Protein Protocols Protein Protocols

734 Wang and Chait
Fig. 1. The principle of N-terminal protein ladder sequencing. A stepwise Edman degradation
is carried out in the presence of small amount of terminating reagent (phenylisocyanate,
PIC) in the coupling step. The resulting phenylcarbomyl (PC) peptides are stable to
trifluoroacetic acid (TFA) used to cleave the terminal amino acid from the phenylthiocarbomyl
(PTC) peptides in the cleavage step. A ladder peptide mixture is formed as the result of
PC-peptide accumulation in successive Edman cycles. Each black block represents an amino
acid residue.
1. The amino acid sequence is read-out from one spectrum. Thus, optimally, all members of
the sequence-defining fragments of the peptide, each differing by one amino acid residue,
are simultaneously examined. The sequence of the peptide is deduced from the set of
fragmentation products. Such a data set contains mutually interdependent information that
determines the identity and the order of each amino acid residue in the parent peptide.
Carry-over resulting from incomplete sequencing reactions cause no ambiguities in the
present method.
2. The method can be used to obtain sequence information from peptide mixtures.
3. Direct detection of posttranslational modifications can be made with ladder sequencing (5).
4. The ladder generation chemistry does not require complicated apparatus and can be done
in any chemistry laboratory. However, the current procedure requires at least 100 pmol of
peptide, since sample loss generally occurs in the liquid-phase extraction.