Retention of Native Protein Structures in the Absence of Solvent: A Coupled Ion Mobility and Spectroscopic Study

Abstract

Can the structures of small to medium-sized proteins be conserved after transfer from the solution phase to the gas phase? A large number of studies have been devoted to this topic, however the answer has not been unambiguously determined to date. A clarification of this problem is important since it would allow very sensitive native mass spectrometry techniques to be used to address problems relevant to structural biology. A combination of ion-mobility mass spectrometry with infrared spectroscopy was used to investigate the secondary and tertiary structure of proteins carefully transferred from solution to the gas phase. The two proteins investigated are myoglobin and β-lactoglobulin, which are prototypical examples of helical and β-sheet proteins, respectively. The results show that for low charge states under gentle conditions, aspects of the native secondary and tertiary structure can be conserved.

Keywords: IR spectroscopy; gas-phase reactions; mass spectrometry; protein folding; protein structures.

Figure 1

Condensed‐phase structures of a) myoglobin, a 153 amino acid protein with a native secondary structure that is mostly α‐helical and which contains a non‐covalently attached heme group (PDB ID: 1MBN) and b) β‐lactoglobulin, a 162 residue protein that has abundant β‐sheet secondary structure (PDB ID: 3BLG).

Figure 2

Mass spectra and collision cross‐sections for myoglobin and β‐lactoglobulin. a, b) Mass spectra obtained when spraying from buffered (ammonium acetate, pH≈7) aqueous or water/methanol solutions (green and blue, respectively). Prominent peaks are labeled, and in the case of myoglobin, they correspond to the holo form. For low charge states of β‐lactoglobulin, adducts with palmitic acid can be observed (labeled 7+′, 8+′ and 9+′). c, d) Cross‐sections as a function of charge. Solid green circles stem from ions sprayed from buffered aqueous solution, solid blue circles from ions from water/methanol solution, and open blue circles from ions from water/methanol solution with 1 % formic acid.

Figure 3

Infrared spectra for gas‐phase and condensed‐phase myoglobin and β‐lactoglobulin. a, b)Spectra for ions sprayed from water/methanol solutions. The positions and shapes of the amide‐I bands around 1655 cm⁻¹ indicate helical secondary structures. c) Spectra for samples sprayed from aqueous solutions (green lines) and solution phase FTIR spectra (gray lines; data reproduced from Ref. 18 for β‐lactoglobulin and Ref. 19 for myoglobin). The amide‐I band for myoglobin 8+ indicates a helical secondary structure, while that of β‐lactoglobulin shows a clear β‐sheet signature.