Downsizing improves sensitivity 100-fold for hydrogen exchange-mass spectrometry

This paper describes a method that substantially improves the sensitivity of high-performance liquid chromatography hydrogen exchange-mass spectrometry (HPLC HX MS). The success of this method relies on using a capillary HPLC column (0.1mm IDx5cmL) to increase the sensitivity of electrospray ionization, while keeping analysis times short to minimize hydrogen/deuterium (H/D) exchange. A small, immobilized pepsin column and a capillary C18 trap were included in the capillary HPLC MS system to provide rapid digestion, peptide concentration, and desalting while maintaining slow H/D exchange conditions. To minimize the analysis time, dead volumes and capacities of all components were optimized. Fully deuterated cytochrome c and its fully deuterated peptic peptides were used to evaluate deuterium recovery at amide linkages. The deuterium recovery measured at low flow rates using this system spanned a range of 66-77% (average of 71%), which was similar to the range measured for a much larger system (67-80%, average 75%). Signal levels of most peptides for the downsized system increased by about 100-fold compared with the signal for the larger system. These results greatly strengthen the HPLC HX MS technique for studies where the quantity of protein is small.