Ladder sequencing

Top) peptide ladder sequencing principle. Phenyl isothiocyanate (PITC) produces phenylthiohydantoin (PTH) of the terminal amino acid and a new peptide with one less amino acid. Phenyl isocyanate (PIC), in low quantity, produces N-terminal phenylcarba-mate (PC) from a small fraction of each peptide. (Bottom) example of sequencing of [Glu1]fibrinopeptide B. Reproduced (modified) from Chait B.T., Wang R., Beavis R.C. and Kent S.B.H., Science, 262, 89, 1993, with permission. 

Thiede, B., Salnikow, J. and WittmannLiebold, B. (1997) C-terminal ladder sequencing by an approach combining chemical degradation with analysis by matrix-assisted-laser-desorption ionization mass spectrometry. Eur. J. Biochem., 244 (3), 750 4. 

Figure 2 A ladder sequence through 6 cycles of a synthetic peptide (CD28-3PY) carried out on a total of 17.5 picomoles of starting peptide. The peptide is especially interesting in that it contains both a proline and phosphorylated tyrosine residue. Both residues undergo the sequencing chemistry satisfactorily.

Figure 3 A ladder sequence through 6 cycles of [Glul] fibrinopeptide B carried out on a total of 17.5 picomoles of starting peptide.

In the ladder sequencing method, the oligonucleotide is enzymatically digested to subsequently remove either 3 - or 5 -terminal residues. The sequence can be derived from the mass change after each cleavage. Ladder sequencing was demonstrated using either MALDI-MS or ESI-MS [27]. 

Sequence information can also be obtained using Edman degradation to remove amino-terminal residues from a peptide, to produce a collection of peptides. MALDI-TOF can then be used to obtain the peptides masses, and the sequence determined by mass difference between consecutive peptides. This methodology is called protein ladder sequencing, and allows information to be obtained for up to 30 residues. This method is useful for the identification of posttranslational modifications, such as phosphorylated amino acid residues.24... 

The following ion-activation techniques have been used at one time or other to sequence peptides (1) fast atom bombardment (FAB) ionization, (2) CID—tandem MS (MS/MS), (3) ESI in-source CID, (4) MALDI ion-source decay, (5) MALDI postsource decay (PSD), (6) electron-capture dissociation (ECD) and electron-transfer dissociation, and (7) peptide ladder sequencing. Because of the lack of space, only (2) and (4) will be discussed further. 

BT Chait, R Wong, RC Beavis, SBH Kent. Protein ladder sequencing. Science... 

The de novo method of sequencing that uses enzymes to digest the terminal amino acids from a peptide is called peptide ladder sequencing. In this technique, an enzyme such as carboxypetidase-Y is used to digest one amino acid at a time from the C-terminus. No additional fragment peaks are formed. This method, unlike the MS-MS de novo technique, requires that the peptide be pure. In addition to the need for a pure peptide, another drawback of this technique is the somewhat lengthy sample preparation. The major advantage of this technique is the ease with which the mass spectrum can be interpreted and the partial sequence determined. 

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