affinity resins and methods aimed at efficient enrichment of phosphorylated peptides from complex biological mixtures. Given the heterogeneity of phosphopeptides and the overlap in chemical properties between phospho- and unmodified peptides, it is likely that the use of multiple resins will provide the best combination of specificity, yield, and coverage for large-scale proteomics studies. Recently titanium and zirconium dioxides have been used successfully for enrichment of phosphopeptides. Here we report the first demonstration that niobium pentoxide (Nb2O5) provides for efficient enrichment and recovery (∼50−100%) of phosphopeptides from simple mixtures and facilitates identification of several hundred putative sites of phosphorylation from cell lysate. Comparison of phosphorylated peptides identified from Nb2O5 and TiO2 with sequences in the PhosphoELM database suggests a useful degree of divergence in the selectivity of these metal oxide resins. Collectively our data indicate that Nb2O5 provides efficient enrichment for phosphopeptides and offers a complementary approach for large-scale phosphoproteomics studies.信号级联反应的蛋白质组学分析，依赖于日益丰富的亲和树脂及相关方法，旨在从复杂生物基质中高效富集磷酸化肽段。鉴于磷酸化肽段的异质性，以及磷酸化肽与未修饰肽之间化学性质的重叠性，采用多种亲和树脂联用，有望为大规模蛋白质组学研究实现特异性、回收率与覆盖度的最优组合。此前，二氧化钛与二氧化锆已被成功应用于磷酸化肽段的富集流程。本研究首次证实，五氧化二铌（niobium pentoxide，Nb₂O₅）可从简单混合物中高效富集并回收磷酸化肽段（回收率约50%~100%），并能从细胞裂解液中鉴定出数百个潜在磷酸化位点。将五氧化二铌与二氧化钛（TiO₂）所鉴定得到的磷酸化肽段，与磷酸化位点数据库（PhosphoELM）中的序列进行比对后发现，这两类金属氧化物树脂的选择性存在具有应用价值的差异。综合本研究的全部数据可知，五氧化二铌可实现磷酸化肽段的高效富集，为大规模磷酸化蛋白质组学研究提供了一种互补的技术方案。