Gas-Phase Ion/Ion Reactions of Biologically Relevant Molecules

Stella Katheryn Betancourt, Purdue University

Abstract

Mass spectrometry (MS) is widely used for the analysis of complex mixtures. MS techniques have been developed to isolate and probe, via fragmentation, analytes of interest in these complex mixtures. Through fragmentation, such as ion-trap collision induced dissociation (CID) ions can be activated to form fragments that could be used to determine the original structure of the analyte. Various techniques have also been used to enhance the analysis of molecules of interest. One such technique is solution-phase derivatization reactions that covalently alter the analyte prior to introduction into an MS instrument. Solution-phase derivatization is commonplace and can provide various benefits in the analysis of biologically-relevant molecules. These reactions have helped to determine structural information and increase ionization efficiency. However, solution-phase derivatization is a timely method and there is no guarantee that the entire analyte population is modified. Post-source ion manipulation, done via gas-phase ion/ion reactions, is fast, efficient, and can provide insight in the functional groups attached to unknown ions. Here, work will be presented showing the benefits of ion/ion interactions to improve upon current analytical techniques and in some work, go as far as determining structural information depending on connectivity or identifying the functional group of the analyte. Modified, commercially-available instruments are used to effectively manipulate the ion-type formed upon ionization. In doing so, the charge state of the ion is changed and, in some cases, covalent modification is performed. Ion manipulation, without covalently modifying the analyte, can be facilitated by changing solution conditions. For instance, lowering the pH of a solution to obtain mostly protonated analyte ions. However, due to other polar compounds that could be found in solution, changing solution conditions will not result in 100% formation of the preferred analyte ion-type. However, gas-phase ion/ion reactions can be used to convert an entire ionic population to the desired analyte type, such as obtaining a solely singly charged population. Polysorbate 80, commercially known as Tween® 80, is a polydispersed polymer that is conventionally synthesized with a multitude of fatty acyl chains and due to synthesis byproducts can have different head-groups. Since aqueous solutions of polymers inherently spray adducted to metal cations via electrospray ionization (ESI). There is a multitude of ions and charge states formed for each component of the polymer, thus resulting in a very congested spectrum. Here, gas-phase ion-ion reactions are used to adduct a weakly coordinating anion to these cationic components, then subsequent fragmentation results in a singly charged, singly adducted peak for each component. In doing so, the resulting spectrum is less congested and the peak capacity is significantly increased allowing for the determination of the components found in a sample of Tween® 80. Functionalities can also be deduced based on gas-phase ion/ion reaction between a reagent and an analyte of interest. Small molecules can be analyzed by spraying a mixture via ESI, isolating the analyte ion, and perform CID on that ion. However, the fragmentation patterns do not always confirm the functionality that could be found in the analyte molecule. In this work, small molecules containing aldehyde functionalities are reacted with primary amines found on biological molecules that serve as reagents. The overall reaction of these molecules results in a Schiff Base formation that is identified by a signature water (18 Da) loss from the complex. This reaction can selectively identify the aldehyde functional group, providing more characterization than the original CID spectrum. Gas-phase ion/ion reactions can also be applied to an existing shotgun lipidomics platform. Shotgun lipidomics is when an extract of a biological sample is subjected to ESI and analyzed for its lipid composition without the use of any chromatographic technique prior to ionization. For this particular work, glycerophospholipids (PL) are subjected to ion/ion reactions to manipulate their charge state, more specifically charge inverting PL cations formed via ESI to PL anions. These reactions are undertaken by reacting PL cations with a dicarboxylate reagent. In doing so, phosphatidylethanolamine (PE) and phosphatidylcholine (PC) isomers are chemically separated. This is effected by the different reactivity between the PLs and the dicarboxylate. PCs, for instance, form an electrostatically-bound complex, and can transfer a methyl group and a proton upon CID, resulting in a demethylated PC anion [PC - CH3]–. PEs, on the other hand, do not form a dominant complex and transfer two protons to the dicarboxylate reagent resulting in a deprotonated PE anion, [PE - H] –. These two products effectively separate these isomeric cations by charge inversion and separation in the mass-to-charge (m/z) domain. This technique can also be applied to the analysis of a complex mixture. (Abstract shortened by ProQuest.)

Degree

Ph.D.

Advisors

McLuckey, Purdue University.

Subject Area

Chemistry|Analytical chemistry

COinS