Structure evaluation of protected synthetic peptides by ESI and FAB high resolution MS/MS

Sequence information of the human genome and that of few organisms are nowadays available. The next challenge regards the developing of proteomics, and in particular the understanding of the structure and the function of proteins encoded by particular genomes for the progress of therapeutic interventions against genetic diseases. Mass mapping and/or sequence analysis of peptides from enzymatic cleavage (either in gel or in solution and or without subsequent separation) is at present time a common way to identify unknown proteins in the protein sequence databases. A reliable confirmation of peptide structures, in the case of a novel protein, could require the use of peptides built by fast-automated synthesis. In the chemical synthesis of natural and modified peptide chains, mass spectrometry plays a relevant role in order to check the exact structure of the sequences. FAB and ESI have been proposed as strategies not only to provide a tool for assessing molecular weight of fully protected peptide, but also to allow either the control of the built-up sequence or the monitoring of the protection grade. However, the extremely acid-labile groups employed for the side-chain protection could be easily lost either by gas phase or surface degradation of the analytes, limiting the usefulness of the information obtained by FAB MS. Besides, the high lypophilicity typical of fully protected peptides represents a consistent limit to their analysis under ESI conditions. The applicability of FAB and ESI MS/MS in the full structure determination of N-Fmoc protected peptides containing acid-labile masking groups has therefore been exploited with the sequences 10-16, 17-24 and 25-28 of porcine calcitonin as Fmoc-Ser(Trt)-Ala-Tyr(tBu)-Trp(tBu)-Trp-Arg(Pmc)-Asn(Trt)-Leu-OH, Fmoc-Asn-Asn(Trt)-Phe-His(Trt)-Arg(Dmtr)2-Phe-Ser(Trt)-Gly-OH, and Fmoc-Met-Gly-Phe-Gly-OH. A series of fragment ions due to the releasing of protecting groups, stepwise degradation of the peptide back-bone (sequencing) and side-chain elimination reaction, present both in the stable and metastable (CID)(-) FAB spectra, provide a complete structure elucidation. Sequence ions are formed either from the intact or from the [(M-Fmoc)H]- peptides. Similar and complementary information can be obtained from MS and MS/MS ESI spectra of mono and double charged molecular ions.