On-line mass spectrometry: membrane inlet sampling
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Significant insights into plant photosynthesis and respiration have been achieved using membrane inlet mass spectrometry (MIMS) for the analysis of stable isotope distribution of gases. The MIMS approach is based on using a gas permeable membrane to enable the entry of gas molecules into the mass spectrometer source. This is a simple yet durable approach for the analysis of volatile gases, particularly atmospheric gases. The MIMS technique strongly lends itself to the study of reaction flux where isotopic labeling is employed to differentiate two competing processes; i.e., O2 evolution versus O2 uptake reactions from PSII or terminal oxidase/rubisco reactions. Such investigations have been used for in vitro studies of whole leaves and isolated cells. The MIMS approach is also able to follow rates of isotopic exchange, which is useful for obtaining chemical exchange rates. These types of measurements have been employed for oxygen ligand exchange in PSII and to discern reaction rates of the carbonic anhydrase reactions. Recent developments have also engaged MIMS for online isotopic fractionation and for the study of reactions in inorganic systems that are capable of water splitting or H2 generation. The simplicity of the sampling approach coupled to the high sensitivity of modern instrumentation is a reason for the growing applicability of this technique for a range of problems in plant photosynthesis and respiration. This review offers some insights into the sampling approaches and the experiments that have been conducted with MIMS.
KeywordsMembrane-inlet mass spectrometry Oxygenic photosynthesis Water-splitting Carbonic anhydrase Water binding Artificial photosynthesis
Electron impact (or electron ionization)
Equilibrium isotope effect
Kinetic isotope effect
Membrane inlet mass spectrometry
Mass spectrometry overview
Mass spectrometry (MS) is an analytical technique that provides selectivity in mass for charged molecules or complexes in gas phase. Based on the initial gas ionization work of Wilhelm Wien in 1898 (Audi 2006), the concept of mass spectrometry using magnetic fields was further developed by Thomson (1913). He observed that a stream of ionized Ne+ ions passing through an electromagnetic field would take two different trajectories and concluded that Ne was composed of atoms of two different atomic masses (i.e., 20Ne and 22Ne). This provided the first evidence for the existence of stable isotopes. Since then, mass spectrometry has advanced to be a versatile and important analytical tool in science and engineering for purposes ranging from analyzing single atoms and small molecules to studying organisms up to the cell level (Kaltashov and Eyles 2005).
Ionization in the modern era includes techniques such as Electro Spray Ionization (ESI) and Matrix Assisted Laser Desorption Ionization (MALDI). These advances provide users with the possibility to study intact proteins with no apparent mass limitation. John Fenn and Koichi Tanaka were honored with the Nobel Prize in Chemistry (2002) for the discovery of ESI-MS. The ESI technique uses a capillary inlet operated with high voltage (~3–4 kV) to create a stream of evaporating charged solvent/analyte droplets that enter the vacuum of the mass spectrometer. The MALDI technique uses typically a pulse laser to a mixture of organic matrix and analyte molecules. The former technique is ideal for liquids, while the latter is suitable for solids such a proteins embedded in films or tissues (Kaltashov and Eyles 2005; Konermann et al. 2008).
Mass analyzer and ion detection
In order to separate and analyze ions of different mass there are two basic approaches: time or magnetic deflection. To separate ions of different weight by time, the Time-of-Flight (TOF) instrumentation uses the time it takes for ions to fly across an evacuated tube for analysis, while magnetic/electric sector field instruments intercept specific ion trajectories under the influence of an external magnetic/electric field. Both types of instrumentation enable separation of ions according to their individual m/z ratio with very high accuracy—the resolution is measured as a few parts per million.
The detector elements for isotope ratio instruments use simple faraday cups to collect the ion currents. The current per M•+ ion is one coulomb and this is converted via high gain amplification into a voltage for readout. Such cups have very long life and can be packed close together in arrays for simultaneous detection of multiple ions. Other detectors used in different mass spectrometers include electron multipliers and micro channel plates, but these are not used in isotope ratio magnetic sector instruments.
The key component of MIMS is a membrane that is typically 10–100 μm thick and can be a few cm2 in size. To prevent collapse it requires support from a porous supporting material that does not impose a significant diffusion barrier. Porous plastic sheeting or thin metal supports with fine holes can provide this function. To prevent water vapor entering the mass spectrometer, particularly as result of a membrane puncture, a cryogenic trap is installed between membrane and ion source. In addition to trapping water vapor the trap can be used to differentially remove other organics or gasses by choosing the trap temperature. The trap may be filled for example with dry ice/ethanol (~200 K) or liquid nitrogen (77 K).
