Screening combinatorial libraries for optimal enzyme substrates by mass spectrometry.
Screening combinatorial libraries for optimal enzyme substrates by mass spectrometry.
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通过质谱法筛选最佳酶底物的组合文库。
DOI：
10.1002/rcm.360
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Pei,D
中科院分区：
文献类型：
--
作者：
Wang,P;Snavley,DF;Freitas,MA;Pei,D
已经开发出一种从组合文库中快速鉴定最佳酶底物的方法。该方法通过筛选 361 个成员 N 末端甲酰化三肽库 f-XXR（X = 19 个不同氨基酸）来验证大肠杆菌肽去甲酰基酶 (PDF) 的最佳底物。该文库是通过分池合成方法在固相上合成的。在二环己基碳二亚胺存在下，用 HCO2H 和 DCO2D 的 1:1（mol/mol）混合物处理树脂，添加 N 末端甲酰基。在质谱中，文库的每个成员都产生一个双峰（间隔为 1.0063 Da）。 E 对该库的有限处理。 coliPDF 导致那些作为酶最有效底物的肽发生去甲酰化。由于质量简并甲酰基的损失，去甲酰化产物各自在质谱中产生一个单线峰。因此，PDF 产物峰很容易通过串联质谱法进行鉴定和测序。结果表明，PDF 强烈偏好正亮氨酸，并在较小程度上偏好苯丙氨酸作为 N 端残基，而它在倒数第二个位置几乎没有选择性。该结果与文献数据非常吻合，因此证明该方法是鉴定最佳酶底物的有效方法。该方法应该普遍适用于其他酶以及合成催化剂。版权所有 © 2001 约翰·威利父子有限公司
A method has been developed for the rapid identification of optimal enzyme substrates from combinatorial libraries. This methodology was validated by screening a 361‐member N‐terminally formylated tripeptide library, f‐XXR (X = 19 different amino acids), for optimal substrates ofEscherichia colipeptide deformylase (PDF). The library was synthesized on a solid phase via the split‐pool synthesis method. The N‐terminal formyl group was added by treating the resin with a 1:1 (mol/mol) mixture of HCO2H and DCO2D in the presence of dicyclohexylcarbodiimide. In a mass spectrum, each member of the library produced a doublet peak (separated by 1.0063 Da). Limited treatment of this library withE. coliPDF resulted in the deformylation of those peptides that are the most efficient substrates of the enzyme. The deformylated products, due to loss of the mass‐degenerate formyl group, each generated a singlet peak in the mass spectrum. Thus, the PDF product peaks were readily identified and sequenced via tandem mass spectrometry. The results showed that PDF strongly prefers a norleucine and, to a lesser extent, a phenylalanine as the N‐terminal residue, whereas it has little selectivity at the penultimate position. This result is in excellent agreement with the literature data and therefore demonstrates the methodology as an effective approach to the identification of optimal enzyme substrates. This method should be generally applicable to other enzymes as well as synthetic catalysts. Copyright © 2001 John Wiley & Sons, Ltd.
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