A Comparison of UHPLC MS/MS Peptide Identifications and Particle Size Diameter: Smaller particles sizes at Ultrahigh Pressures Yield More Peptide IDs.

Description:
In the medical and pharmaceutical industries, ‘omic’ (i.e., lipidomics, proteomics, metabolomics) analyses are increasingly important in informing drug design and disease diagnosis. However, multiomic analyses are inherently complex due to the wide range of structures and sizes present among the different analytes and the biological nature of the analytes. Much work is done to develop analytical techniques, methods, and sample preparation to handle biological samples for multiomic analysis via LC-MS, IM-MS, or GC-MS. The separation component of these techniques is essential for reducing the overall complexity of the sample as it allows for different classes of analytes to be separated based on their defining structural and mass differences. In liquid chromatography, metabolites, lipids, peptides, and proteins can be separated based on polarity and hydrophobicity differences. Understanding the biological difference between unique phenotypic traits via identification (ID) of metabolites, lipids, peptides, and proteins is crucial. Enhancing separation efficiency (H) can help increase the number of IDs corresponding to analytes in a complex biological matrix. Decreasing the stationary phase particle diameter (dp) leads to higher H. However, as dp decreases, pressure increases exponentially. Columns with sub 2 µm particles require systems capable of ultrahigh pressures. We investigated how decreasing dp affects peptide identification via LC-MS/MS experiments to enhance the proteomic coverage of a multiomic approach. Capillary columns were packed with different sizes of BEH C18 particles (1.1, 1.5, and 1.7 µm) and used to characterize a standard peptide mixture. We used a home-built UHPLC that can reach pressures as high as 45 kpsi allowing these separations to be done at 35 kpsi; a pressure predicted to give higher H than the commercially available 20 kpsi pressure limit. Preliminary results suggest that smaller particles and higher pressures increase peptide IDs.

Speaker: Hayley Herderschee - University of Michigan, Ann Arbor
Hayley Herderschee is currently a 4th year Ph.D. candidate working with Dr. Robert Kennedy at the University of Michigan. Her research focuses on using ultrahigh pressure liquid chromatography for improved efficiency for biological molecule separations and also investigates novel capillary column packing using sub 2 µm particles. She received her Master's in chemistry from the University and her Bachelor's in chemistry from Gustavus Adolphus College, where she conducted 4 years of research focused on applications of 2D-LC-MS with Dr. Dwight Stoll. Hayley has also done extensive 2D-LC work with Dr. Imad Haidar-Ahmad and Dr. Erik Regalado via an internship at Merck & Co., Inc. in Rahway, NJ during the summer of 2019.