ProC-TEL: Profiling of Protein C-Termini by Enzymatic Labeling.
Proteins are frequently processed by proteases in cell signaling pathways to perform their biological functions in response to environmental stimuli. Identification of the exact cleavage sites provides necessary information for the study of their biological functions. Although proteomic approaches for profiling of protein N-termini have been developed extensively in the past few years, the N-terminal profiling strategy has its inherent disadvantages. Therefore, C-terminal profiling approaches might be a complementary approach for the identification of protein cleavages although it has similar shortcomings as N-terminal profiling methods. In this protocol, we describe an approach, termed ProC-TEL: Profiling of Protein C-Termini by Enzymatic Labeling, for affinity labeling of protein C-termini for a protein or proteome. This method uses the transpeptidase activity of carboxypeptidase Y to label protein C-termini with an affinity biotin tag for subsequent isolation with avidin beads and identification by mass spectrometer. It is complementary to the N-terminal profiling approaches and can be used to identify proteolytic cleavages for a specific protease or in cell signaling events, such as apoptosis.
Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China.
Wenwen
W
Duan
Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China. gux2002@suda.edu.cn.|Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, Soochow University, Suzhou, Jiangsu, China. gux2002@suda.edu.cn.|College of Pharmaceutical Sciences, Soochow University, 199 Ren'ai Road, Yunxuan Building, Room 1328, Suzhou, Jiangsu, 215123, China. gux2002@suda.edu.cn.
Guoqiang
G
Xu
D043402:Cathepsin A / Q000378:metabolism; D002853:Chromatography, Liquid; D006868:Hydrolysis; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D059748:Proteolysis; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D012984:Software; D013194:Staining and Labeling; D013223:Statistics as Topic / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods
D010455:Peptides; D011506:Proteins; D020543:Proteome; D043402:Cathepsin A
Biocytinamide; Carboxypeptidase Y (CPY); Enzymatic labeling; Mass spectrometry (MS); ProC-TEL