Correlation of cleavage techniques with side-reactions following solid-phase peptide synthesis

Solid-phase peptide synthesis is routinely used in research ranging from the elucidation of chemical mechanisms to the development of potential therapeutics. This chapter discusses problems in peptide assembly versus cleavage by amino acid analysis (AAA), RP-HPLC, Edman degradation sequence analysis, electrospray mass spectrometry (ESMS), and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). RP-HPLC, using a C₁₈ column appears to provide an accurate estimate of the complexity of the peptide samples; however, its accuracy was diminished by the presence of scavengers and side-chain protecting group adducts. The chapter suggests that efficient characterization of synthetic peptides is best obtained by a combination of RP-HPLC and MS, with sequencing by either Edman degradation or tandem MS being used to identify the positions of modifications and deletions. Proper peptide characterization is essential, especially in light of the lack of correlation between product quality and cleavage reagents or work-up protocols. Thus, these results suggest that laboratory technique plays an important role in peptide synthesis and hence product integrity should not be taken for granted.