Standardisation of rapid in-gel digestion by mass spectrometry
Standardisation of rapid in-gel digestion by mass spectrometry
复制标题
DOI：
10.1002/pmic.200600607
复制
发表时间：
2007-03-01
期刊：
影响因子：
3.4
通讯作者：
Eichacker, Lutz Andreas
中科院分区：
文献类型：
--
作者：
Granvogl, Bernhard;Gruber, Patrick;Eichacker, Lutz Andreas
凝胶内消化已标准化使用聚乙烯（丙烯）一次性。我们设计了一个四步快速和简单的凝胶内消化方案，该方案在一个独立的反应管中进行，避免了角蛋白污染。为了量化凝胶内消化的效率，我们开发了一种快速柱上肽乙酰化方案。结果表明，胰蛋白酶在凝胶内摄取增加，凝胶内消化在15分钟内完成90%。我们进一步表明，谱质量，肽产率和序列覆盖范围的质谱分析得到了提高。我们利用大麦光系统II的2-D PAGE分离来证明该协议有助于鉴定高度疏水的膜蛋白。
in-gel digestion has been standardised using a poly(propylene) disposable. We designed a four-step rapid and simple in-gel digestion protocol which is carried out in one self-contained reaction tube avoiding keratin contamination. In order to quantify the efficiency of in-gel digestion, we developed a rapid on-column peptide acetylation protocol. Results show that trypsin in-gel uptake is increased and in-gel digestion is 90% complete within 15 min. We further show that spectrum quality, peptide yield and sequence coverage for mass spectrometric analysis are enhanced. We utilise 2-D PAGE separation of photosystem II from barley to demonstrate that the protocol facilitates identification of highly hydrophobic membrane proteins.