Chain Ejection Model for Electrospray Ionization of Unfolded Proteins: Evidence from Atomistic Simulations and Ion Mobility Spectrometry

Abstract

The ion evaporation model (IEM) and the charged residue model (CRM) represent cornerstones of any discussion related to the mechanism of electrospray ionization (ESI). Molecular dynamics (MD) simulations have confirmed that small ions such as Na are ejected from the surface of aqueous ESI droplets (IEM), while folded proteins in native ESI are released by water evaporation to dryness (CRM). ESI of unfolded proteins yields [M + zH] ions that are much more highly charged than their folded counterparts. A chain ejection model (CEM) has been proposed to account for the protein ESI behavior under such non-native conditions (Konermann, L., et al. Anal. Chem. 2013, 85, 2-9). The CEM envisions that unfolded proteins are driven to the droplet surface by hydrophobic and electrostatic factors, followed by gradual ejection via intermediates where droplets carry extended protein tails. Thus far, it has not been possible to support the CEM through MD simulations using realistic protein models and atomistic force fields. Such endeavors require much larger droplets than in previous MD studies. Also, the incorporation of CEM-related H migration is difficult. This work overcomes these challenges in MD simulations on unfolded apo-myoglobin (aMb) in droplets with a 5.5 nm radius (∼22500 water molecules). We focused on solutions at pH ∼4 where the aMb solution charge coincides with the charge on some of the electrosprayed ions (22+ to 27+), such that H migration could be neglected. Na ions were added to ensure a droplet charge close to the Rayleigh limit. We found that 16 of 17 MD runs on various protonation patterns produced [M + zH] ions via chain ejection. The predicted stretched-out aMb conformations were consistent with experimental collision cross sections. These results support the view that unfolded proteins follow the CEM. Overall, the IEM/CRM/CEM triad can account for a wide range of ESI scenarios involving various types of analytes.

Ultrafast enzymatic digestion of proteins by microdroplet mass spectrometry

“…They also proposed a chain ejection mode (CEM) to account for the protein ESI behavior 67 and afterwards found evidence from atomistic simulation and ion mobility spectrometry in 2018 43 . Through CEM, unfolded proteins are driven to the droplet surface by hydrophobic and electrostatic factors, which may act synergistically with surface accumulation effects in microdroplets 20 to improve protein digestion.…”

“…The results demonstrate the advance and potential significance of microdroplets in protein identification, including a dramatic decrease in digestion time-from overnight with traditional methods or several minutes with other accelerated devices-to less than a millisecond for the complete cleavage of peptide bonds at the C-terminal side of lysine or arginine residues except when followed by proline; and an increase in sequence coverage from 60 to 100%. The possible mechanism driving this acceleration is discussed, including the surface concentration effect in microdroplets, the spontaneous generation of hydroxyl radicals or hydrogen peroxide, gas bubbles induced by ammonium bicarbonate, and the chain ejection model (CEM) during the ESI process 43 to drive the proteins to the microdroplet surface. To further demonstrate the practical utility of this technology, the sequence of the therapeutic antibody trastuzumab (~148 kDa), also known as Herceptin, is confirmed by ESSI-MS with a sequence coverage of 100% for light chains and 85% for heavy chains.…”

Comparison of the pH‐dependent formation of His and Cys heptapeptide complexes of nickel(II), copper(II), and zinc(II) as determined by ion mobility‐mass spectrometry

“…In positive ion mode (Figure S4), the same behavior of Cu(II)‐bound dimer formation was also exhibited with the increase of one to four chelated Cu(II) ions also observed as pH increased from pH 4.6 to 10. This suggests that two of the binding sites can be protonated, possibly during the positive ion electrospray process, and the chelation of Cu(II) is still stable via three sites. The bond lengths from the amide nitrogens to the Cu(II) are 1.94 to 1.96 Å, whereas, the bond distance of the imidazoles or C‐terminus to Cu(II) are 2.01 to 2.06 Å.…”

“…The positive ion analyses ( Figure S3) also exhibited a similar pHdependent Ni(II)-binding as the negative ion analyses, especially for species A, B, and C. The positive ion pH-dependent behavior was not as distinct as the negative ion analyses and was probably affected by excess protonation of the complex via electrochemical reactions and droplet evaporation during the positive ion electrospray process. 58 However, the results still indicate that the Ni(II) chelation was dependent on the weak acidic and basic Cys and His substituent groups. and results in the His 1 , Cys 2 , Cys 7 , and C-terminus carboxylate coordination of Ni(II) (Figure 4).…”

A Quantitative MALDI-MSI Study of the Movement of Molecules in Biological Systems

“…This model suggests that an unfolded protein migrates to the surface of the droplet due to the exposure of hydrophobic residues and one chain terminus get partially ejected from the droplet into the gas phase. This is followed by further ejection of the rest of the protein, which will result in highly charged ions (Konermann et al, 2013;Metwally, Duez and Konermann, 2018). A schematic illustration of the main mechanisms responsible of ion formation by ESI is provided in Figure 1.3.…”