Peptide ladders, 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. 

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.

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. 

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. 

Analysis of the mixture using matrix-assisted desorption ionization mass spectrometry (section 5.3) allows for direct sequence determination from the snccessive mass differences of the peptide ladder. The application of the volatile trifluoroethyl isothiocyanate results in a significant optimization of this procedure and allows for peptide sequencing at the femtomole level (Bartlet-Jones et al., 1994). C-terminal ladder sequencing uses ammonium thiocyanate in acetic anhydride coupled with mass spectrometric analysis of truncated peptides (Thiede et al, 1997). Matrix-assisted desorption ionization instruments with delayed extraction (Brown and Leimon, 1995) allow for the discrimination of aU amino acids, except Leu and He. 

Bartlet-Jones, M., et al. (1994). Peptide Ladder Sequencing by Mass Spectrometry Using a Novel, Volatile Degradation Reagent, Rapid Communications, in Mass Spectrometry 8 737—742. 

The MW determination by means of MALDI-TOF is so exact that the mass difference between a peptide with n amino acids and the same peptide minus one amino acid allows the identification of the missing amino acid. Even Asp (MW 115.1) and Asn (MW 114.1) can be determined this way. High-end devices even detect whether the peptide is lacking Lys (MW 146.190) or Gin (MW 146.146). However, the peptides should not be too large (maximum 30 amino acid residues). Following Chait et al. (1993), ladder sequencing is based on the accuracy of MALDI-TOF and a modified Edman reaction (Figure 7.10). 

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.

Once you have the peptide ladder, you get the sequence of your peptide within minutes from a MALDI-TOF run. Because you can produce ladders from different peptides (in different tubes) at the same time, sequencing with peptide ladders and MALDI-TOF is faster than with the traditional Edman method Table 7.4. In addition, you need less peptide. This is true only, however, if it works, because as any other method ladder sequencing also has its problems. 

Peptide Ladder Sequencing (Following Bartlet-Jones Et Al.)... 

Example 8.4 The peptide of Example 8.3 (i.e., Thr-Gly-Met-Leu-Phe) is sequenced by the ladder sequencing technique. At what miz values will ions be observed in the MALDI mass spectrum after digestion with carboxypeptidase Y ... 

The sequence of an unknown peptide was determined with the peptide ladder sequencing technique. After treatment with an aminopaptidase and... 

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