A Method To Define the Carboxyl Terminal of Proteins

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• 作者：Salvatore Sechi and Brian T. Chait
• 刊名：Analytical Chemistry
• 出版年：2000
• 出版时间：July 15, 2000
• 年：2000
• 卷：72
• 期：14
• 页码：3374 - 3378
• 全文大小：74K
• 年卷期：v.72,no.14(July 15, 2000)
• ISSN：1520-6882

文摘

Accurate definition of the carboxyl terminal of proteins isnecessary for elucidating posttranslational processing atthe C-terminal and more generally for characterizingprotein primary structures. Here, we describe a strategyfor isolating and characterizing the C-terminal peptide ofa protein after proteolysis with endoprotease Lys-C. Isolation is achieved using anhydrotrypsin, a catalytically inertderivative of trypsin that binds peptides containing lysineor arginine residues at their C-termini without cleavingthem. Rapid, accurate characterization of the isolatedC-terminal peptide is achieved by mass spectrometry.Initial identification of the C-terminal peptide is obtainedby comparing matrix-assisted laser desorption/ionizationtime-of-flight mass spectra of the digest prior to and afterincubation with anhydrotrypsin. Characterization of theC-terminal sequence is achieved by capillary-HPLC electrospray ionization tandem mass spectrometry of theisolated peptide using a quadrupole ion trap mass spectrometer in the selective reaction monitoring mode. Thisstrategy was successfully applied to the characterizationof the C-terminal of proteins with molecular massesranging up to 56 kDa.