Lys-C/Trypsin Tandem-Digestion Protocol for Gel-Free Proteomic Analysis of Colon Biopsies
The protocol presented was specifically optimized for in-depth analysis of the human colon mucosa proteome. After cell lysis in a sodium deoxycholate/urea buffer, a tandem digestion with Lys-C and trypsin was performed. Prior to LC-MS/MS analysis, peptides were TMT-labeled and fractionated by high pH reversed-phase spin columns. This protocol is a powerful, reproducible, sample-saving, and cost-effective option when an in-depth quantitative proteome analysis is desired.
Key wordsColon mucosa proteome Sodium deoxycholate Lys-C TMT labeling High pH reversed-phase fractionation
We thank Ilona Urbarova and Jack-Ansgar Bruun for fruitful discussions, as well as Prof Jon Florholmen and Rasmus Goll for supplying colon biopsies. Our work was supported by a grant from the North Norway Regional Health Authorities.
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