Oral Presentation 24th Annual Lorne Proteomics Symposium 2019

Reversed phase x reversed phase on-line comprehensive two-dimensional liquid chromatography of intact proteins with triple quadrupole mass spectrometric detection (#54)

Kevin Schug 1 , Yehia Z Baghdady 1 , Evelyn H Wang 1 , Durga D Khanal 1
  1. The University of Texas Arlington, Arlington, TEXAS, United States

The interest in analysis of intact proteins has increased significantly over the past several years, especially as it relates to biomarker discovery and biopharmaceutical development. On-line comprehensive two-dimensional liquid chromatography (LCxLC) has only been applied in a very limited fashion for analysis of intact proteins. We set out to create the first LCxLC method based on pH switching between two successive dimensions of reversed phase chromatography. Reversed phase x reversed phase LCxLC offers significant advantages in terms of availability of column chemistries, good compatibility of solvents between the two dimensions, and good compatibility of eluent mobile phases for mass spectrometric detection. Significant efforts succeeded to develop high pH separation conditions on a Water Acquity BEH wide pore C4 column using a triethylammonium bicarbonate buffer (pH = 10). Selectivity for a series of model proteins was demonstrated to be different and complementary than for a standard low pH reversed phase separation on an AMT Halo protein C4 column using a combined formic acid/trifluoroacetic acid-based mobile phase. LCxLC was accomplished by combining the high pH separation in the first dimension with the low pH separation in the second dimension on a Shimadzu Nexera-e instrument,t interaced with a LCMS-8050 triple quadrupole mass spectrometer. Several proteins in the model mixture, unresolved in the first dimension were successfully resolved in the 1 minute separation in the second dimension. A complex E. coli proteome sample was used to demonstrate a high degree of orthogonality and good coverage of the two-dimensional separation space. The effective peak capacity of the LCxLC-MS analysis was 2268, with a peak production rate of 37.8 peaks per minute over the entire 60-minute analytical run.