Fig 2. Purification and characterization of monoPEGylated human amylin.

a) MonoPEGylated human amylin was synthesized in organic solvent by conjugation human amylin (5 mg/mL) with mPEGsc5k (5:1 molar excess) and purified by C18-RP-HPLC with a 30–70% CH₃CN gradient (in the presence of 0.1% TFA) up to 20 min. b) MALDI-ToF-MS of the fractions from the purification step showing the mono PEGylated human amylin corresponding to the peak comprising the elution time 11 min–13 min. The panel is rotated in order to align the PAGE-like display of the mass spectra with the respective original fractions in the chromatogram (upper panel, A). c) Trypsin-digestion of PEGylated human amylin. Purified monoPEGylated human amylin was subjected to trypsin digestion and submitted to MALDI-ToF-MS for identification of the products. The 2748 m/z ion coincides with the expected monoisotopic mass for the sodium adduct of the non-modified (non-PEGylated) human amylin 12–37 fragment (amylin 12–37, amide at C-terminus; monoisotopic mass 2,725.3 Da)