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.
The Dow Chemical Company, Analytical Sciences, 1897 Building, Midland, MI 48667, USA. SJulka@dow.com
Samir
S
Julka
Demetrius
D
Dielman
Scott A
SA
Young
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