Anal. Chem. 1998, 70, 3206-3212
Direct Analysis of Single Rat Peritoneal Mast Cells with Laser Vaporization/Ionization Mass Spectrometry Eliza N. Fung and Edward S. Yeung*
Ames LaboratorysUSDOE and Department of Chemistry, Iowa State University, Ames, Iowa 50011
A linear time-of-flight mass spectrometer was used as a detector for flow cytometry. These two techniques were coupled by a laser vaporization/ionization interface. The estimated mass detection limit of the combined system was 20 amol of serotonin standard with one laser pulse. An aqueous buffer at physiological pH was used to ensure compatibility with cells. Rat peritoneal mast cells (RPMCs) were dispensed into the mass spectrometer in a single file confined within a 20-µm-i.d. capillary. By using the mass spectrometer as a detector, no precolumn staining or derivatization is required. Determination of serotonin and histamine in individual cells was demonstrated. With this method, hundreds of cells can be analyzed within a few minutes. The average amounts of histamine and serotonin per RPMC were found to be 0.75 ( 0.33 and 0.11 ( 0.06 fmol, respectively. No correlation was found between the amounts of the two amines in each cell.
Cells are the fundamental units of living organisms. Complex organisms such as mammals have different types of cells in different organs. Each cell has a complex mixture of biomolecules such as proteins and DNA. The direct analysis of individual cells and substructures within individual cells is rapidly becoming a powerful tool with which to investigate questions related to cellular metabolism, signal transduction, and differentiation. The techniques that are used must be very sensitive because there is only a minute amount of sample present in a cell. In addition, the ability to provide fast analysis is a desirable feature. Flow cytometry is a well-known and effective tool in the analysis of single cells.1-6 In flow cytometry, a flowing stream of cells is lined up in single file with the aid of a sheath liquid. (1) Van Dilla, M. A., Dean, P. N., Laerum, O. D., Melamed, M. R., Eds. Flow Cytometry: Instrumentation and Data Analysis; Academic Press: London, 1985. (2) Pfeffer, F. I.; Colvin, R. B. In Analysis and Sorting by Flow Cytometry: Applications to the Study of Human Disease; Pretlow, T. G., II, Pretlow, T. P., Eds.; Academic Press: London, 1987; pp 311-340. (3) Melamed, M. R., Mullaney, P. F., Mendelsolu, M. L., Eds. Flow Cytometry and Sorting, 2nd ed.; Wiley: New York, 1990. (4) Shapiro, H. M. Practical Flow Cytometry, 3rd ed.; Wiley: New York, 1995. (5) Papavasilion, F.; Casellas, R.; Suh, H.; Qin, X.; Besmer, E.; Pelanda, R.; Nemazee, D.; Rajewsley, K.; Nussenzweig, M. C. Science 1997, 278, 298300.
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Interesting analytes in cells are usually labeled with fluorescent dyes that bind specifically to the analytes. The fluorescence signal is detected when the cell passes the focal point of the laser. The distinct advantage of flow cytometry includes high-speed acquisition of information on the cell population as well as on individual cells. However, the applicability of this technique is subject to the availability of a suitable fluorescence probe. Mass spectrometry (MS), on the other hand, provides definitive information on the identity of biomolecules.7-11 In