Under non-denaturing solvent conditions, the mean charge state of a multiply charged protein ion formed by electrospray is linearly correlated with the macromolecular surface

Electrospray mass spectrometric studies in native folded forms of several proteins in aqueous solution have been performed in the positive and negative ion modes. The mass spectra of the proteins show peaks corresponding to multiple charge states of the gaseous protein ions. The results have been analyzed using the known crystal structures of these proteins. Crystal structure analysis shows that among the surface exposed residues some are involved in hydrogen-bonding or salt-bridge interactions while some are free. The maximum positive charge state of the gaseous protein ions was directly related to the number of free surface exposed basic groups whereas the maximum negative charge state was related to the number of free surface exposed acidic groups of the proteins. The surface exposed basic groups, which are involved in hydrogen bonding, have lower propensity to contribute to the positive charge of the protein. Similarly, the surface exposed acidic groups involved in salt bridges have lower propensity to contribute to the negative charge of the protein. Analysis of the crystal structure to determine the maximum charge state of protein in the electrospray mass spectrum was also used to interpret the reported mass spectra of several proteins. The results show that both the positive and the negative ion mass spectra of the proteins could be interpreted by simple consideration of the crystal structure of the folded proteins. Moreover, unfolding of the protein was shown to increase the positive charge-state because of the availability of larger number of free basic groups at the surface of the unfolded protein.

Section: Correlation Of the Number Of Exposed Free Basic Residues Witsupporting

confidence: 88%

Section: Correlation Of the Number Of Exposed Free Basic Residues Witsupporting

confidence: 72%

Section: Correlation Of the Number Of Exposed Free Basic Residues Witmentioning

confidence: 99%

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Direct correlation of the crystal structure of proteins with the maximum positive and negative charge states of gaseous protein ions produced by electrospray ionization

Prakash

Mazumdar

2005

“…Denatured calmodulin also sprays with higher charge states than does the wild type [82]. Others suggest, however, that the higher charged states correlate only with surface area and not the number of exposed side chains [83]. Conformation 2: A folded, native state of the protein with normally folded EF-hand sequences.…”

Section: Spectroscopymentioning

confidence: 99%

Metal-binding properties of human centrin-2 determined by micro-electrospray ionization mass spectrometry and UV spectroscopy

Craig

Benson

Bergen

et al. 2006

We analyzed the metal-binding properties of human centrin-2 (HsCen-2) and followed the changes in HsCen-2 structure upon metal-binding using micro-electrospray ionization mass spectrometry (ESI-MS). Apo-HsCen-2 is mostly monomeric. The ESI spectra of HsCen-2 show two charge-state distributions, representing two conformations of the protein. HsCen-2 binds four moles calcium/mol protein: one mol of calcium with high affinity, one additional mol of calcium with lower affinity, and two moles of calcium at low affinity sites. HsCen-2 binds four moles of magnesium/mol protein. The conformation giving the lower charge-state HsCen-2 by ESI, binds calcium and magnesium more readily than does the higher charge-state HsCen-2. Both conformations of HsCen-2 bind calcium more readily than magnesium. Calcium was more effective in displacing magnesium bound to HsCen-2 than vice versa. Binding of a peptide from a known binding partner, the xeroderma pigmentosum complementation group protein C (XPC), to apo-HsCen-2, occurs in the presence or the absence of calcium. Near and far-UV CD spectra of HsCen-2 show little difference with addition of calcium or magnesium. Minor changes in secondary structure are noted. Melting curves derived from temperature dependence of molar ellipticity at 222 nm for HsCen-2 show that calcium increases protein stability whereas magnesium does not. ⌬25 HsCen-2 behaves similarly to HsCen-2. We conclude that HsCen-2 binds calcium and magnesium and that calcium modulates HsCen-2 structure and function by increasing its stability without undergoing significant changes in secondary or tertiary

“…Black circles indicate this study. White squares show data from Hatreux et al [74]. Gray triangles show data from Hogan et al [68].…”

Section: Mechanism Of Protein Electrospray Ionizationmentioning

confidence: 99%

Ion Mobility Measurements of Nondenatured 12–150 kDa Proteins and Protein Multimers by Tandem Differential Mobility Analysis–Mass Spectrometry (DMA-MS)

Hogan

Mora

2011

The mobilities of electrosprayed proteins and protein multimers with molecular weights ranging from 12.4 kDa (cytochrome C monomers) to 154 kDa (nonspecific concanavalin A hexamers) were measured in dry air by a planar differential mobility analyzer (DMA) coupled to a time-offlight mass spectrometer (TOF-MS). The DMA determines true mobility at atmospheric pressure, without perturbing ion structure from that delivered by the electrospray. A nondenaturing aqueous 20 mM triethylammonium formate buffer yields compact ions with low charge states, moderating polarization effects on ion mobility. Conversion of mobilities into cross-sections involves a reduction factor ξ for the actual mobility relative to that associated with elastic specular collisions with smooth surfaces. ξ is known to be 1.36 in air from Millikan's oil drop experiments. A similar enhancement effect ascribed to atomic-scale surface roughness has been found in numerical simulations. Adopting Millikan's value ξ=1.36 and assuming a spherical geometry yields a gas-phase protein density ρ p =0.949±0.053 g cm −3 for all our protein data. This is substantially higher than the 0.67 g cm −3 found in recent low-resolution DMA measurements of singly charged proteins. DMA-MS can distinguish nonspecific protein aggregates formed during the electrospray process from those formed preferentially in solution.The observed charge versus diameter relation is compatible with a protein charge reduction mechanism based on the evaporation of triethylammonium ions from electrosprayed drops.