Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan; Proteome Homeostasis Research Unit, RIKEN Center for Integrative Medical Sciences, Yokohama, Kanagawa, Japan.
Mol Cell Proteomics. 2023 May;22(5):100535. doi: 10.1016/j.mcpro.2023.100535. Epub 2023 Mar 22.
We have successfully developed a bioinertized nanoflow LC/MS/MS (nanoLC/MS/MS) system for the highly sensitive analysis of phosphopeptides by depleting metal ions from the mobile phase. We found that not only direct contact of phosphopeptides with metal components, but also indirect contact with nanoLC pumps through the mobile phase causes significant losses during the recovery of phosphopeptides. Moreover, electrospray ionization was adversely affected by the mobile phase containing multiple metal ions as well as by the sample solvents contaminated with metal ions used in immobilized metal ion affinity chromatography for phosphopeptide enrichment. To solve these problems, metal ions were depleted by inserting an online metal ion removal device containing metal-chelating membranes between the gradient mixer and the autosampler. As a result, the peak areas of the identified phosphopeptides increased an average of 9.9-fold overall and 77-fold for multiply phosphorylated peptides with the insertion of the online metal ion removal system. This strategy would be applicable to the highly sensitive analysis of other phosphorylated biomolecules by microscale-LC/MS/MS.
我们成功开发了一种基于纳米液相色谱/串联质谱（nanoLC/MS/MS）的生物惰性化系统，可通过从流动相中去除金属离子来实现对磷酸肽的高灵敏度分析。我们发现，不仅磷酸肽与金属成分的直接接触，而且通过流动相与 nanoLC 泵的间接接触，都会导致在回收磷酸肽时发生显著损失。此外，含有多种金属离子的流动相以及用于磷酸肽富集的固定化金属离子亲和色谱的样品溶剂中的金属离子会对电喷雾电离产生不利影响。为了解决这些问题，我们在梯度混合器和自动进样器之间插入了一个在线金属离子去除装置，其中包含金属螯合膜，以去除金属离子。结果，插入在线金属离子去除系统后，鉴定出的磷酸肽的峰面积总体平均增加了 9.9 倍，对于多磷酸化肽则增加了 77 倍。该策略将适用于通过微尺度 LC/MS/MS 对其他磷酸化生物分子进行高灵敏度分析。