The choice of membrane depends on the experiment. If high sensitivity is required then a highly permeable membrane and a large inlet area are advantageous to facilitate a higher rate of gas sampling. It may also be possible in some circumstances to operate with a higher vacuum to influence greater gas transmission. In contrast, if long term sampling is required with near constant background gas concentrations, then a low consumption (i.e., thicker) membrane is required and/or use of a small sampling area. Most membranes have a good chemical resistance and if measurements are undertaken at elevated pressure (e.g., 20 bar) a supported membrane with an embedded metal grid can be used. A range of membranes suitable for MIMS applications are the following: silicone membranes (MEM-213, Mem Pro); Teflon films such as FET or AF (DuPont); silicone rubber; oxygen electrode membranes3; HDPE plastic films (various sources); silicon membranes with embedded metal grid (Franatech GmbH, Germany). Thus, the choice of MIMS sensitivity versus gas concentration stability is an important factor in the experimental design.
Stable isotopes that are important for isotope ratio MS and their levels of natural abundance
Mass of atom (u)
Practical applications of MIMS
Whole leaf photosynthesis and respiration
Photosynthesis and respiration are important biological processes which involve the flux of O2 and CO2 species into and out of biological tissues, particularly leaves. The benefits of studying whole leaf photosynthesis and respiration by net CO2 and O2 concentrations have been a strong incentive for using field instrumentation such as the infrared gas analyzers like the LI-6400 (LI-COR Environmental, Nebraska USA). However, when it comes to the separation of in vivo CO2 and O2 fluxes mass spectrometry is the technique of choice because of its ability to monitor CO2 and O2 species with one instrument and to selectively analyze all isotopes of these gases. The unique fact that makes isotopic approaches particularly useful in photosynthetic organisms is that the O2 evolved from PSII has the isotopic signature of water while the oxygen uptake reactions consumes the gaseous oxygen. Thus, measurement of gross oxygen evolution and gross oxygen uptake can be achieved by the use of enriched 18O2 atmospheres and H 2 16 O (Radmer and Kok 1976). Although there are obvious issues with field deployment, mass spectrometry has been crucial in resolving O2 and CO2 fluxes in plants and algae that can be brought into the laboratory. The first experiments with algae (Radmer and Kok 1976; Radmer and Ollinger 1980b) and leaves (Canvin et al. 1980) answered many important questions regarding CO2 and O2 metabolism in plants.
Liquid-phase measurements of photosynthesis in solution (i.e., algae, chloroplasts) are equivalent in concept to leaf gas exchange (Badger and Andrews 1982; Espie et al. 1988; Hanson et al. 2003), except that there are different solubilities of the gases which alter measurement sensitivities. Thus, O2 is measured with greater sensitivity while CO2 may be less sensitive due to the fact that CO2 equilibrates with hydrogencarbonate (formerly termed bicarbonate) in solution and CO2 may be only a small fraction of the total inorganic carbon used for photosynthesis. The ratio of CO2/hydrogen carbonate will depend on the pH of the assay reaction and will decrease at alkaline pH. Liquid-phase measurements are particularly useful for studying aquatic photosynthesis, since for such systems there are no other techniques which allow for detailed examinations of both CO2 and O2 fluxes associated with photosynthesis (Badger et al. 1994; Palmqvist et al. 1994; Woodger et al. 2005; Rost et al. 2006).
The assay of CA activity by MIMS has several advantages compared to other techniques: it is rapid and accurate over a wide temperature ranges, but a unique feature is that enzymatic activity is obtained at chemical equilibrium—i.e., under conditions of equilibrated CO2 and HCO3 − concentration. Other CA assays in contrast, using 14C labeling or pH transients, are reliant on rapid changes in the equilibrium that are slowed on ice and are not obtained at chemical equilibrium.
It is also possible to express isotopic exchange more qualitatively as the change in 18O enrichment (18α) as given by Eq. 6. When the enrichment is plotted as the natural log(18α) for CO2 versus time (Mills and Urey 1940) the slope of the line gives a measure of the pseudo first-order rate constant for hydration of CO2 by the CA reaction, see Fig. 5b. The rate of this reaction is commonly compared to the uncatalysed rate in the same assay solution (Badger and Price 1989; Von Caemmerer et al. 2004; Clausen et al. 2005).
Real time isotopic fractionation
Substrate water exchange in PSII
Isotopic exchange of water-derived oxygen ligands of the oxygen-evolving complex (OEC) into O2 has been of long standing interest with PSII, because it contains information of how, when, and where substrate-water is bound to the OEC and in what manner it is oxidized to molecular O2—e.g. via: (1) a sequence of oxidation steps involving partial water oxidation intermediates; or (2) a concerted reaction mechanism during the S3 → S0 transition. A MIMS approach to this question was first employed by Radmer and Ollinger (Radmer and Ollinger 1980a). They attempted to determine the rate of appearance of 18O in the O2 products of water splitting by PSII samples suspended in 18O-enriched water. The experiment is analogous to stop-flow experiments and requires rapid injection/mixing of isotopically labeled water into the suspension of photosynthetic samples followed by a series of light flashes to photogenerate O2. This first MIMS experiment indicates that water exchanges rapidly and by inference conceded that there are no non-exchangeable stable water oxidation products (e.g., bound peroxides) up to the S3 state of the OEC. This work and others that followed (Radmer and Ollinger 1980a, 1986; Bader et al. 1993) were limited by mixing/stabilization times of >30 s, and it wasn’t until more rapid mixing techniques were developed that also strongly reduced the O2 background rise from the injection of the labeled water that more specific information about water binding could be learned (Messinger et al. 1995).
