Anal. Chem. 1999, 71, 2003-2013
Protocol for the Characterization of Oceanic Particles via Flow Cytometric Sorting and Direct Temperature-Resolved Mass Spectrometry Elizabeth C. Minor* and Timothy I. Eglinton†
Department of Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543 Jaap J. Boon‡
FOM Institute for Atomic and Molecular Physics, 1098SJ Amsterdam, The Netherlands Robert Olson§
Biology Department, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543
This paper presents a protocol for applying flow cytometric (FC) sorting and direct temperature-resolved mass spectrometry (DT-MS) to oceanic particulate organic matter (POM) samples. Flow cytometric sorting allows the physical separation of particle subclasses on the basis of morphological and chemical criteria including size, shape, and autofluorescence characteristics. Direct temperatureresolved mass spectrometry is a rapid (∼2-min) measurement that provides fingerprints of molecular-level characteristics across a wide range of compound classes, including both desorbable and pyrolyzable components. The resulting DT-MS data, therefore, can bridge the gap between information available from bulk measurements, such as elemental analysis, and information derived from detailed but laborious compound-class-specific analyses. In addition, the sensitivity of DT-MS (which requires only microgram quantities of sample) allows it to be used in conjunction with flow cytometric sorting. In this study, the sample-handling procedures required by these techniques are described in detail and shown to yield useful, representative results. The study of particulate organic matter (POM) in the oceans is complicated by heterogeneity occurring in at least two different organizational levels. At the population level, POM consists of many different types of living and nonliving particles including phytoplankton, phytodetritus, zooplankton, fecal material, aeolian dust, etc. At the molecular level, it consists of many different classes of organic compounds, including (but not restricted to) major biochemical constituents of autochthonous and allochthonous organic matter (OM) inputs such as polysaccharides, * Corresponding author. Current address: FOM-AMOLF, Kruislaan 407, 1098 SJ Amsterdam, The Netherlands. Tel.: ++31 0(20) 6081234. E-mail: l.minor@ amolf.nl. † Tel.: (509)457-2000. E-mail:
[email protected]. ‡ Tel.: ++31 0(20) 6081234. E-mail:
[email protected]. § Tel.: (508)457-2000. E-mail:
[email protected]. 10.1021/ac9811726 CCC: $18.00 Published on Web 04/13/1999
© 1999 American Chemical Society
proteins, lipids, and lignin. How the heterogeneity within these two organizational levels is connected is not yet well-understood. In other words, the general molecular-level characteristics of phytoplankton, phytodetritus, fecal material, and other subclasses within field samples of POM have not yet been determined. Molecular-level work has been performed on marine phytoplankton, zooplankton, and bacterial cultures, but the applicability of results from culture studies to natural populations in oceanic environments has been called into question by many researchers. Flow cytometry1 (FC) can improve our understanding of POM at the population level by distinguishing phytoplankton from nonphytoplankton particles on the basis of chlorophyll fluorescence. In conjunction with staining techniques, it can also be used to identify additional populations of particles or to probe POM chemical composition.2 In the latter case, however, it is difficult to obtain detailed molecular-level compositional information on more than a handful of compounds at a time. By coupling flowcytometric sorting with selected analytical chemistry techniques, much more detailed chemical information can be obtained on each sorted subpopulation within a POM sample. Direct temperature-resolved mass spectrometry (DT-MS) can improve our understanding of POM heterogeneity at the molecular level since it provides rapid molecular-level fingerprinting across a range of compound classes.3,4 In this technique, microgram quantities of unextracted, underivatized organic matter are placed on a platinum/rhodium sample wire and inserted directly into the ion source of a mass spectrometer. The wire is resistively heated following a controlled-heating program that allows desorbed material to elute off the wire before extensive pyrolysis occurs. Therefore, information on both desorbable and pyrolyzable (1) Herzenberg, L. A.; Sweet, R. G. Sci. Am. 1976, 243, 108-112. (2) Moreira-Turcq, P.; Martin, J. M.; Feury, A. Mar. Chem. 1993, 43, 115126. (3) Boon, J. J. Int. J. Mass. Spectrom. Ion Processes 1992, 118/119, 755-787. (4) Eglinton, T. I.; Boon, J. J.; Minor, E. C.; and Olson, R. J. Mar. Chem. 1996, 52, 27-54.
