BIOC 2580 Lecture Notes - Lecture 5: Arginine, Valine, Threonine
8 Dec 2017
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Synopsis: 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. N terminal tagging works because the n terminal a nucleophile under mildly basic conditions (+nh3 , the normal state at ph 7 is not a nucleophile, but by increasing to ph 9 (pka. The nucleophilic n terminal :nh2 will then react by displacing hf from the reagent fluorodinitrobenzene. Thus, the bright yellow dinitrophenyl group becomes bonded to the n terminal amino acid.
