The ABRF has ~ 130 member laboratories engaged in peptide synthesis and structural analysis of peptides and proteins as a service in academic, government and research institutions, and private industry. An initial study in 1991 by the Peptide Synthesis/MS Committee requested the synthesis of a test peptide. This year a similar study was designed whereby problems in peptide assembly versus cleavage were evaluated by an array of analytical techniques. Participating ABRF laboratories were asked to synthesize the following peptide by the methodology most commonly used in their facility:
H-Gly-Val-Arg-Gly-Asp-Lys-Gly-Asn-Pro-Gly-Trp-Pro-Gly-Ala-Pro-Tyr-OH
A total of 58 crude and 33 purified peptide samples and 42 peptide-resins were returned to the committee. Sixteen of the crude peptides were synthesized by Boc chemistry (28%) and 42 by Fmoc chemistry (72%). For the prior ABRF study, 18 of the crude peptides were synthesized by Boc chemistry and 18 by Fmoc chemistry. Hence, the fraction of peptides synthesized by Fmoc chemistry significantly increased this year. As estimated by electrospray mass spectrometry (ESMS), 30 of the 58 crude samples (52%) and 26 of the 33 purified samples (79%) contained >75% of the desired product. This represents a considerable improvement over the prior study, where the percentage of crude and purified products containing ~ 75 % of the desired material were 28 and 65 %, respectively. Possible reasons for these improved results are any combination of: (i) a peptide sequence less susceptible to side-reactions during peptide-resin cleavage (no Cys or Met present), (ii) a larger portion of peptides synthesized by Fmoc chemistry where cleavage conditions are less harsh, and (iii) more care in laboratory techniques following last year's results. The vast majority of poor quality peptides in the present study were due to peptide assembly difficulties, while most problems in the prior study were side-reactions during peptide-resin cleavage.
The range of peptide quality allowed us to evaluate critically the merits of different analytical techniques for peptide characterization. Reverse phase HPLC provided an accurate estimate of the complexity of the peptide samples and, in situations such as this where a sample of the desired product was available, an accurate estimate of the content of the desired product. The only exception was the failure to detect a co-eluting Asn deletion peptide. Since a sample of the desired product is not usually available, additional analyses are typically required. Capillary electrophoresis provided little useful information in this study. Amino acid analysis (AAA) was the best technique for absolute amino acid quantitation, but was not helpful for detecting modifications of amino acid residues. Preview sequence analysis had the same strengths and weaknesses as AAA and the advantage of determining residue position. The mass spectrometric techniques examined here [ESMS, fast-atom-bombardment mass spectrometry (FABMS), plasma-desorption mass spectrometry (PDMS), and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)] allowed for identification of peptide modifications, including residual protecting groups. MS as used in this study did not distinguish between products of the same mass, nor allow for assignment of the positions of residue deletions and/or modifications. HPLC, ESMS, FABMS, and PDMS were usually in agreement in estimating the % desired product, with all four estimating that the mean % desired product in 56 crude samples was 65-70%. Only 40% of the ABRF laboratories responding to our survey had MS available at their institutions and used it regularly for peptide characterization. The major reason for not using mass spectrometry was lack of on-site availability. Our experience in the present and prior study suggests that efficient characterization of synthetic peptides is best obtained by a combination of HPLC, AAA, and MS, with sequencing by either Edman degradation or tandem MS being used to identify the positions of modifications and deletions.
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