About the Electrospray Ionization Source in Mass Spectrometry: Electrochemistry and On-chip Reactions

The present work shows that the electrochemical properties of electrospray ionization (ESI) can be used to add functions to the process. As example, we show how the choice of the electrode material can be used to study interactions between metal ions and biomolecules in mass spectrometry (MS). In positive ionization MS, an electrospray device acts as anode, which implies oxidation reactions. Sacrificial electrodes (made of copper or zinc) are used to supply the electrospray current and to produce cations that are able to react on-line with compounds of interest. Thus, the interactions between copper ions and ligands or peptides were investigated by using a copper electrode. Another example is the in situ electrogeneration of a dinuclear zinc(ii) complex for the mass tagging of phosphopeptides when working with a zinc electrode. In order to perform these reactions on the same microchip, a dual-channel microsprayer was used, where one channel was dedicated to the tag elec- trogeneration and the other to the infusion of a phosphopeptides solution. Finally, this dual-channel microsprayer was used to study complexation at liquid-liquid interfaces in biphasic ESI-MS, such as thioether crowns and lead ions or peptides and phospholipids complexes. These examples illustrate the use of electrochemistry and on-chip reactions in ESI-MS analysis.

Polymer nanospray interfaces for on-line electrochemically induced modifications

Tatiana Rohner

The aim of this work has been to design, and fabricate a miniaturized electrospray-type interface for mass spectrometry analysis, which allows the nebulization of solutions by applying a high potential difference between the source and the mass spectrometer. The fabrication of the nanospray emitter is based on UV-laser photoablation of polymer substrates. Thick-film channel flow electrodes can be integrated in the microfluidic system, allowing the application of a high voltage to generate electrospray from the open outlet of the microchannel. The polymer-based interface first presented is characterized by a flat edge interface. However, a V-shaped nozzle has been found to present higher stability and ease of use. This design also allows the generation of spray by applying high voltage through an electrode located in a solution reservoir, thereby inducing an electroosmotic pumping. Taking advantage of the intrinsic electrolytic behavior of electrospray, a specific electrochemically induced tagging of free cysteine residues located in proteins can be performed on-line. A p-hydroquinone buffer is used to perform a continuous infusion of protein solution. When in contact with the high-voltage microelectrode, p-hydroquinone undergoes oxidation, thereby producing protonated p-benzoquinone. The latter reacts with free cysteine residues, following a Michael-type addition. The modification of the protein can be followed thanks to MS analyses. A complete study of the mechanisms involved in the electrochemically induced tagging with hydroquinone has been carried out by cyclic voltammetry and digital simulation. The process of the tagging consists of an ECE mechanism, since the formed adduct is stabilized in the reduced state, and is thus free to be oxidized. The rate constant of the chemical reaction has been extracted, by performing cyclic voltammetry studies and modeling the whole system by digital simulations. Finite element simulations have also permitted to highlight several mechanistic aspects of the electrotagging. The nanospray interface has been modeled as a channel-flow electrode cell. After validation of the model with respect to the experimental results, kinetics and mass transport conditions have been investigated. The flow profile, as well as the kinetic rate constant, has been shown to be of great importance in the electrochemically induced tagging. The use of a sacrificial electrode as metallic ion source to generate electrospray has also been investigated. Transition metal electrodes, i.e. copper, zinc, nickel and iron, as well as silver, were used to get on-line complexation with model peptides. It has been demonstrated that the use of in-reservoir sacrificial electrode was an efficient method to generate metal ions in order to form complexes with peptides, without the addition of metallic salts. These studies have demonstrated that polymer nanospray interfaces can be advantageously used as analytical devices, together with their primary function, i.e. ionization sources for protein analyses by mass spectrometry.