Analytical Chemistry, Vol. 71, No. 10, May 15, 1999 2003
substances are obtained. The temperature or time axis can be used to determine whether the ion of interest is a desorption or pyrolysis product (or both). As DT-MS only requires microgram quantities of unextracted, underivatized sample, it can also provide molecular-level information on subpopulations of POM identified by flow cytometry and physically isolated by flow-cytometric sorting. Our goal, therefore, is to combine these different approaches to study POM in a manner that reveals detailed compositional information within and between particle populations. The application of flow-cytometric sorting and DT-MS to the study of oceanic organic matter imposes certain nontraditional sample-handling challenges. Samples must be suspended in liquid for introduction to the flow-cytometry flow cell and for application to the DT-MS sample probe. In addition, flow cytometry and DTMS have two somewhat contradictory requirements. For flow cytometry, the particles must remain intact; for DT-MS analysis, the salt content of the samples must be minimized. Therefore, the introduction of osmotic pressure during the desalting of DTMS samples must occur after flow-cytometric analysis. In addition, potential cell lysis should not lead to significant organic-matter losses prior to mass spectrometry. With these requirements, conventional methods for the recovery of particles (e.g., the use of glass-fiber filters) are inappropriate. The use of DT-MS for broad-band molecular-level characterization imposes some additional considerations. Electron-impact ionization (the most commonly used ionization mode) provides information on a wide range of compound classes, including a large number of contaminants. Interferences from these contaminants must, therefore, be minimized. While one of the main advantages of DT-MS is its sensitivity, small sample sizes increase the difficulty in analyzing representative portions of heterogeneous samples, such as large-particle (>53 µm) POM. Finally, the presence of sea salt interferes with DT-MS, often swamping the detector with inorganic species (e.g., HCl, m/z 36, and SO2, m/z 64) and changing the dissociation pathways of organic molecules.5 Effective desalting is, as mentioned above, a prerequisite for obtaining informative mass-spectrometric data. Although flow cytometry and DT-MS are not yet widely used as analytical tools in marine chemistry, it is likely that these techniques will see increasing application as instrumentation improves in flexibility, availability, and ease of use. These techniques can also be used to meet the increasing demands for the generation of dense, information-rich, and statistically relevant data that are required to develop and constrain models of ocean biogeochemistry. Therefore, this paper offers a protocol for sample collection, processing, and measurement which yields reproducible, informative results. The type and breadth of information attainable from DT-MS analyses of POM have been the focus of a previous study.4 EXPERIMENTAL SECTION Analytical Method. The analytical method is outlined in Figure 1. Seawater is collected via Niskin bottle, diaphragm pump, or peristaltic pump. It is prefiltered through a nylon 53-µm screen to separate large-particle POM from small-particle POM (the 53µm cutoff has been used in previous studies to separate an (5) van der Kaaden, A.; Haverkamp, J.; Boon, J. J.; de Leeuw, J. W. J. Anal. Appl. Pyrolysis 1983, 5, 199-220.
2004 Analytical Chemistry, Vol. 71, No. 10, May 15, 1999
Figure 1. Scheme for isolating and analyzing oceanic large-particle POM, small-particle POM, “phytoplankton”, and “detritus.”
approximation of sinking POM from an approximation of suspended POM).6 Tangential flow filtration or TFF (0.8-µm Fluoro centrasette and ultrasette, Filtron, driven by Masterflex peristaltic pumps) is used to concentrate the smaller particles while keeping them suspended in seawater. The 0.8-µm Fluoro filter membrane was selected for several reasons. Its nominal pore size is similar to that of a commonly employed filter in oceanographic studies (the Whatman GF/F filter), its chemical characteristics limit the potential for sorption of sample material to the surface, and it is compatible with cleaning agents necessary for preventing sample crossover. For a typical sampling station, 4-100 L of seawater are concentrated to approximately 100 mL of >0.8 µm retentate. The larger volume samples are initially processed via Filtron centrasette (inlet pressure 1 µm obtained from Coulter Counter determinations14). While this discrepancy may result from filtration effects, it must be remembered that the flowcytometry counts were made on samples from a different location at a different time and that only a few milliliters of seawater (or the equivalent) were actually analyzed. The 0.8-53 µm coastal (wh) particle sample shows a 13-fold reduction in both detritus (14) McCave, I. N. Deep-Sea Res. 1975, 22, 491-502.
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Figure 4. (a) Flow cytometric analysis, reported as counts/mL of seawater of unfiltered and filtered aliquots of two seawater samples (wh from Great Harbor, Woods Hole, MA and s from the northwest Atlantic Ocean, 37°15.03′N, 73°3.03′W) and 0.8 rd and >2.0 rd indicate, respectively, 0.8-53 µm and 2.0-53 µm filtered seawater aliquots rediluted to their original concentrations. (b) Same FC analyses as in (a), but reported as a ratio of phytoplankton counts to phytoplankton plus detritus counts.
counts/mL of seawater and phytoplankton counts/mL of seawater when compared to unfiltered wh. The 2.0-53 µm aliquot of wh differs little from the 0.8-53 µm aliquot in either phytoplankton or detrital counts/mL of seawater. The open-ocean sample (s) detrital counts decrease 2-fold after tangential-flow filtration, while the phytoplankton counts decrease 4-fold. The decreases in phytoplankton and detritus counts after tangential-flow filtration could result from flow-cytometry sensitivity to particles that would pass through our 0.8-µm TFF membrane. Forward angle light scatter is merely a proxy for particle size and is affected by particle shape and composition. In addition, the forward angle light scatter detector was optimized for viewing phytoplankton populations rather than for limiting detection to fluorescent bead standards in size ranges greater than 0.8 µm. Therefore, we could merely be seeing the effect of imposing a size distinction. Given the strong bias of oceanic particles toward smaller sizes,14 the inclusion of a small percentage of the total
