BIOC 2580 Lecture Notes - Lecture 5: Edman Degradation, Deprotonation, Hydrolysis

• School: University of Guelph
• Department: Biochemistry
• Course: BIOC 2580
• Professor: Enoka Wijekoon

Document Summary

Early! methods! to! determine! the! amino! acid! sequence! of! a! protein! relied! on! cycling! between! basic! and! acidic! environments! to! change! the! reactivity! of! the! peptide. ! In! practice,! proteins! need! to! hydrolysed! into! shorter! peptides! for! sequencing. ! Selective* hydrolysis! of! the! polypeptide! chain! by! proteases,! or! with! chemicals,! cuts! very! long! polypeptides! into! specific! fragments! of! more! manageable! sizes. ! Using! tandem! mass* spectrometry,! proteins! can! be! sequenced!and!their!identity!determined!by!searching!protein!databases!and!using!search!tools! like!blast. ! Nc terminal! a! nucleophile! under! mildly! basic! conditions! (+nh3n,! the! normal! state! at! ph! :nh2n*will!then! react! by! displacing* hf! from! the! reagent* fluorodinitrobenzene. Thus,* the* bright* yellow* dinitrophenyl* group* becomes* bonded*to*the*nnterminal* amino* acid. * the! tagged! protein! is! then! hydrolysed! to! its! constituent! amino! acids,! and!the!labeled! (yellow)!ncterminal!amino! acid!can!easily!be!separated!and!identified! by!chromatography. Sanger"s! method! requires! complete* hydrolysis* of* the* peptide* chain* to* recover* the*tagged*amino*acid,!and!this!destroys!the!rest!of!the!peptide!chain!so!that!amino!acids!#2,!#3! etc!are!not!easily!identified. !this!creates!a!random! mixture! of! dipeptides! and! tripeptides! (short! chains! of! Per* edman! solved! the! problem! of! hydrolyzing! the! complete! peptide! to! recover! the! tagged! amino! acid! in!