Extensive deuterium back-exchange in certain immobilized pepsin columns used for H/D exchange mass spectrometry
Extensive deuterium back-exchange in certain immobilized pepsin columns used for H/D exchange mass spectrometry
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DOI：
10.1021/ac0518497
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发表时间：
2006-03-01
影响因子：
7.4
通讯作者：
Engen, JR
中科院分区：
文献类型：
--
作者：
Wu, Y;Kaveti, S;Engen, JR
在质量分析之前的胃蛋白酶消化增加了氢交换质谱实验的空间分辨率。使用固定化胃蛋白酶的在线消化是有利的，原因有几个，包括更好的消化效率。我们已经发现，某些固定化胃蛋白酶柱引起大量的氘反交换，使数据不可用。当将固定在波罗斯支持物上的胃蛋白酶用于在线消化时，反交换在预期范围内，并且类似于通过溶液中胃蛋白酶消化产生的氘代肽的反交换。然而，当胃蛋白酶固定到选定的聚苯乙烯-二乙烯基苯支持用于在线消化相同的系统，氘损失是非常高的。该效应似乎与用于缀合胃蛋白酶的固体支持物的性质有关。
Pepsin digestion prior to mass analysis increases the spatial resolution of hydrogen exchange mass spectrometry experiments. Online digestion with immobilized pepsin is advantageous for several reasons including better digestion efficiency. We have found that certain immobilized pepsin columns cause substantial deuterium back-exchange, rendering the data unusable. When pepsin immobilized on a POROS support was used for online digestion, back-exchange was within the expected range and was similar to the back-exchange of deuterated peptides produced by in-solution pepsin digestion. However, when pepsin immobilized onto selected polystyrene-divinylbenzene supports was used for online digestion with the same system, deuterium loss was extremely high. The effect seems linked to the properties of the solid support used to conjugate the pepsin.