EPFL2003

Electrospray ionization mass spectrometry

Michel Prudent

Metal ions are essential elements in all living systems. Their detections and identifications in biological processes require different methodologies. Mass spectrometry is one of the commonly used techniques in bioinorganic analyses to fulfill these requirements. Electrospray ionization mass spectrometry (ESI-MS) is a method of choice and the present work takes benefit of the electrochemical aspects of ESI and of the source design flexibility for studying the interactions between metal ions and biomolecules. In positive ionization mass spectrometry, an electrospray device acts as an anode, which implies oxidation reactions. Thus, transition metal electrodes can be used to generate metal ions in addition of sustaining the electrospray process. Sacrificial copper electrodes, coupled to polyimide microchips, are used first in order to study copper complexes. Firstly, it is shown that, when using different chelating agents for copper(II) and copper(I) ions, the electrodissolution of copper leads to the formation of the respective complexes that are directly analyzed in MS. This methodology offers a direct route to synthesize copper(I) complexes in aqueous solutions. Following this concept, the interactions between copper ions and peptides are investigated. On-line electrogeneration of copper-peptide complexes is achieved either by using a sacrificial copper electrode or by mixing the peptide solution with a copper(II) salt by applying a high voltage via a platinum electrode. The latter yields mainly copper(II) complexes with histidine residues or with the peptide backbone (copper(I) complexes can be also formed due to gas-phase reactions), whereas the former can generate a mixture of both copper(I) and copper(II) complexes that yields different complexation patterns. In addition, the influence of these cations on peptide fragmentation is reported. The role of copper in cysteine-containing peptide oxidation is studied with the same procedure. Using peptides containing one, two and three cysteines, we have compared the different chemical and electrochemical oxidation pathways of cysteine (RS-IIH) to cysteine (RS-IS-IR) and to sulfenic, sulfinic and sulfonic acid (RS0OH, RSIIO2H and RSIVO3H, respectively). In the absence of copper ions, intra-molecular reactions are the most abundant, whereas inter-molecular reactions are found to be enhanced by the presence of copper ions. These cations favor the formation of 2:1 (peptide : copper) complexes compared to 1:1 complexes, thus enhancing the formation of inter-molecular bridges. This study highlights the importance of the cysteines position inside a peptide in the disulfide bridges formation. The concept of sacrificial electrodes is then employed to tag molecules on-line instead of studying interactions. Phosphopeptides tagging reactions by dinuclear zinc(II) complexes are performed with a dual-channel microsprayer in ESI-MS. This device consists in a polyimide microchip with two microchannels etched on each side of the structure and connecting only at the tip of the microchip. The reaction is first studied ex situ and analyzed with a commercial electrospray source. On-chip reactions (i.e. inside the Taylor cone) are achieved with a dual-channel microsprayer both with the tag synthesized chemically before the experiments and with the tag electrogenerated by an in situ oxidation of a zinc electrode, also used to apply the electrospray current. It is thus demonstrated that mixing two solutions with different physico-chemical properties inside the Taylor cone can be used to selectively tag target molecules. Finally, a biphasic electrospray ionization (BESI) source has been built in the goal of probing interfacial complexes in MS. Based on the dual-channel microsprayer design, the two channels are filled with immiscible liquids and put in contact only within the Taylor cone. Two types of interfacial complexation reactions have been studied: the interfacial complexation of aqueous lead ions by thioether crown molecules, and the interfacial complexation of an aqueous dipeptide by dibenzo-18-crown-6 as ionophore. The mass spectra also give valuable information on the stoichiometry of the complexation reaction. BESI is also applied to the study of phospholipids, which are known to adsorb at the liquid-liquid interfaces, like their interactions with metal ions and peptides. Metal ion-phospholipid complexes show the formation of clusters containing one metal ion, where the size depends on the valence of the ion. As for the peptide-phospholipid complexes, it could be formed under these conditions and the stability of these non-covalent complexes are related to their structure. Hence, BESI sources offer new analytical opportunities in different research fields.

EPFL2009

Copper(I) and copper(II) binding to β-amyloid 16 (Aβ16) studied by electrospray ionization mass spectrometry

Hubert Girault, Yu Lu, Michel Prudent, Liang Qiao

Copper-b-amyloid 16 (Aβ16) complexes were investigated by electrospray ionization mass spectrometry (ESI-MS). Copper(I) and (II) complexes were formed on-line in a microchip electrospray emitter by using a sacrificial copper electrode as the anode in positive ionization mode. In the presence of ascorbic acid in the peptide solution, the amount of Cu(I)-Aβ16 generated electrochemically was even higher. A kinetic model is proposed to account for the generation of copper complexes. The structure of Cu(I)-Aβ16 was investigated by tandem mass spectrometry (MS/MS), and the binding site of Cu(I) to Aβ16 was identified at the His13, His14 residues. Cu(II)-Aβ16 was also investigated by MS/MS and, based on the unusual observations of a-ions, the two binding residues of His13 and His14 of Aβ16 to Cu(II) were also confirmed. This approach provides direct information on Cu(I)-Aβ16 complexes generated in solution from metallic copper and gives evidence that both His13 and His14 are involved in the coordination of both Cu(I)- and Cu(II)-Aβ16 complexes.