High Resolution Peptide Mapping Separations with MS-Friendly Mobile Phases and Charge Surface Modified C18
Matthew Allen Lauber , Stephan M Koza , Scott A McCall , Bonnie A Alden , Pamela C Iraneta , and Kenneth J Fountain
Anal. Chem., Just Accepted Manuscript
DOI: 10.1021/ac401481z
Publication Date (Web): June 17, 2013
Copyright © 2013 American Chemical Society
Ionic analytes, such as peptides, can be challenging to separate by reversed-phase chromatography with optimal efficiency. They tend, for instance, to exhibit poor peak shapes, particularly when eluted with mobile phases preferred for electrospray ionization mass spectrometry. We demonstrate that a novel charged surface C18 stationary phase alleviates some of the challenges associated with reversed-phase peptide separations. This column chemistry, known as CSH (charged surface hybrid) C18, improves upon an already robust organosilica hybrid stationary phase, BEH (ethylene bridged hybrid) C18. Based on separations of a nine peptide standard, CSH C18 was found to exhibit improved loadability, greater peak capacities, and unique selectivity compared to BEH C18. Its performance was also seen to be significantly less dependent on TFA-ion pairing, making it ideal for MS applications where high sensitivity is desired. These performance advantages were evaluated through application to peptide mapping, wherein CSH C18 was found to aid the development of a high resolution, high sensitivity LC-UV-MS peptide mapping method for the therapeutic antibody, trastuzumab. From these results, the use of a C18 stationary phase with a charged surface, like CSH C18, holds significant promise for facilitating challenging peptide analyses.
Keyword: Peptide Inhibitors