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Identification of site‐specific heterogeneity in peptide drugs using intact mass spectrometry with electron transfer dissociation
Author(s) -
Gucinski Ashley C.,
Boyne Michael T.
Publication year - 2014
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.6957
Subject(s) - chemistry , peptide , electron transfer dissociation , chromatography , protamine , mass spectrometry , tandem mass spectrometry , protamine sulfate , protein mass spectrometry , peptide sequence , bottom up proteomics , biochemistry , gene , heparin
RATIONALE Protamine sulfate is a peptide drug product consisting of multiple basic peptides. As traditional high‐performance liquid chromatography (HPLC) separation methods may not resolve these peptides, as well as any possible peptide‐related impurities, a method utilizing top‐down mass spectrometry was developed for the characterization of complex peptide drug products, including any low‐level impurities, which is described in this study. METHODS Herring protamine sulfate was used as a model system to demonstrate the applicability of the method. Direct infusion mass spectrometry and tandem mass spectrometry (MS/MS) on a high‐resolution, mass accurate instrument with electron transfer dissociation (ETD) were used to identify all the species present in the herring protamine sulfate sample. Identifications were made based on mass accuracy analysis as well as MS/MS fragmentation patterns. RESULTS Complete sequence coverage of the three abundant herring protamine peptides was obtained using the top‐down ETD‐MS/MS method, which also identified a discrepancy with the published herring protamine peptide sequences. Additionally, three low‐abundance related peptide species were also identified and fully characterized. These three peptides had not previously been reported as herring protamine peptides, but could be related to the published sequences through amino acid additions and/or substitutions. CONCLUSIONS A method for the characterization of protamine, a complex peptide drug product, was developed that can be extended to other complex peptide or protein drug products. The selectivity and sensitivity of this method improves a regulator's ability to identify peptide impurities not previously observed using the established methods and presents an opportunity to better understand the composition of complex peptide drug products. Published in 2014. This article is a U.S. Government work and is in the public domain in the USA.