In order to evaluate the S-state dependence of the 18O exchange rates, the sample is preset in the various S states with appropriate pre-flash protocols. The sample chamber is optically coupled to a bank of three xenon flash lamps via a 3-to-1 fiber optic to enable fast turnover sequences to be initiated. The 18O-water injection can be accomplished with a t½ ~5 ms and subsequent Xe turnover flashes given 5–10 ms apart to photogenerate O2. Since the actual instrumental response time is relatively slow (~10 s due to the diffusion of the O2 gas across the semi-permeable membrane into the inlet line), the flash spacing of a subsequent flash sequence that is used to normalize the oxygen signals is increased, typically to 20 s. As such, in order to retard the deactivation reactions of the higher S states, the temperature of the sample is reduced (usually to 10°C). Details of the set-up have been published earlier (Messinger et al. 1995; Hillier and Wydrzynski 2000, 2004).
Photobiological hydrogen production
There are three types of photobiological H2 producing processes in nature: (i) oxygenic photosynthesis coupled to hydrogenases, (ii) oxygenic photosynthesis coupled to nitrogenases, and (iii) non-oxygenic photosynthesis coupled to nitrogenases (for review see Ghirardi et al. 2009). Understanding these biological processes on the level of whole cell metabolism and elucidating the reaction mechanisms of the involved enzymes is expected to allow optimizing the yields of the biological processes and constructing efficient artificial systems (Melis and Happe 2004; Lubitz et al., 2008). A key aspect in these endeavors is the detailed characterization of the H2 production under different conditions, for example at different oxygen levels. Two prominent methods for this are the electrochemical characterization of hydrogenases (Armstrong, this issue) and the online recording of H2 production/consumption rates and of the rates of H/D exchange between D2 and H2O by MIMS (Hemschemeier, Melis and Happe, this issue; Vignais 2005). The experimental set-up for the MIMS reactions is very similar to that described above, only that conditions are applied (e.g. larger sample volume, smaller inlet, thicker membrane) that reduce the gas consumption rates of the mass spectrometer (for details see Vignais 2005).
Synthetic model systems
With the dramatic anthropogenic increase in atmospheric CO2 concentration considerable interest has been created in the development of artificial water-splitting and hydrogen-forming catalysts. These can be either molecular devices that are directly driven by light, or compounds covering an electrode surface that is eventually powered by electricity created in solar panels. If the catalysts are made of earth-abundant materials, such an approach can provide the means for producing hydrogen from water in a sustainable way (Lubitz et al. 2008).
We hope that we were able to demonstrate in this short overview article that since its development in the early 1960s Membrane Inlet Mass Spectrometry has become an important technique for the study of gases, particularly those associated with photosynthetic reactions. But it is also seen as increasingly useful for testing catalytic enzymatic activity and catalysts for artificial water-splitting and hydrogen generation. The technique through the years has essentially remained unchanged in terms of the basic sampling design. However, the mass spectrometers have advanced tremendously both in terms of sensitivity and stability and additionally are increasingly equipped with multiple-ion collector arrays for detection of multiple ion signals. Such developments have opened up some tremendous new insights and MIMS has significant advances in terms of kinetic analysis and sample throughput. While we have concentrated here on examples closely related to photosynthesis, it is worth noting that this technique has had also a significant impact on many other fields, and has found essential applications in many different areas of research that involve gas evolution or consumption (for a recent review see Konermann et al. 2008).
Databases with fragmentation patterns of numerous molecules, including biopolymers are available at e.g. http://webbook.nist.gov/chemistry/mw-ser.html; MS companies additionally provide library software.
The permeability is a product of the diffusion constant (D) and solubility coefficient of the gas in the membrane.
YSI provides a 12.5 µm high sensitivity and a 25.5 µm standard sensitivity Teflon membrane, Hansatech a 25 µm Teflon membrane.
Molecular oxygen is somewhat simplified as there is also a 0.0374% enrichment of 17O at natural abundance. This can be taken into consideration by expansion of the Eq. 4. However, molecular oxygen species from 17O at m/z = 33, 34 and 35 at natural abundance are very small (0.07462, 0.00001, and 0.00015% respectively) and for MIMS approaches can practically be ignored.
HC18O3 − is prepared by incubating NaHCO3 in >95% 18O-water. Isotopic equilibration is ~24 h at room temperature and converts the hydrogencarbonate to triply 18O labeled species.
Support for this work was provided by the Australian Research Council DP0770149 (to WH & TW) and the ARC center of excellence in Plant Energy Biology (to MRB), the Max-Planck Gesellschaft and the Wallenberg-Foundation (to JM).
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