particular, time-of-flight mass spectrometers (TOF-MS) with scan time of much less than 1 ms are well-suited for coupling with high-speed separation methods. If these two techniques are coupled for the analysis of single cells, then it may prove to be an effective disease-screening method. Finally, the elimination of fluorescence labeling can significantly reduce labor requirements. Serotonin (5-hydroxytryptamine) and histamine are two important biogenic amines in mammalian and nonmammalian signal transduction systems.12-14 They are believed to be mediators of allergic reactions.15 Mast cells in rats are rich in these two amines.16 The mast cells are important target cells in IgE antibody-mediated allergic reaction and they release a variety of mediators upon challenge with specific antigens. Rat peritoneal mast cells (RPMCs) have been widely used as a model to study the release of allergic mediators.17,18 A number of methods have (6) Jacquemum-Salon, A., Ed. Flow Cytometry: New Developments; SpringerVerlag: Berlin, 1993. (7) (a) Hofstadler, S. A.; Swanek, F. D.; Gale, D. C.; Ewing, A. G.; Smith, R. D. Anal. Chem. 1995, 67, 1477-1480. (b) Hofstadler, S.; Severs, J. C.; Smith, R. D.; Swanck, F. D.; Ewing, A. G. Rapid Commun. Mass Spectrom. 1996, 10, 919-922. (8) Valaskovic, G. A.; Kelleher, N. L.; McLafferty, F. W. Science 1996, 273, 1199-1202. (9) Xue, Q.; Foret, F.; Dunayevskiy, Y. M.; Zavracky, P. M.; McGruer, N. E.; Karger, B. L. Anal. Chem. 1997, 69, 426-430. (10) Nedved, M. L.; Habibi-Goudarzi, S.; Ganem, B.; Henion, J. D. Anal. Chem. 1996, 68, 4228-4236. (11) Dunayevskiy, Y. M.; Vouros, P.; Winters, E. A.; Shipps, G. W.; Carell, T.; Rebek, J., Jr. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 6152-6157. (12) Whitaker-Azmitia, P. M., Peroutka, S. J., Eds. The Neuropharmacology of Serotonin; Ann. N.Y. Acad. Sci. 1990, 600. (13) Galli, S. J.; Wershill, B. K. Nature 1994, 381, 21-22. (14) Meltzer, H. Br. J. Psychol. 1989, 155 (Suppl. 8), 25-31. (15) Colten, H. R. Nature 1994, 371, 474-475. (16) Helander, H. F.; Bloom, G. D. J. Microsc. 1974, 100, 315-321. (17) Padawer, J. Ann. N.Y. Acad. Sci. 1963, 103, 87-138. (18) Langunoff, D.; Chi, E. Y. Cell Biology of Mast Cells and Basophils. In The Cell Biology of Inflammation; Glynn, L. E., Houck, J. C., Weissmann, G., Eds.; Elsevier/North-Holland: New York, 1980; pp 217-265. S0003-2700(98)00290-X CCC: $15.00
© 1998 American Chemical Society Published on Web 06/12/1998
been developed to analyze the contents of an individual RPMC and its exocytotic release of mediators including histochemistry,16 patch clamp techniques,19 capillary electrophoresis coupled with laser-induced fluorescence (LIF),20 UV imaging,21 and electrochemical detection.22 In this research, a laser vaporization/ionization interface is used to couple flow cytometry and mass spectrometry. RPMCs are lined up in a single file, confined within a 20-µm-i.d. capillary, and moved hydrodynamically toward the mass spectrometer. Once a cell reaches the focal point of the laser beam, its content is vaporized, ionized by the laser, and subsequently extracted into the flight tube of the time-of-flight mass spectrometer. Serotonin and histamine are detected. Hundreds of cells can be analyzed in this way within 5 min. Thus, information on the cell population can be obtained as well as information on individual cells.
