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 th e characterization of a potent growth hormone releasing hormone (GHRH) analog, paramethylhippuroyl derivative of the N-terminus of a precurs or porcine-GHRH (2-76) (pMHpGHRH) derived by recombinant DNA technolog y. Tryptic digestion can be completed within one hour at room temperat ure. All tryptic peptides can be separated by reversed phase high perf ormance liquid chromatography (RPHPLC) on a Vydac C18 protein column w ith trifluoroacetic acid (TFA)-acetonitrile (ACN) gradient elution. El even single fragments obtained in tryptic peptide mapping have been id entified by coupled Ionspray mass spectrometry. Combined fragments T2- 3 and T4-5 were also observed andverified by Ionspray mass spectrometr y and microsequencing analysis. A stability study indicated that the d igestion solution will remain stable for 9 days at 5 degrees C. The re tention time variations for the tryptic peptides found to be 1.9% rela tive standard deviation (RSD).