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Development and application of open tubular and open tubular capillary IMAC devices for phosphopeptide enrichment and characterization.

机译：用于磷酸肽富集和表征的开放式管状和开放式管状毛细管IMAC装置的开发和应用。

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摘要

Immobilized metal-ion affinity chromatography (IMAC) is a useful method for enrichment of phosphopeptides from enzymatic digests of proteins. Detection and identification of phosphopeptides is thereby greatly facilitated, since most non-phosphorylated peptides are eliminated from the resulting mass spectra. In this study, IMAC ligands were attached directly to the silanol groups of open glass tubing and fused silica capillaries. The resulting IMAC devices make for more effective and sensitive affinity capture than commercially available sepharose-based IMAC. The sensitivity advantage is especially pronounced for capillary-based IMAC, owing to the small elution volumes in the miniaturized IMAC device. Application of open tubular IMAC to the human Na+/H+ exchanger protein His182 allowed for mapping of 5 phosphorylation sites by MALDI-MS/MS. The miniaturized open tubular capillary IMAC has been shown to be able to detect phosphopeptides from gel-separated proteins at the sub-pmol level. Furthermore, it is also shown that following methylation of carboxylate residues in peptides, application of the negative-ion mode is an interesting way to selectively ionize phosphopeptides.
机译：固定金属离子亲和色谱法（IMAC）是从蛋白质的酶消化物中富集磷酸肽的有用方法。由于从产生的质谱图中消除了大多数非磷酸化的肽，因此大大简化了磷酸肽的检测和鉴定。在这项研究中，IMAC配体直接连接到开放式玻璃管和熔融石英毛细管的硅烷醇基上。与市售的基于琼脂糖的IMAC相比，所得的IMAC设备可实现更有效和敏感的亲和捕获。基于毛细管的IMAC的灵敏度优势尤其明显，这是因为小型化的IMAC设备的洗脱体积很小。将开放的管状IMAC应用于人类Na + / H +交换蛋白His182可以通过MALDI-MS / MS定位5个磷酸化位点。小型化的开放式管状毛细管IMAC已显示出能够以亚pmol水平检测来自凝胶分离蛋白的磷酸肽。此外，还显示出在肽中的羧酸根残基甲基化之后，负离子模式的应用是选择性离子化磷酸肽的有趣方式。

著录项

  • 作者

    Stupak, Jacek.;

  • 作者单位

    University of Alberta (Canada).;

  • 授予单位 University of Alberta (Canada).;
  • 学科 Chemistry Analytical.
  • 学位 M.Sc.
  • 年度 2005
  • 页码 111 p.
  • 总页数 111
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 化学;
  • 关键词

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