EXPERIMENTAL SECTION Materials. Capillaries were obtained from Polymicro Technologies Inc. (Phoenix, AZ). Sprague-Dawley rats were purchased from Laboratory Animal Resources (Iowa State University, Ames, IA). The chemicals used were purchased from several vendors: the CO2-independent medium and L-glutamine from Gibco BRL; histamine and metrizamide from Sigma (St. Louis, MO); fatty acid-free bovine serum albumin and ammonium chloride from ICN Biochemicals (Aurora, OH); serotonin hydrochloride and copper(II) chloride from Aldrich (Milwaukee, WI). All chemicals were used as received without any further purification. Deionized water was purified by Milli-Q Synthesis and Elix 5 (Millipore, Bedford, MA). The ammonium chloride solution was adjusted to the desired pH by adding a NaOH solution. Laser Desorption/Ionization Interface. The interface is based on a previous design with some modifications.23 It is briefly outlined here. A 50-cm capillary (20 µm i.d. and 360 µm o.d.) was glued onto a 0.635-cm glass rod, which was fed into the vacuum region of the mass spectrometer through a Quick Connect/Disconnect adapter (MDC, Hayward, CA). A KrF waveguide excimer laser (Model GX-1000, Potomac Photonic, Lanham, MD) was used to vaporize and ionize the eluent from the capillary. The 248-nm output provided a maximum of 60 µJ/ pulse with a pulse width of 15 ns. A 1.22-m focal length lens was used to collimate the laser output, which was directed into the MS chamber by a mirror. A 25.4-cm focal length lens was used to focus the laser beam to a 50-µm-diameter spot at the tip of the capillary. The laser energy measured at the capillary tip was 34 µJ, which corresponds to a power density of 1.38 × 106 W/cm2. The laser was operated at a repetition rate of 80 Hz. CuCl2 was added to the buffer solution to couple the laser energy for the vaporization and ionization processes. Ions were formed and extracted immediately into the TOF flight tube. To obtain a stable operation of the laser vaporization/ionization interface, a match of the rate of laser vaporization and solution flow rate is required. (19) deTeledo, A.; Fernandez-Chacon R.; Fernandez, J. M. Nature 1993, 363, 554-557. (20) Lillard, S. J.; Yeung, E. S. Anal. Chem. 1996, 68, 2897-2904. (21) Lillard, S. J.; Yeung, E. S. J. Neurosci. Methods 1997, 75, 103-109. (22) Pihel, K.; Hsieh, S.; Jorgenson, J. W.; Wightman, R. W. Anal. Chem. 1995, 67, 4514-4521. (23) Chang, Y.; Yeung, E. S. Anal. Chem. 1997, 69, 2251-2259.
The detection limit and sensitivity are routinely calibrated by a serotonin standard solution. The inlet end of the capillary is put in a sample chamber, which is pumped by a mechanical pump to the desired vacuum (which is higher than the pressure at the ion source) to provide a stable hydrodynamic flow. TOF Mass Spectrometer. A home-built linear time-of-flight mass spectrometer was used for ion detection. The ion optics are the Wiley-McLaren dual-grid configuration (R. M. Jordan, Grass Valley, CA). The repeller was set at 4.6 kV. The two acceleration plates are spaced 1.8 cm apart from the repeller. The voltage of the first grid was adjusted to focus the ion beam, and the second one was set at ground. A pair of X- and Y-deflectors was used to adjust the ion trajectories. The flight tube was 1 m long. The system was pumped by a M-6 diffusion pump (Varian, Lexington, MA) to a vacuum of 1 × 10-6 Torr. The ions generated were detected by a triple microchannel plate detector (MCP) (Galileo Electrooptics, Sturbridge, MA), which provides a gain of 5 × 107. Data Acquisition System. Ion current from the MCP was measured by a digital oscilloscope (LeCroy 9310AM, Chestnut Ridge, NY). Each measurement was triggered by the electrical signal generated by a photodiode (model 3399, Hamamatsu) that was irradiated by the scattered light from the laser pulse. The ion signals derived from individual laser pulses were stored in either a memory card (with a maximum memory of 512 kbyte) or a PC computer with a Pentium processor. The data were transferred to the computer through a GPIB interface card (GPIB PCII/IIA, National Instrument, Austin, TX). An in-house program (LabVIEW, National Instrument) was used to control the data transfer. Traces are acquired in the sequence mode (each sequence consists of 40 traces) and transferred to the computer. The acquisition rate was 40 traces/s with a time resolution of 10 ns/point. Acquisition parameters such as the sampling rate can be controlled at the front panel of the oscilloscope or remotely by the computer with the in-house program. Isolation of Mast Cells. A male Sprague-Dawley retired breeder rat was anesthetized by ether and decapitated. A lavage saline solution was prepared by adding 0.0587 g of L-glutamate and 0.10 g of acid-free BSA to 100 mL of CO2-independent medium; the solution was filtered through a 0.22-µm filter. The lavage saline (50 mL) was injected into the peritoneal cavity of the rat. After massaging the rat for 5 min to promote mixing, an incision was made into the peritoneal cavity. The saline injected was withdrawn by a transfer pipet. The cells were pelleted by centrifuging at 700g at 10 °C for 10 min. The supernatant was discarded. The cells were resuspended in 4 mL of the lavage saline and placed on top of a 22.5% metrizamide gradient in the lavage saline. The solution was centrifuged at 1400g at 10 °C for 20 min to remove contaminants such as macrophages. The supernatant was discarded, and the cells were rinsed with fresh lavage saline. The purified cells were resuspended in fresh lavage saline. Mast Cell Analysis. The cells were rinsed two times with fresh CO2-independent medium (without L-glutamate or BSA) and resuspended in 200 mL of 2 mM ammonium chloride and 0.1 mM CuCl2 (pH 7.3) solution. The solution was allowed to flow into Analytical Chemistry, Vol. 70, No. 15, August 1, 1998
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Figure 1. Averaged mass spectrum (400 laser pulses) of 100 µM serotonin in 2 mM NH4Cl at pH 7.3 buffer solution. Peaks: 1, Na+ ion; 2, K+ ion; 3, [serotonin - CH2NH2]+ (m/z ) 147); 4, [serotonin + H]+ (m/z ) 177).
