Castillo Mary Joan, Reynolds Kristy J, Gomes Alexander, Fenselau Catherine, Yao Xudong
Department of Chemistry, University of Connecticut, Storrs, Connecticut.
Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland.
Curr Protoc Protein Sci. 2014 Apr 1;76:23.4.1-23.4.9. doi: 10.1002/0471140864.ps2304s76.
This unit describes the stepwise procedure for differential oxygen isotope labeling of peptides and mass spectrometric quantification of relative protein levels in comparative proteomic experiments. The [¹⁸O] labeling of peptides happens at the peptide C-terminus and is achieved via the enzymatic oxygen exchange of tryptic peptides via catalysis of immobilized trypsin. Experimental considerations in effective incorporation and stabilization of the oxygen labels are discussed. Methods for mass spectrometric quantification of peptides with differential [¹⁶O] and [¹⁸O] isotopes are presented.
本单元描述了在比较蛋白质组学实验中对肽段进行差异氧同位素标记以及对相对蛋白质水平进行质谱定量的逐步程序。肽段的[¹⁸O]标记发生在肽段的C末端，通过固定化胰蛋白酶催化的胰蛋白酶肽段的酶促氧交换来实现。讨论了有效掺入和稳定氧标记的实验注意事项。介绍了用差异[¹⁶O]和[¹⁸O]同位素对肽段进行质谱定量的方法。
