Microsequence analysis of peptides and proteins. VI. A continuous flow reactor for sample concentration and sequence analysis.
Microsequence analysis of peptides and proteins. VI. A continuous flow reactor for sample concentration and sequence analysis.
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肽和蛋白质的微序列分析。
DOI：
10.1016/0003-2697(87)90257-0
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发表时间：
1987
影响因子：
2.9
通讯作者：
Ronk,M
中科院分区：
文献类型：
--
作者：
Shively,JE;Miller,P;Ronk,M
我们设计并测试了用于肽和蛋白质微序列分析的连续流动反应器 (CFR)。 CFR 形成肽或蛋白质底物固定和自动化 Edman 化学的位点。 CFR 由 0.125 英寸外径、0.0625 英寸内径构成。聚四氟乙烯管（长度 2-3 厘米）含有 5-10 毫克聚凝胺涂层球形多孔二氧化硅（粒径 100-200 微米）。二氧化硅被保留在 CFR 中，并在床底部和任选的床顶部设有多孔聚四氟乙烯过滤器 (Zitex)。身份证号当 0.0625 英寸外径时，选择 CFR 的紧密压配合。 Teflon 线插入 CFR 的顶部和底部。这种设计允许用 CFR 替换传统微测序仪中现有的盒/玻璃纤维盘，只需进行最少的更改。与之前的设计相比，CFR 的优点包括背景或噪音水平较低，并且在样品应用之前无需预循环 Polybrene，而且整个装置价格低廉，因此是一次性的。我们认为，噪声的减少，尤其是常见的二苯基硫脲峰的减少，是由于更直接的流路和 CFR 中未扫掠区域的相对缺乏。在 CFR 中对几种标准肽和蛋白质进行了测序，以证明改进的结果。与墨盒/玻璃纤维盘设计的直接比较表明，CFR 的背景更少，初始产量和重复产量更高。另一个优点是能够将样品直接浓缩在含有反相填料的 CFR 上。我们已成功将 1.0 ml 样品 (200 pmol) 浓缩到 5 mg 辛基癸基甲硅烷基衍生二氧化硅上，收率达到 95–100%。将 Polybrene 涂层二氧化硅添加到 CFR 后，对所得样品进行微测序，具有较高的初始产率和重复产率。该方法有望改善低皮摩尔量肽和蛋白质的样品处理和微序列分析。
We have designed and tested a continuous flow reactor (CFR) for microsequence analysis of peptides and proteins. The CFR forms the site for immobilization of the peptide or protein substrate and automated Edman chemistry. The CFR was constructed from 0.125-in.-o.d., 0.0625-in.-i.d. Teflon tubing (length 2–3 cm) containing 5–10 mg of Polybrene-coated, spherical, porous silica (100–200-μm particle size). The silica is retained in the CFR with porous Teflon filters (Zitex) at the bed bottom and optionally at the bed top. The i.d. of the CFR was selected for a tight press fit when 0.0625-in.-o.d. Teflon lines are inserted at the top and bottom of the CFR. This design allows the replacement of the existing cartridge/glass fiber disk found in conventional microsequencers with a CFR with a minimal amount of changes. The advantages of the CFR over the previous design include a lower background or noise level and no need to precycle Polybrene before sample application, and the entire unit is inexpensive and therefore disposable. We believe that the decrease in noise, especially the decrease in the commonly observed diphenylthiourea peak, is due to the more direct flow path and relative absence of unswept area in the CFR. Several standard peptides and proteins were sequenced in the CFR to demonstrate the improved results. A direct comparison to the cartridge/glass fiber disk design demonstrated less background and higher initial and repetitive yields for the CFR. An additional advantage is the ability to directly concentrate samples on CFRs containing reverse-phase packing. We have successfully concentrated 1.0-ml samples (200 pmol) onto 5 mg of octyldecylsilyl-derivatized silica in yields of 95–100%. The resulting samples were microsequenced after addition of Polybrene-coated silica to the CFR with high initial and repetitive yields. This methodology promises to improve sample handling and microsequence analysis of low picomole amounts of peptides and proteins.