Detection of C-terminal peptide of proteins using isotope coding strategies.
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) O18 (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.
Julka
Samir
S
The Dow Chemical Company, Analytical Sciences, 1897 Building, Midland, MI 48667, USA. SJulka@dow.com
Dielman
Demetrius
D
Young
Scott A
SA
D000215:Acylation; D002853:Chromatography, Liquid; D004951:Esterification; D010103:Oxygen Isotopes / Q000737:chemistry; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization
D010103:Oxygen Isotopes; D010455:Peptides; D011506:Proteins