Assembly-enhanced differentiation of isomers by ion mobility mass spectrometry

Abstract

Isomerism is widely observed in basic biomolecules such as saccharides, amino acids, nucleotides, which are the building blocks of important macromolecules in nature. Isomers are also commonly found in isolated and synthesized products in organic and pharmaceutical studies. The structural differences of isomers can lead to significant differences in their physical and chemical properties, and thus their biological and pharmaceutical activities. Differentiation of isomers is essential in chemistry, biology, pharmaceutical science, medicine, etc. Ion mobility mass spectrometry (IM-MS) is an emerging technique for differentiation of isomers. In this thesis, we report the development and applications of assembly-based approach for differentiation of isomers by IM-MS. Leucine (Leu), isoleucine (Ile) and allo-isoleucine (allo-Ile) are isomeric amino acids that are difficult to be distinguished by IM-MS due to their very small size difference. In this study, by optimizing the ESI conditions, the types of additive metal ions and their concentration ratios with the amino acids, we have been able to observe higher degrees of assemblies of Leu, Ile or allo-Ile (M) in the presence of copper ion, including [Cu4(M)6]2+, [Cu4(M)7]+, [Cu5(M)8]2+, [Cu6(M)10]2+, [Cu7(M)12]2+, etc. Interestingly, it was found that the difference of the isomer assemblies in drift time and collision cross sections (CCSs) in IM-MS become larger with the increased number (n) of the assembled amino acids. From n = 1 to 12, the CCS difference (∆CCS) was increased from 1.6 to 15.2 Å2 and from 0.6 to 10.2 Å2 between the assemblies of Leu and Ile and between the assemblies of allo-Ile and Ile, respectively, indicating that the isomeric differences could be accumulated and amplified in the assembly process for enhanced discrimination of isomers in IM-MS. The combination of IM-MS, MS/MS and computational modelling have been employed to obtain the structure information of the assemblies and understand their gradually increased differentiation. The preliminary results showed that the assemblies might be formed with the alkyl chains of amino acids protruded outwards and the slight differences between the alkyl chains could be accumulated to eventually cause significant differences in the assemblies. Further investigation on the analogue compounds (e.g., peptides, amino alcohols, keto acids and fatty acids) by IM-MS proved the enhanced differentiation of isomers in the assemblies and supported the structural models. Monosaccharide and disaccharide isomers including glucose (Glc), fructose (Fru), mannose (Man), galactose (Gal) and maltose (Mal), cellobiose (Cel), isomaltose (IsoMal), lactose (Lac) and sucrose (Suc) with different compositions, connectivity and configurations were selected in this study. With addition of NiCl2 or ZnCl2 at the optimal ratio, multimeric saccharide (M), i.e., [Ni(M)n]+/2+ or [Zn(M)n]+/2+, could be detected using ESI-MS with n =1-2 for monosaccharides and n =1-8 for disaccharides. The IM separation for the assemblies of disaccharides were found to be enhanced and the increased ∆CCS allowed better differentiation between composition isomers Lac and Gal, between connectivity isomers IsoMal and Mal, and between configuration isomers Mal and Cel. Propranolol (Prp) is a chiral drug with the S-form more active than the R-form. Chiral phenylalanine (Phe) was chosen as the chiral selector to form complexes with Prp in the presence of copper ion. IM study showed that the ∆CCS for the Prp enantiomers was 0 in the dimeric complex ion [Cu(Phe)(Prp)]+ and increased to 6.41Å2 for the trimeric complex ion [(Cu)2(Phe)2(Prp)]+, indicating the enhanced differentiation for the enantiomers in higher assembly. Further study and the structural interpretation of the complexes for the enantiomer differentiation are still ongoing. In summary, isomers including Leu, Ile and allo-Ile, Glc, Fru, Man, Gal, Mal, Cel, IsoMal, Lac and Suc, S- and R-Prp, which are constitutional, configurational, and chiral isomers, respectively, were investigated for their differentiations by IM-MS in this project. With the presence of metal ions and optimized ESI conditions, various multimeric assemblies of isomers were observed in ESI-MS. Investigation by IM-MS revealed that, in general, higher assembly could significantly enhance the differentiation of isomers. Structural investigation suggested that the initial isomeric differences could be accumulated in the assembly and lead to significant differences in higher assemblies of the isomers. This study proposes a new strategy for differentiation of isomers and provides new insights into the structures and applications of molecular assemblies in the gas phase.