Reduced Back-Exchange by Modified RPLC Mobile Phase Separation of Proteolytic Peptides for MS Analysis of H/D Exchange of Solution-Phase Proteins

摘要

Novel method for decreasing back-exchange in H/D exchange by partly replacing water in the mobile phase with polar organic modifiers. Increased chromatographic separation of peptide fragments with modified solvents due to slower elution, yet yielding less backexchange. DMF-modified solvents seem to be superior due to lower viscosity and higher number of fragments relative toNMP modified solvents. Average decrease in back-exchange for the DMF-modified solvents was 24percent relative to conventional LC mobile phases. Further work is in progress to optimize the use of modified solvent systems with complex protein mixtures inHDXexperiments.