Ladder Sequencing of Peptides

The laborious parts of the method of Chait et al. (1993) are the extraction and the washing and drying processes. In addition, you lose peptide. Bartlet-Jones et al. (1994) work more elegantly. They also create the peptide ladder via successive cleaving of the N-terminal amino 

Remove basic buffer and TFEITC under vacuum, and then cleave off N-terminal amino acid with heptafluorobutyric acid. 

Remove heptafluorobutyric acid under vacuum and add fresh peptide. 

Repeat steps 1 through 10 in Table 7.3 n-1 times for n cycles. After the last cycle, add 2 il peptide and 5 il water. Then dry for at least 1 h over NaOH pills. 

Dissolve the peptide ladder in 3 to 5 ml 50% aq. acetonitrile/0.1% (v/v) TFA and ultrasonicate for 5 minutes. Pipette several aliquots (0.3 to 0.5 al) one after the other onto the carrier and let them air dry for 5 minutes each. Finally, pipette 0.3 il matrix solution (1% (w/v) a-cyano-4-hydroxy cinnamic acid in 50% aq. acetonitrile/0.1% (v/v)/TFA) onto the dried sample and air dry again. Insert this preparation into the MALDI mass spectrometer and analyze it. Important  

---

Figure 7.10. Ladder sequencing of peptides (following Chait et al.) (A) Peptide is coupled with PICT and PIC. TFA cleaves the PICT-coupled amino acid. The PIC-derivatized peptide remains unchanged. (B) Several cycles (here three) of PICT/PIC coupling and subsequent acid cleavage generate a peptide ladder with PIC N-termini, a residue peptide with free N-terminus, and the PICT-derivatized amino acids. For the analysis of the peptide ladder, you also block the residue peptide with PIC.

---