Shimadzu ASMS 2017 Monday Breakfast Seminar 

"Lessons from Doctor Strange: Exploring Alternate Dimensions for Intact Protein Separations and Detection using Liquid Chromatography-Tandem Mass Spectrometry"

              Proteins are inherently more complex to analyze than small molecules.  Yet, the increasing interest in biopharmaceuticals and biomarkers has created a demand for new methods to characterize protein analytes.  In most cases, a one-size-fits-all method does not exist to comprehensively evaluate all desired attributes.  A multitude of methods are needed to evaluate the large number of proteoforms that exist.  One area where development has been lacking is in the direct quantification of intact proteins.  We have recently demonstrated the ability to use multiple reaction monitoring (MRM) mode on a triple quadrupole mass spectrometer to directly quantify a protein without prior digestion into constituent peptides (J. Amer. Soc. Mass Spectrom. 2016, 27, 886-896).  This approach has acknowledged advantages and limitations.  Among interesting aspects, we have more recently investigated how intact protein ion transmission in QQQ-MS differs than that for small molecules.  Ion scattering, charge transfer, and mass resolution settings can all affect the degree to which diagnostic product ions of multiply-charged intact protein precursor ions can be sensitively detected.  We hypothesize that top-down protein quantitation may have benefits in terms of addressing the wide dynamic range of proteins in biological systems that have to be addressed.  Chromatographic separations will certainly be an integral part to such a strategy.  Interfacing liquid chromatography with tandem mass spectrometry for intact protein determination requires some important considerations for maximizing both separation and ionization efficiency (J. Sep. Sci. 2016, 39, 3716-3727). To accommodate greater complexity, we are exploring on-line comprehensive two-dimensional liquid chromatography (LCxLC).  The number of instances combining LCxLC with tandem mass spectrometry for intact protein analysis to date have been very limited.  Exploring this approach has required close collaboration with experts in industry and academia in order to make headway.  In this talk, our recent developments, the challenges, and the advantages and limitations of intact protein quantitation by LC-MS will be discussed.

Kevin Schug, Ph.D.,

Director, CLEAR (http://clear.uta.edu) 

Department of Chemistry & Biochemistry

The University of Texas at Arlington