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.

Quantitative Mapping of Protein Structure by Hydroxyl Radical Footprinting-Mediated Structural Mass Spectrometry: A Protection Factor Analysis

“…It has been previously demonstrated that the footprinting rate k fp determined from synchrotron radiolysis experiments has a qualitative correlation with local solvent accessibility (8,31,(37)(38)(39). A similar correlation was also observed in other measurements with hydroxyl radicals generated from either electrical discharge (40) or lasers (14,41,42). From a quantitative perspective, however, such a comparison is still insufficient, given that only identical segments could be compared across different conformational states.…”

Corona discharge-induced reduction of quinones in negative electrospray ionization mass spectrometry

“…22 On the other hand, higher spray voltage will result in more vigorous CD, based on which Downard's group have done intensive proteomics-related research. [31][32][33] Therefore, the results indicated that a more sever CD through raising spray voltage or using metal spray emitter facilitated the formation of Mc À ions.…”

Concentration of hydroxyl radicals during electrical discharge for radical probe protein footprinting mass spectrometry

“…The electrical discharge approach has been exploited in numerous studies of proteins and their interactions, including investigations of the impact of sustained oxidation of these proteins and complexes of significance to oxidative damage in disease pathogenesis. It has also been implemented in conjunction with the deposition of samples onto a large format matrix‐assisted laser desorption/ ionisation (MALDI) plate to aid high‐throughput analysis.…”

“…This results in the production of hydroxyl radicals within the solution at the needle tip, such that the electrosprayed droplets release oxidised protein in addition to its unmodified counterpart. While the level of oxidised to unoxidised protein can be reliably quantified by mass spectrometry (at typically between 10–30% under optimal conditions for footprinting experiments), the concentration of hydroxyl radicals in solution required to effect this has not yet been studied.…”

“…An aqueous solution of hen egg white lysozyme (10 μM) in 10 mM sodium cacodylate (pH 7.4) containing 1 mM indigo carmine was passed through the needle held at 8.5 kV at between 10–15 μA of a dedicated ESI source using a sheath gas of oxygen at 1–2 μL/min. Solution droplets were collected into grounded sample vials centrally positioned 1–2 mm under the vertically mounted needle as previously described . The flow rate of the solution was adjusted down incrementally from 20 to 1 μL/min, with a common volume of 20 μL collected in each case by varying the spray time from 1 to 20 min accordingly.…”