Electrothermal Supercharging in Mass Spectrometry and Tandem Mass Spectrometry of Native Proteins

Abstract

Electrothermal supercharging of protein ions formed by electrospray ionization from buffered aqueous solutions results in significant increases to both the maximum and average charge states compared to native mass spectrometry in which ions are formed from the same solutions but with lower spray potentials. For eight of the nine proteins investigated, the maximum charge states of protonated ions formed from native solutions with electrothermal supercharging is greater than those obtained from conventional denaturing solutions consisting of water/methanol/acid, although the average charging is slightly lower owing to contributions of small populations of more folded low charge-state structures. Under these conditions, electrothermal supercharging is slightly less effective for anions than for cations. Equivalent sequence coverage (80%) is obtained with electron transfer dissociation of the same high charge-state ion of cytochrome c formed by electrothermal supercharging from native solutions and from denaturing solutions. Electrothermal supercharging should be advantageous for combining structural studies of proteins in native environments with mass spectrometers that have limited high m/z capabilities and for significantly improving tandem mass spectrometry performance for protein ions formed from solutions in which the molecules have native structures and activities.

Mechanistic and Kinetic Study of Singlet O2 Oxidation of Methionine by On-Line Electrospray Ionization Mass Spectrometry

“…As a result, the stainless steel emitter could not be used in negative ESI. Cassou et al reported negative nano-ESI of water solution by using borosilicate capillary with an ESI spray potential of -0.7 kV and a 3-mm distance between the emitter and the counter electrode [53]. We were not able to achieve stable ESI with the same field strength, presumably because of the higher flow rate we used.…”

Direct Analysis of Intact Biological Macromolecules by Low-Energy, Fiber-Based Femtosecond Laser Vaporization at 1042 nm Wavelength with Nanospray Postionization Mass Spectrometry

“…41 Cytochrome c has been well-studied by mass spectrometry not only for chemical analysis in proteomics 30,31,42,43 but also for protein conformation studies in structural biology. [44][45][46][47] ESI-MS of a given protein usually results in a charge state distribution (CSD) corresponding to multiply charged ions and the CSD shifts to higher charge states (low m/z) when the protein unfolds under extreme conditions such as an acidic or heated solution. 48 While there has been some debate concerning the use of charge states to determine the conformation of protein molecules in mass spectrometry, 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 12 folded conformations with detection of a small amount of unfolded protein.…”

Intact protein mass spectrometry and top-down proteomics

“…Kathrin Breuker (University of Innsbruck, Innsbruck, Austria) considered the relationship between the rate of gas-phase unfolding of a protein versus the efficiency of dissociation after electron capture events, illustrating the benefits of unfolding. Evan Williams (University of California at Berkeley, CA, USA) described the use of reagents that modify the electrospray ionization process for supercharging of protein ions, and introduced electrothermal supercharging [7].…”