On-plate deposition of oxidized proteins to facilitate protein footprinting studies by radical probe mass spectrometry.

ABSTRACT

The on-plate deposition of oxidized proteins is described to advance footprinting applications by radical probe mass spectrometry (RP-MS). An electrospray ionization (ESI) needle assembly mounted vertically over a 384-target matrix-assisted laser desorption/ionization (MALDI) plate enabled the limited oxidation of proteins as they were released in the charged droplets ahead of their deposition on the plate. This method combined with on-plate proteolytic digestion protocols expedites the analysis of proteins oxidized by RP-MS, and avoids the need to collect and reconstitute samples prior to analysis by MALDI mass spectrometry. Oxidation of peptides from solutions in water as well as an ammonium bicarbonate solution was investigated to test the optimal conditions required for on-plate oxidation of proteins. These comprised of peptides with a wide range of reactive amino acids including Phe, Tyr, Pro, His, Leu, Met and Lys that were previously shown to oxidize in both electrospray discharge and synchrotron radiolysis based footprinting experiments. The on-plate deposition of lysozyme oxidized at electrospray needle voltages of 6 and 9 kV were carried out to demonstrate conditions suitable for footprinting experiments as well as those that induce the onset of protein damage.