Journal
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
Volume 874, Issue 1-2, Pages 101-110Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jchromb.2008.09.012
Keywords
C-terminal detection; Isotope coding; O-18; Acylation; Esterification; LC-ESI-MS
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The determination of C-terminal peptide sequence is critical since the C-terminal peptide contains biologically relevant information and often undergoes post-translational processing. Another important application is in estimating purity of the biopharmaceuticals, especially for determining the presence of ragged processed ends and for N-terminally blocked polypeptides and proteins. In this paper, different isotope coding strategies in combination with reversed phase chromatography (RPC) coupled with electrospray ionization-mass spectrometry (ESI-MS) were evaluated to detect the C-terminal peptide from proteolytic digests. These were (i) O-18 (ii) acylation and (iii) esterification based isotope coding strategies. Using reversed phase chromatography, the C-terminal peptide was resolved from other internal peptides. The isotope coding approaches specifically rendered a characteristic MS signature to the C-terminal peptide, thereby facilitating its detection. The unique MS signature, along with accurate mass data for the C-terminal peptide was found to be sufficient for its detection and identification. The advantages and limitations of the three approaches will be discussed. (C) 2008 Elsevier B.V. All rights reserved.
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