Voyager DE/Ligations

From ChemicalProteinSynthesis

Monitoring of Native Chemical Ligation reactions by directly spotting on the maldi plate is not possible because the presence of 6 M guanidine hydrochloride interfers with sample spotting (spot does not dry completely) and efficient ionization of the peptides. Fortunately, this problem can be overcome with a few simple procedures.

Dilution

• Dilute an aliquot of the ligation reaction 1:20 or 1:40 with 50% H₂O / 50% ACN + 0.1% TFA
• Spot this sample as described in plate spotting

Because of the sensitivity of the Maldi-TOF instrument, peptides diluted 1:20 or 1:40 from the original 1-5 mM concentration of the ligation reaction can be detected effectively. The dilution of the guanidine hydrochloride from 6 M to 300 mM seems to remove most of the negative effects of the salt.

Desalting

• Desalt an aliquot of the ligation reaction using a zip tip or small scale dialysis should also serve to eliminate the disruptive effects of the 6 M guanidine hydrochloride on maldi analysis of a ligation reaction.