Characterization of Tryptic Peptides of a Potent Growth Hormone Releasing Hormone Analog by Reversed Phase High Performance Liquid Chromatography-Ionspray Mass Spectrometry

A highly reproducible tryptic digestion procedure was developed for the characterization of a potent growth hormone releasing hormone (GHRH) analog, paramethylhippuroyl derivative of the N-terminus of a precursor porcine-GHRH (2-76) (pMHpGHRH) derived by recombinant DNA technology. Tryptic digestion can be completed within one hour at room temperature. All tryptic peptides can be separated by reversed phase high performance liquid chromatography (RPHPLC) on a Vydac C18 protein column with trifluoroacetic acid (TFA)-acetonitrile (ACN) gradient elution. Eleven single fragments obtained in tryptic peptide mapping have been identified by coupled Ionspray mass spectrometry. Combined fragments T2-3 and T4-5 were also observed andverified by Ionspray mass spectrometry and microsequencing analysis. A stability study indicated that the digestion solution will remain stable for 9 days at 5 degrees C. The retention time variations for the tryptic peptides found to be 1.9% relative standard deviation (RSD).