the mass spectrometer. The pressure in the sample box was ∼400 Torr, which resulted in a flow rate of 1 nL/s.
RESULTS AND DISCUSSION Cell Viability. Cells are very sensitive to environmental factors such as pH and ionic strength. They are usually suspended in balanced salt solutions, which contain more than 100 mM total concentration of salts, to maintain cell viability. However, as noted by other groups, such high salt content is detrimental to mass spectrometric analysis due to the clogging of the capillary tips and the formation of adducts.7,8 In addition, undesirable accumulation of nonvolatile material can occur inside the mass spectrometer. The development of a buffer system that can maximize the cell life while maintaining the resolution and LOD of the mass spectrometric measurement is, therefore, a necessary step in single-cell analysis with mass spectrometry. The morphologies of intact, degranulated, and lysed cells differ significantly. Under 100× magnification, intact cells appear round and smooth, degranulated cells look roughened and porous, and lysed cells do not even have well-defined cell membranes. We examined through a microscope the viability of the mast cells in different buffer solutions. A number of buffer solutions, including Tris, Li2CO3, NaCl, NH4Cl, and isotonic glucose solution at different concentrations and pH values, were studied. We found that the cells agglutinated at pH 100 µm during the minute the cell is being transported. The same diffusional broadening applies if the cell lysed during transport. Many consecutive mass spectra would have been recorded, with a Gaussian distribution in signal levels that resembles capillary electrophoresis.20 This confirms that the cells were able to maintain their integrity in this buffer composition and that we are doing true whole-cell measurements. We have demonstrated the determination of serotonin and histamine in single rat peritoneal mast cells by combining flow cytometry and mass spectrometry. Other detection schemes such as fluorescence or scattering can be added to the present system (before the mass spectrometer inlet) so that more information can be obtained. In addition, it will still be desirable to further improve the detection limit of the system so that other minor constituents such as histidine and tryptamine (precursors of histamine and serotonin, respectively) can be analyzed to gain more insight into the biochemistry and signal transduction. It is conceivable that even more sophisticated experiments of this type can be done. If the cells are lysed at the entrance of the capillary (e.g., cross-flow of a reagent), then one can have electrophoretic separation before mass spectrometry. The penalty of such an Analytical Chemistry, Vol. 70, No. 15, August 1, 1998
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experiment is a lower cell concentration must be used to make room for the individual electropherograms. The throughput will thus be lower. ACKNOWLEDGMENT The authors thank Dr. Ho-ming Pang and Ms. Andrea Ho for helpful discussions. We also thank Dr. Michael McCloskey in the Department of Zoology at Iowa State University for his help in isolating the mast cells. The Ames Laboratory is operated for the
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U.S. Department of Energy by Iowa State University under Contract W-7405-Eng-82. This work was supported by the Director of Energy Research, Office of Basic Energy Sciences, Division of Chemical Sciences.
Received for review March 12, 1998. Accepted May 12, 1998. AC980290Q
