Anne E. Counterman

Conformer-Dependent Proton-Transfer Reactions of Ubiquitin Ions

The conformations of ubiquitin ions before and after being exposed to proton transfer reagents have been studied by using ion mobility/mass spectrometry techniques. Ions were produced by electrospray ionization and exposed to acetone, acetophenone, n-butylamine, and 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene. Under the conditions employed, the +4 to +13 charge states were formed and a variety of conformations, which we have characterized as compact, partially folded, and elongated, have been observed. The low charge state ions have cross sections that are similar to those calculated for the crystal conformation. High charge states favor unfolded conformations. The ion mobility distributions recorded after ions have been exposed to each base show that the lowest charge state that is formed during proton-transfer reactions favors a compact conformation. More open conformations are observed for the higher charge states that remain after reaction. The results show that for a given charge state, the apparent gas-phase acidities of the different conformations are ordered as compact < partially folded < elongated.

A database of 660 peptide ion cross sections: Use of intrinsic size parameters for bona fide predictions of cross sections

An ion trap/ion mobility/time-of-flight mass spectrometry technique has been used to measure collision cross sections for 660 peptide ions generated by tryptic digestion of 34 common proteins. Measured cross sections have been compiled into a database that contains peptide molecular weight and sequence information. The database is used to generate average intrinsic contributions to cross section (size parameters) for different amino acid residues by solving systems of equations that relate the unknown contributions of individual residues to the sequences and cross sections of database peptides. Size parameters are combined with information about amino acid composition to calculate cross sections for database peptides. Bona fide cross section predictions (made prior to measurement) for peptides observed in tryptic digests of sperm whale myoglobin and yeast enolase are made. Eight of 10 predicted cross sections are within 2% of the experimental values and all 10 are within 3.2%. The utility of size parameters for cross section prediction is explored and discussed.

Influence of solvent composition and capillary temperature on the conformations of electrosprayed ions: unfolding of compact ubiquitin conformers from pseudonative and denatured solutions

Abstract High-resolution ion mobility/mass spectrometry methods have been used to examine the influence of solvent composition and capillary temperature on the gas-phase conformations of ubiquitin ions (+6 to +13) formed during electrospray ionization. Three general conformer types are observed: compact forms (favored for the +6 and +7 charge states); partially folded conformers (favored for the +8 and +9 ions); and, unfolded conformers (favored for the +10 to +13 charge states). The populations of different conformers are highly sensitive to solvent composition and capillary temperature used for electrospray ionization. Electrospray of “pseudonative” solutions leads to formation of some conformers that are more compact than those observed for “denatured” solutions. Studies as a function of capillary temperature show that as the capillary temperature is increased, compact and partially folded states undergo unfolding transitions. Compact states from pseudonative solutions unfold at higher temperatures than compact states from denatured solutions. The products of unfolding transitions have similar cross sections for both solution types.

High-order structure and dissociation of gaseous peptide aggregates that are hidden in mass spectra

Injected-ion mobility and high-pressure ion mobility techniques have been used to examine the conformations of bradykinin, insulin chain A, and several other peptide ions in the gas phase. Under the experimental conditions employed, evidence for multimer formation in the mass spectra of peptides is minimal or absent altogether. However, ion mobility distributions show that aggregates of peptides (containing a single charge per monomer unit) are observed at the same mass-to-charge ratios as the singly charged parent ions. Collision cross sections for these clusters show that they have tightly packed roughly spherical conformations. We have bracketed the average density as 0. 87 ρ < ρ < 1. 00 g cm−3. In some cases, specific stable aggregate forms within a cluster size can be distinguished indicating that some high order structures are favored in the gas phase. Multimer formation between different sizes of polyalanine peptides shows no evidence for size specificity in aggregate formation. Collisional and thermal excitation studies have been used to examine structural transitions and dissociation of the multimers. Aggregates appear to dissociate via loss of singly charged monomers. The observation that peptide multimers can be concealed in mass spectral data requires that fragmentation patterns and reactivity studies of singly charged monomers be undertaken with care.

Gas-phase separations of protease digests

A mixture of peptides from a complete tryptic digest of ubiquitin has been analyzed by ion mobility/time-of-flight mass spectrometry techniques. All components of the mixture were electrosprayed and ions were separated in the gas phase based on differences in their mobilities through helium before being dispersed into a time-of-flight mass spectrometer for mass-to-charge analysis. The data show that ions separate into families primarily according to differences in their charge states and, to a lesser extent, differences in conformation. This approach reduces spectral congestion typically associated with electrosprayed mixtures and provides charge assignments for mass-to-charge ratio data. Gas-phase separations of ions appear to provide a new physical basis for characterizing components of biological mixtures.

Formation of peptide aggregates during ESI: Size, charge, composition, and contributions to noise

Ion mobility/time-of-flight techniques have been used to examine the onset of aggregation in model systems of Gly-Xxx (where Xxxx = Ala, Asn, Asp, Gln, Glu, His, Leu, Ser, Thr, and Trp) dipeptides. Under the experimental conditions employed, there is evidence that simple binary and quaternary mixtures of these dipeptides produce clusters containing as many as 16 to 75 peptide units (and 1 to 7 charges). In some systems, cluster compositions appear to come about largely from statistical association of peptide units; other dipeptide mixtures (and generally for small clusters) show evidence for nonstatistical behavior which could arise from some differences in gas-phase or solution thermochemistry. The minimum aggregate size appears to be largely determined by the charge state. Average thresholds for aggregate formation in the z = 2, 3, and 4 charge state families occur at m/z ∼500, 660, and 875, respectively. We briefly consider the idea that aggregates formed during electrospray ionization (ESI) may contribute to the background signal observed in the analysis of complex peptide mixtures.

Large anhydrous polyalanine ions: substitution of Na+ for H+ destabilizes folded states

Abstract The conformations of a series of anhydrous sodiated polyalanine ions ([Alan + 3Na]3+, where n = 18–36) have been examined in the gas phase by ion mobility measurements and molecular modeling simulations. The experimental results indicate that these ions exist as highly extended conformations. There is no strong evidence for a folded state, observed previously for a series of analogous protonated polyalanines ([Alan + 3H]3+, where n = 24–41) (A.E. Counterman, D.E. Clemmer, J. Am. Chem. Soc., submitted). Molecular dynamics simulations for the [Alan + 3Na]3+ ions also indicate that extended structures are favored. The simulations show that extensive helical regions are present; however, near the sites where Na+ ions are attached, helical regions appear to be substantially disrupted by intramolecular charge solvation of the Na+. Simulations of some [Alan + 3Na]3+ charge site assignments show evidence for structures that are similar to folded structures observed for analogous [Alan + 3H]3+ ions; however, the calculated energy gap between the folded and unfolded states for the triply sodiated system is slightly greater than the gap in analogous protonated polyalanines. We propose this as a possible explanation for the absence of experimental evidence for a folded state in the sodiated system. Keywords: Polyalanine; Ion mobility; Sodium; Stability; Electrospray