From the ‡Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute of Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.
From the ‡Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute of Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands
Mol Cell Proteomics. 2018 May;17(5):1028-1034. doi: 10.1074/mcp.TIR117.000518. Epub 2018 Feb 15.
Here we demonstrate that biomolecular contaminants, such as nucleic acid molecules, can seriously interfere with immobilized metal ion affinity chromatography (IMAC)-based phosphopeptide enrichments. We address and largely solve this issue, developing a robust protocol implementing methanol/chloroform protein precipitation and enzymatic digestion using benzonase, which degrades all forms of DNA and RNA, before IMAC-column loading. This simple procedure resulted in a drastic increase of enrichment sensitivity, enabling the identification of around 17,000 unique phosphopeptides and 12,500 unambiguously localized phosphosites in human cell-lines from a single LC-MS/MS run, constituting a 50% increase when compared with the standard protocol. The improved protocol was also applied to bacterial samples, increasing the number of identified bacterial phosphopeptides even more strikingly, by a factor 10, when compared with the standard protocol. For we detected around 1300 unambiguously localized phosphosites per LC-MS/MS run. The preparation of these ultra-pure phosphopeptide samples only requires marginal extra costs and sample preparation time and should thus be adoptable by every laboratory active in the field of phosphoproteomics.
在这里，我们证明生物分子污染物，如核酸分子，可以严重干扰基于固定化金属离子亲和层析（IMAC）的磷酸肽富集。我们解决了这个问题，并开发了一种稳健的方案，在 IMAC 柱加载之前，采用甲醇/氯仿蛋白质沉淀和苯甲酸钠酶解，有效地降解所有形式的 DNA 和 RNA。这种简单的方法大大提高了富集的灵敏度，使得在单个 LC-MS/MS 运行中，能够鉴定出人源细胞系中的约 17000 个独特的磷酸肽和 12500 个明确定位的磷酸化位点，与标准方案相比增加了 50%。改进后的方案也应用于细菌样本，与标准方案相比，鉴定的细菌磷酸肽数量增加了 10 倍。对于 ，我们检测到每个 LC-MS/MS 运行中约 1300 个明确定位的磷酸化位点。这些超纯磷酸肽样品的制备仅需要少量额外的成本和样品制备时间，因此应该被磷酸蛋白质组学领域的每个实验室采用。
