Peptide ladders

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. 

Peptide ladder samples were analyzed on a matrix-assisted laser desorption time-of-flight mass spectrometer constructed at The Rockefeller University and described elsewhere (8,9). The individual peptides (9 residues to 18 residues in one vial and 19 to 32 residues in another vial) were mixed in approximately equal amounts and dissolved in water. The ladder mixtures were added to the matrix material (4-hydroxy-a-cyano-cinnamic acid (4HCCA) in formic acid/water/isopropanol 1 3 2) to a final concentration of 1-5 pM for each peptide component. The complete peptide ladder, which ranged from the 9 mer to the 32 mer (except 16 mer and 28 mer) was measured from 4HCCA in water/acetonitrile 2 1. The final concentration of each peptide component was in the range of 0.2-1 pM. Bovine insulin emd substance P were used as internal calibrants. 

Table II. A tabular presentation of the peptide ladder mass spectrometry. The data was obtained from two mixtures. The first mixture contained the individual peptides from the 9 mer to the 18 mer and the second mixture from the 19 mer to the 32 mer.

Stepwise analysis is an efficient and direct method to monitor the progress of side reactions in Fmoc chemistry. The analysis involves stepwise micro-scale TFA cleavage in conjunction with HPLC and MS. Micro-scale TFA cleavage can be conveniently carried out on a small amount of sample. MS analysis of peptide ladders provides a rapid method for monitoring and identification of side reactions in peptide synthesis. 

Figure 4. Partial view of the matrix-assisted laser desorption mass spectrum of the synthetic peptide ladder from 9 to 32 residues. The formation of aspartimide (loss of water, -18u) and the piperidine adduct (+67n) are strongly observed after the synthesis of 13 residues. The weak intensity of the peak corresponding to the 14 mer is due to the low amount of 14 mer added.

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. 

My recommendation protect the N-terminal ends during purification. But what if the N-terminus is already blocked You can try sequencing C-terminally (see Section 7.6.5) or with the mass spectrometer (see Section 7.6.6). If you cannot do this or do not want to, you have no choice but to give up and cleave the protein into peptides, and separate and sequence them. Which of the three methods is to be preferred depends on the local climate. If you can find someone in the institute who knows a lot about sequencing by mass spectrometer, enlist the services of this person. For one or a few sequences it is not worth studying the peptide ladder technique for yourself (see Section 7.6.6), especially if you have to purchase a mass spectrometer first. 

The most popular method is to completely digest the proteins after the blot. For this, you identify your protein on the (unblocked ) blot by means of protein stain, cut out the blot piece, and add a selective protease. This creates peptides that are separated by HPLC. You can sequence these peptides following Edman (see Section 7.6.4) or by means of a peptide ladder and MALDI-TOF (see Section 7.6.6). 

The cycle can be repeated at will. The result is a ladder of PIC peptides with n, n-1, n-2, n-3 amino acids and a residue peptide with a free N-terminus. This one also becomes blocked with PIC after the last cycle. Now, the MALDI-TOF separates the PIC peptide ladder and measures the MW of the individual PIC peptides. By their MW differences, you can identify the cut-off amino acids. Then, the peptide sequence can be read directly from the spectrum (Figure 7.10). 

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... 

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.

Figure 7.11. Peptide ladder following Bartlet-Jones et al.

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 ... 

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.)... 

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

Peptide Ladder Sequencing The treatment of a glycopeptide with a carboxypeptidase or aminopeptidase to generate peptide ladders, followed by MALDI-MS analysis of those ladders, is another feasible approach to identifying the site of glycosylation. This approach may not be effective with multiglycosylation sites because the enzymatic activity of these proteases is impaired at or near the site of glycosylation [84]. 

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