Biomolecular Mass Spectrometry and Proteomics, Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands; Netherlands Proteomics Center, Padualaan 8, 3584 CH Utrecht, The Netherlands.
J Proteomics. 2013 Oct 8;91:331-7. doi: 10.1016/j.jprot.2013.07.024. Epub 2013 Aug 3.
Analysis of tyrosine (Tyr) phosphorylation by mass spectrometry (MS)-based proteomics remains challenging, due to the low occurrence of this post-translational modification compared to serine and threonine phosphorylation events in mammalian systems. Conventional metal-based affinity chromatography methods used to enrich phosphopeptides can nowadays isolate over 10,000 phosphopeptides. However, these approaches are not particularly suitable for the selective enrichment of low abundant Tyr phosphorylated peptides as the higher abundant co-enriched serine (Ser) and threonine (Thr) phosphorylated peptides typically obscure their detection. Therefore, a more targeted approach based on immuno-affinity precipitation at the peptide level has been introduced for the specific analysis of Tyr phosphorylated species. This method typically leads to the detection of a few hundreds of phosphopeptides, albeit typically over 70% of those are Tyr phosphorylated. Here, we evaluated and compared phosphotyrosine peptides enriched by a phospho-Tyr immuno-affinity enrichment (employing pY99 antibodies) and a multidimensional approach consisting of metal-affinity based enrichment (Ti(4+)-IMAC) followed by hydrophilic interaction liquid chromatography (HILIC) fractionation. Our aim was to assess differences and similarities in the set of Tyr phosphorylated peptides detected by each approach. Our data suggest that both strategies are not redundant but complementary and should ideally be combined for a more comprehensive view at phosphotyrosine signaling.
Here we evaluated enabling tools for the global analysis of phosphotyrosine phosphorylation. Phosphotyrosine phosphorylation is a key protein modification driving cellular response also involved in disease/cancer molecular pathways.
由于与哺乳动物系统中丝氨酸和苏氨酸磷酸化事件相比，酪氨酸（Tyr）磷酸化的这种翻译后修饰发生的频率较低，因此基于质谱（MS）的蛋白质组学分析酪氨酸磷酸化仍然具有挑战性。如今，用于富集磷酸肽的传统基于金属的亲和色谱方法可以分离出超过 10000 种磷酸肽。然而，这些方法并不特别适合选择性富集低丰度 Tyr 磷酸化肽，因为较高丰度的共富集丝氨酸（Ser）和苏氨酸（Thr）磷酸化肽通常会掩盖它们的检测。因此，已经引入了一种基于肽水平的免疫亲和沉淀的更具针对性的方法，用于 Tyr 磷酸化物种的特异性分析。该方法通常可检测到几百种磷酸肽，尽管其中超过 70%的磷酸肽是 Tyr 磷酸化的。在这里，我们评估并比较了通过磷酸化 Tyr 免疫亲和富集（使用 pY99 抗体）和多维方法（包括基于金属亲和的富集（Ti(4+)-IMAC）和随后的亲水相互作用液相色谱（HILIC）分级）富集的磷酸酪氨酸肽。我们的目的是评估每种方法检测到的 Tyr 磷酸化肽之间的差异和相似之处。我们的数据表明，这两种策略不是冗余的，而是互补的，理想情况下应结合使用，以更全面地了解磷酸酪氨酸信号。
在这里，我们评估了用于全面分析磷酸酪氨酸磷酸化的工具。磷酸酪氨酸磷酸化是一种关键的蛋白质修饰，可驱动细胞反应，也参与疾病/癌症分子途径。
