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Engineered ChymotrypsiN for Mass Spectrometry-Based Detection of Protein Glycosylation.
ACS Chemical Biology ( IF 4 ) Pub Date : 2019-11-25 , DOI: 10.1021/acschembio.9b00506
Balakrishnan Ramesh 1 , Shaza Abnouf 1 , Sujina Mali 2 , Wilna J Moree 2 , Ujwal Patil 2 , Steven J Bark 2 , Navin Varadarajan 1
Affiliation  

We have engineered the substrate specificity of chymotrypsin to cleave after Asn by high-throughput screening of large libraries created by comprehensive remodeling of the substrate binding pocket. The engineered variant (chymotrypsiN, ChyB-Asn) demonstrated an altered substrate specificity with an expanded preference for Asn-containing substrates. We confirmed that protein engineering did not compromise the stability of the enzyme by biophysical characterization. Comparison of wild-type ChyB and ChyB-Asn in profiling lysates of HEK293 cells demonstrated both qualitative and quantitative differences in the nature of the peptides and proteins identified by liquid chromatography and tandem mass spectrometry. ChyB-Asn enabled the identification of partially glycosylated Asn sites within a model glycoprotein and in the extracellular proteome of Jurkat T cells. ChymotrypsiN is a valuable addition to the toolkit of proteases to aid the mapping of N-linked glycosylation sites within proteins and proteomes.

中文翻译:

工程化的ChymotrypsiN,用于基于质谱的蛋白质糖基化检测。

我们已经设计了胰凝乳蛋白酶的底物特异性,可以通过对底物结合口袋进行全面改造而创建的大型文库的高通量筛选来对Asn进行切割。工程变体(chymotrypsiN,ChyB-Asn)表现出改变的底物特异性,并且对含Asn的底物具有更大的偏好。我们证实蛋白质工程学不会通过生物物理表征损害酶的稳定性。比较野生型ChyB和ChyB-Asn在HEK293细胞的分析裂解物中,证明了通过液相色谱和串联质谱鉴定的肽和蛋白质的性质在质量和数量上都存在差异。ChyB-Asn能够鉴定模型糖蛋白和Jurkat T细胞的细胞外蛋白质组中部分糖基化的Asn位点。ChymotrypsiN是蛋白酶工具包的宝贵补充,可帮助绘制蛋白质和蛋白质组中N-联糖基化位点的图谱。
更新日期:2019-11-28
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