B American Society for Mass Spectrometry, 2019 J. Am. Soc. Mass Spectrom. (2019) DOI: 10.1007/s13361-019-02183-w SHORT COMMUNICATION Ultra-High Resolution Elemental/Isotopic Mass Spectrometry (m/Δm > 1,000,000): Coupling of the Liquid Sampling-Atmospheric Pressure Glow Discharge with an Orbitrap Mass Spectrometer for Applications in Biological Chemistry and Environmental Analysis Edward D. Hoegg,1 Simon Godin,2 Joanna Szpunar,2 Ryszard Lobinski,2 David W. Koppenaal,3 R. Kenneth Marcus1 1Department of Chemistry, Clemson University, Clemson, SC 29634, USA 2CNRS, Institute for Analytical & Physical Chemistry of the Environment & Materials, UPPA, IPREM, UMR 5254, Helioparc 2, Av Pr Angot, F-64053, Pau, France 3Pacific Northwest National Laboratory, EMSL, 902 Battelle Blvd, Richland, WA 99354, USA Abstract. Many fundamental questions of astro- physics, biochemistry, and geology rely on the ability to accurately and precisely measure the mass and abundance of isotopes. Taken a step further, the capacity to perform such measure- ments on intact molecules provides insights into processes in diverse biological systems. De- scribed here is the coupling of a combined atomic and molecular (CAM) ionization source, the liquid sampling-atmospheric pressure glow discharge (LS-APGD) microplasma, with a commercially available ThermoScientific Fusion Lumos mass spectrometer. Demonstrated for the first time is the ionization and isotopically resolved fingerprinting of a long-postulated, but never mass-spectrometrically observed, bi-metallic complex Hg:Se-cysteine. Such a complex has been impli- cated as having a role in observations of Hg detoxification by selenoproteins/amino acids. Demonstrated as well is the ability to mass spectrometrically-resolve the geochronologically important isobaric 87Sr and 87Rb species (Δm ~ 0.3 mDa, mass resolution m/Δm ≈ 1,700,000). The mass difference in this case reflects the beta-decay of the 87Rb to the stable Sr isotope. These two demonstrations highlight what may be a significant change in bioinorganic and atomic mass spectrometry, with impact expected across a broad spectrum of the physical, biological, and geological sciences. Keywords: High resolution, Microplasma, Orbitrap, Isotope analysis, Metallobiochemistry, Mercury:selenoamino acid Received: 11 January 2019/Revised: 27 January 2019/Accepted: 3 March 2019 Introduction nology, metabolism, nuclear forensics, and nutrition are just a few of the areas where the ability to determine or monitor he need to assess the isotopic composition of materials isotopic compositions are of relevance. Mass spectrometry of Ttouches many fields of basic and applied sciences. Ques- one form or another is the method of choice for the determina- tions of the origins of the universe, atomic physics, geochro- tion of stable and long-lived radioisotopes. Methods to deter- mine isotopic composition lie in the realm of atomic mass spectrometry, wherein the ultimate function of the ionization Correspondence to: R. Marcus; e-mail: [email protected] source is to produce monoatomic ions, usually to the exclusion E. D. Hoegg et al.: Coupling of a CAM Ionization Source with Orbitrap Analyzer of latent molecular information. Thermal ionization mass spec- At present, the device has been coupled to over one-half trometry (TIMS) and inductively coupled plasma mass spec- dozen MS platforms; all of which were equipped with standard trometry (ICP-MS) are the standard bearers in this genre. ESI interfaces. The Orbitrap Fusion Lumos 1M analyzer plat- Elemental signatures (usually metals) are becoming form is designed to provide ultrahigh mass resolution (m/Δm ≈ increasingly relevant as the roles of metals in biochemical 1,000,000 @ m/z = 200) in conjunction with ESI ionization and environmental systems become more evident. Herein towards applications in proteomics. The coupling of the CAM lies the continuously expanding area of metallomics. The ion source with the Orbitrap Lumos provides unique capabili- efficacy of a metal in any biological system, or its trans- ties in terms of diversity of analytes, ultrahigh mass resolution, port in the environment, is dictated by its chemical form. and high precision isotopic analysis. The two demonstrations In this regard, it is not sufficient to identify the presence here highlight what may be a step function change in atomic of a metal in a specimen, but more significant is the and bioinorganic mass spectrometry, with impact expected identification of its molecular form. Electrospray ioniza- across a broad spectrum of the physical, biological, and geo- tion (ESI) has been a workhorse in the ability to produce logical sciences. intact molecular species; so long as the chemistry of the entity is amenable to its ionization in a suitable solution