Structural Resolution of 4-Substituted Proline Diastereomers with Ion Mobility Spectrometry via Alkali Metal Ion Cationization

Abstract

The chirality of substituents on an amino acid can significantly change its mode of binding to a metal ion, as shown here experimentally by traveling wave ion mobility spectrometry-mass spectrometry (TWIMS-MS) of different proline isomeric molecules complexed with alkali metal ions. Baseline separation of the cis- and trans- forms of both hydroxyproline and fluoroproline was achieved using TWIMS-MS via metal ion cationization (Li(+), Na(+), K(+), and Cs(+)). Density functional theory calculations indicate that differentiation of these diastereomers is a result of the stabilization of differing metal-complexed forms adopted by the diastereomers when cationized by an alkali metal cation, [M + X](+) where X = Li, Na, K, and Cs, versus the topologically similar structures of the protonated molecules, [M + H](+). Metal-cationized trans-proline variants exist in a linear salt-bridge form where the metal ion interacts with a deprotonated carboxylic acid and the proton is displaced onto the nitrogen atom of the pyrrolidine ring. In contrast, metal-cationized cis-proline variants adopt a compact structure where the carbonyl of the carboxylic acid, nitrogen atom, and if available, the hydroxyl and fluorine substituent solvate the metal ion. Experimentally, it was observed that the resolution between alkali metal-cationized cis- and trans-proline variants decreases as the size of the metal ion increases. Density functional theory demonstrates that this is due to the decreasing stability of the compact charge-solvated cis-proline structure with increased metal ion radius, likely a result of steric hindrance and/or weaker binding to the larger metal ion. Furthermore, the unique structures adopted by the alkali metal-cationized cis- and trans-proline variants results in these molecules having significantly different quantum mechanically calculated dipole moments, a factor that can be further exploited to improve the diastereomeric resolution when utilizing a drift gas with a higher polarizability constant.

Fast and Effective Ion Mobility–Mass Spectrometry Separation of d-Amino-Acid-Containing Peptides

“…65 If these deviations are unequal for two epimers, metalation can enhance their resolution. 66,67 We have measured single K + adducts generated by spiking the sample with K 2 CO 3 at 70 μM (Figure 3b). While the peak widths barely change, potassiation increases Ω by 0.9% (2.6 Å 2 ) for the D-epimer but 1.6% (4.8 Å 2 ) for the L-epimer, enabling their complete resolution ( r = 1.0) at slow scan rates (Table S1).…”

“…Different metal ions rearrange flexible biomolecules in distinct ways, 65,66 which suggests trying diverse cations to maximize resolution.…”

Experimental and Theoretical Investigation of Sodiated Multimers of Steroid Epimers with Ion Mobility-Mass Spectrometry

“…Additionally, IM-MS offers great potential in rapid separation of isomers that cannot be resolved with mass spectrometry alone. First demonstrated for structural isomers of polycyclic aromatic hydrocarbons [15], this potential has been shown across numerous classes of biological molecules including carbohydrates [16–24], peptides [25, 26], oligonucleotides [19], lipids [19, 27–29], amino acids [29–31], fatty acids [32], glycans and glycopeptides [33], and other small molecules [34]. However, diastereomers and especially epimers, which differ in stereochemistry at a single chiral center, have very subtle structural differences that may contribute to only minor variation in CCS.…”

“…IM-MS experimental results provide information on the charge, atomic composition, and CCS of the molecules, but only a comparison of experimental and theoretical CCS can provide additional molecular structural information, which could not be achieved with experimental results alone. The CCS comparative approach has been used previously to determine structural information for small organic molecules such as crown ethers [52] and carbon clusters [53]; macromolecules such as polyethylene glycol polymers [7]; peptides including bradykinin [54–58]; proteins [7] and cytochrome C [8]; and small biologically-relevant molecules including vitamin D metabolites [51], amino acids [31, 59, 60], and carbohydrates [42]. Comparison of IMS and theoretical results can help identify isomer conformers that can lead to understanding of differences that contribute to separation, improving the potential for these applications.…”

Multiple gas-phase conformations of proline-containing peptides: is it always cis/trans isomerization?

“…Proline-containing peptides, 9-14 small proline-containing proteins, 15-16 and derivatized proline amino acids 17 have been a frequent target of IM-MS studies. Through the creation and analysis of a large database, Counterman and Clemmer were the first to reveal that tryptic peptides containing proline were more likely to display multiple IM peaks than those without proline.…”