Created: 28th February 1999, last updated: 7th April 1999, © 1999 ABRF
The Peptide Synthesis Research Group (PSRG) has completed investigation into the rapid deamidation of one of the test peptides from last year's study. Although the initial aim of the study was to assess the ability of member facilities to identify peptides and solve problems in peptide synthesis, the unexpected occurred. As part of this assessment, a synthetic peptide sample was distributed by the PSRG, and the laboratories were asked to verify whether the "requested" sequence of H-Asp-Glu-Gln-Glu-Ala-Leu-Asn-Arg-Ser-NH2 (1059.5 daltons) was present. The peptide that was sent out for this part of the study had (by design) been synthesized in the reverse direction. Moreover, the requested Ser residue had been replaced with a Thr, and an Asp was incorporated instead of one of the Glu residues. As a consequence, the molecular weight of the test peptide was the same as the one requested, but the sequence was H-Thr-Arg-Asn-Leu-Ala-Asp-Gln-Glu-Asp-NH2. As the study progressed, participants began to report a second high-performance liquid chromatography (HPLC) peak in their analysis of the test peptide. The PSRG therefore initiated stability studies and found that the reverse peptide was very susceptible to deamidation at the C terminus, whereas the requested peptide was quite stable. The molecular weight increase caused by this deamidation could not be detected by low-resolution matrix-assisted laser desorption and ionization/time of flight mass spectrometry (MALDI/TOF-MS) or by low-resolution electrospray ionization mass spectrometry (ESI-MS), but it was readily apparent when a higher-resolution instrument was used or on-line HPLC/ESI-MS was employed. Our results indicate that deamidation can be highly sequence dependent. If deamidation is suspected in a synthetic peptide, the easiest way to assess the level of degradation is through the use of HPLC/ESI-MS. The results of this stability study will be presented at the PSRG workshop at ABRF '99.
The PSRG is in the process of analyzing samples submitted for this year's study on the synthesis of biotinylated peptides. Along with the instructions for sample submission, the study letter supplied the details for a synthetic protocol that can be readily used for preparation of the biotinylated peptide but can also be modified for the covalent attachment of other moieties in addition to biotin. From the results obtained so far, it is clear that variations in the synthetic method can lead to significant differences in peptide purity. Results of the 1998-99 study will be presented in the research group workshop and in the poster session at the ABRF '99 meeting.