phase. In order to make maximum use of high-performance Experimental mass spectrometry platforms, ionization sources which are operationally compatible with the MS, while also While the previous reports of the coupling of the LS-APGD capable of affecting the desired ionization functions, are to Orbitrap platforms have used essentially the same required. The focus of the present communication is the breadboard-based ion source assembly [3, 4], we employed demonstration of the use of a singular, combined atomic here an ion volume cube (constructed from aluminum) to and molecular (CAM) ionization source, coupled to an mount on the Fusion Lumos, along with a new, integrated ultrahigh resolution ThermoScientific Orbitrap Fusion utility control box. The combined atomic and molecular Lumos 1 M mass analyzer towards important problems (CAM) microplasma is struck (10–40 mA; 100–1000 V in bioinorganic chemistry and geochronology; the identi- d.c.) between the surface of the electrolytic solution contain- fication of an intact mercury:seleno amino acid complex ing the analyte solutes and a stainless steel counter elec- and the direct mass spectrometric resolution of 87Sr and trode. The gap between the two electrodes is controlled with 87Rb. Both of these demonstrations are believed to be an adjustable micrometer, with the distance between the the first of their kind. Specifically, the liquid sampling- solution electrode and the entrance to the ion transfer capil- atmospheric pressure glow discharge (LS-APGD) lary fixed at 0.5 cm. A helium sheath gas (0.2–0.5 L min−1) microplasma [1, 2] is employed to efficiently ionize surrounds the capillary delivering the test solution at flow atomic species as well as organometallics, without rates of 10–30 μLmin−1. The sole difference in the opera- changing the ionization source. The LS-APGD has pre- tion of the CAM ionization source between the atomic and viously been shown to provide very high precision mea- molecular implementations is that the former operating with surements of uranium isotope ratios [3, 4], while also 2%HNO3 as the carrier (electrolyte) solution and the latter having the capability to ionize diverse organic species with a 70:30 mixture of methanol:water. With the [5], from small molecules to proteins. The different microplasma operating in the constant-current mode, there Batomic^ and Bmolecular^ operation modes are affected was no discernible difference in maintenance voltage in by a simple change in delivery solvent (solution electro- changing between these solvents. lyte), 5% HNO3 in the former case, and 70:30 The ThermoScientific (San Jose, CA) Orbitrap Fusion methanol:water in the latter. This differential level of Lumos 1M instrument was operated without any modifica- information has been demonstrated explicitly for the case tions [7], other than the replacement of the as-delivered ESI of uranyl acetate, where operation in nitric acid yields a source cube with that of the LS-APGD. Beyond a much spectrum dominated by U+ and UO+, with very minor higher resolution rating, the Lumos has added functionality amounts acetate-related species while the mixed solvent beyond the previous Exactive-series instruments, including yields almost exclusively the ligated uranium [6]. Oper- higher-order collisional activation regions, which are not ation under mixed-solvent (MeOH:H2O) conditions ap- employed here. In these experiments, in-source collisional pears to yield a plasma with lower kinetic temperatures, dissociation was implemented as a means of removing producing spectra that are comparable to atmospheric loosely bound solvent molecules, while high energy colli- pressure chemical ionization (APCI), composed predom- sional dissociation (HCD) was employed for further activa- inately of protonated psuedomolecular ions (M+H)+. tion prior to mass analysis in the Orbitrap cell. The overall Likewise, injections of caffeine (for example) under the Lumos operation was controlled under the TUNE function- acidic electrolyte case result in very low intensity, struc- ality and data acquisition in the Excalibur environment. The turally complex spectra [5], reflecting a more kinetically specific operational parameters of the MS are provided with energetic environment. the figure captions. E. D. Hoegg et al.: Coupling of a CAM Ionization Source with Orbitrap Analyzer Results and Discussion (MeHg) and selenocysteine (Se-Cys) in the presence of the reducing agent dithiothreito, l and a 2% HNO3 final diluent, Application of the CAM Source:Orbitrap with the mixture having a final molar concentration of ~ 3.3 × Combination in Biological Chemistry 10−6 M of each of the reactants. In this case, the use of acid as One of the primary drivers in the development of the CAM the final diluent