Protein Protocols Protein Protocols

738 Wang and Chait
Fig. 2. Amino acid sequence read-out from a mass spectrum of ladder peptides. The vertical
labels are measured m/z values for the peaks in the spectrum. The horizontal numbers are
calculated mass differences between adjacent peaks. The identified amino acid residues are
indicated. The amino-terminal sequence is read from right to left.
4. A detailed discussion on manual Edman chemistry is given in ref. 7.
5. Special care should be taken to remove the coupling reagents and byproducts of the coupling
reaction. Otherwise, nonvolatile salt will form and affect subsequent reactions and
mass spectrometric analysis.
6. It is necessary to perform the conversion twice, since the concentration of PIC is limited
by its solubility and may be too low to yield 100% conversion.
7. The matrix-assisted laser desorption/ionization mass spectrometric analysis of protein and
peptide requires acidic conditions. Unless the pH is lowered, the base used in the coupling
reaction will significantly reduce the sensitivity of the mass spectrometric measurement.
8. It is important to note that the mass spectrometric response of a peptide is dependent on its
amino acid composition. The sensitivity, in general, is enhanced when the peptide contains
basic amino acid residues. On the other hand, the sensitivity is reduced when no
basic amino acid residues are present. The modification on ε-amino groups of lysine
residues by phenylisocyanate may decrease the mass spectrometric response, especially
for peptides that do not contain arginine residues.
9. Leucine and isoleucine have identical residue masses (113 Dalton). These isobaric amino
acid residues cannot be distinguished by the present ladder sequencing method. Lysine
and glutamine have the same nominal residue masses (128 Dalton). However, they can be
distinguished from each other, since the ε-amino group of lysyl residue is modified by PIC
and results in a mass shift of 119 (residue mass of PC-lysine is 247.3 Dalton).
10. To detect a posttranslational modification using ladder sequencing, the modification
should not be labile to the ladder sequencing chemistry. In certain cases, ambiguous results
may result from posttranslationally modified amino acid residues that have similar masses
to natural amino acid residues.
References
1. Chait, B. T., Wang, R., Beavis, R. C., and Kent, S. B. H. (1993) Protein ladder sequencing.
Science 262, 89–92.