Anal. Chem. 2002, 74, 497-503
Single-Neuron Analysis Using CE Combined with MALDI MS and Radionuclide Detection Jason S. Page, Stanislav S. Rubakhin, and Jonathan V. Sweedler*
Department of Chemistry and the Beckman Institute, University of Illinois, Urbana, Illinois 61801
Capillary electrophoresis (CE) has been combined with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and radionuclide detection to assay mass-limited biological samples. Nanovial sampling techniques enable injections into the CE capillary from 50 to 150-nL volume samples; after the separation, nanoliter fraction collection combines the CE effluent with a MALDI matrix and minimizes sample spreading, thus allowing both MALDI MS and radionuclide detection on the CE fractions. MALDI MS complements the elution time information of CE by providing accurate molecular mass data, and radionuclide detection provides zeptomole limits of detection with quantitative information. While MALDI MS detects all fully processed peptides at sufficient concentration, culturing the neuron in media containing 35S-Met provides selective radionuclide detection of newly synthesized methionine-containing peptides. The analysis and detection of the expected neuropeptides and hormones in a single 40-µm bag cell neuron from Aplysia californica with CE/MALDI MS/radionuclide detection demonstrates the ability of this hyphenated approach to work with chemically complex mass-limited samples.
The assay of single cells presents an analytical challenge due to the high chemical complexity and limited amounts of analyte. Adding further significance to many biological samples is the difficulty of cell isolation and the sacrifice of the subject animal to gain the sample. Together, these attributes often make the sample scarce, rare, and precious and the analysis limited to a single measurement. Therefore, an analytical technique that provides a wealth of information is beneficial when the chemical content of a cell is profiled in a single assay. We achieve this by incorporating a nanovial sampling procedure for capillary electrophoresis (CE) followed by multiple detection modes. The introduction of matrix-assisted laser/desorption ionization mass spectrometry (MALDI MS) in the late 1980s expanded the utility of mass spectrometric detection for the analysis of biological samples.1,2 This soft ionization technique allows for the desorption and ionization of higher-mass analytes, such as peptides and * Corresponding author: (phone) (217) 244-7359; (fax) (217) 244-8068; (e-mail)
[email protected]. (1) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (2) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T. Rapid Commun. Mass Spectrom. 1998, 2, 151-153. 10.1021/ac0156621 CCC: $22.00 Published on Web 01/05/2002
© 2002 American Chemical Society
proteins from complex mixtures.3-9 One of the more complex samples directly analyzed by MALDI MS has been intact biological cells. Whole bacteria have been placed into the instrument for classification based on their MS fingerprint.10-12 Single neurons and their individual organelles have been assayed by MALDI MS, providing chemical and spatial information.13-18 Additionally, MALDI MS provides sufficient sensitivity to detect the cellular processing of prohormones in single neurons.18 Cellular analysis by direct MALDI MS is an effective method for determining the peptides and studying the cellular processing in single neurons, and recently, considerable research has been performed utilizing CE for single-cell analyses.19-22 However, enhancements are gained through the coupling of a microseparation technique prior to MS analysis.23-25 In addition to creating an information-rich, two-dimensional analytical technique, a more (3) Burlingame, A. L.; Boyd, R. K.; Gaskell, S. J. Anal. Chem. 1998, 70, 647R716R. (4) Hillenkamp, F.; Karas, M.; Beavis, R. C.; Chait, B. T. Anal. Chem. 1991, 63, 1193A-1203A. (5) Kaufmann, R. J. Biotechnol. 1995, 41, 155-175. (6) Kussmann, M.; Roepstorff, P. Spectroscopy 1998, 14, 1-27. (7) Chaurand, P.; Stoeckli, M.; Caprioli, R. M. Anal. Chem. 1999, 23, 52635270. (8) Sporns, P.; Abell, D. C. Trends Food Sci. Technol. 1996, 7, 187-190. (9) Thiede, B.; Wittmannliebold, B.; Bienert, M.; Krause, E. FEBS Lett. 1995, 375, 65-69. (10) Holland, R. D.; Wilkes, J. G.; Rafii, F.; Sutherland, J. B.; Persons, C. G.; Voorvees, K. J.; Lay, J. O. Rapid Commun. Mass Spectrom. 1996, 10, 12271232. (11) Krishnamurthy, T.; Ross, P. L. Rapid Commun. Mass Spectrom. 1996, 10, 1992-1996. (12) Arnold, R. J.; Reilley, J. P. Rapid Commun. Mass Spectrom. 1998, 12, 630636. (13) Jimenez, C. R.; van Valeen, P. A.; Li, K. W.; Wildering, W. C.; Garaerts, W. P. M.; Tjaden, V. R.; van der Greef, J. J. Neurochem. 1994, 62, 404-407. (14) van Valeen, P. A.; Jimenez, C. R.; Li, K. W.; Wildering, W. C.; Garaerts, W. P. M.; Tjaden, V. R.; van der Greef, J. Org. Mass Spectrom. 1993, 28, 15421546. (15) Li, L.; Garden, R. W.; Romanova, E. V.; Sweedler, J. V. Anal. Chem. 1999, 71, 5451-5458. (16) Li, L.; Garden, R. W.; Floyd, P. D.; Moroz, T. P.; Gleeson, J. M.; Sweedler, J. V.; Pasa-Tolic, L.; Smith, R. D. J. Exp. Biol. 1999, 202, 2961-2973. (17) Rubakhin, S. S.; Garden, R. W.; Fuller, R. R.; Sweedler, J. V. Nat. Biotechnol. 2000, 18, 172-175. (18) Li, L.; Garden, R. W.; Sweedler, J. V. Trends Biotechnol. 2000, 18, 151160. (19) Zabzdyr, J. L.; Lillard, S. J. Trends Anal. Chem. 2001, 20, 467-476. (20) Malek, A.; Khaledi, M. G. Anal. Biochem. 1999, 270, 50-58. (21) Krylov, S. N.; Starke, D. A.; Arriaga, E.; Polakowski, R.; Palcic, M. M.; Dovichi, N. J. Anal. Chem. 2000, 72, 872-877. (22) Han, F.; Lillard, S. J. Anal. Chem. 2000, 72, 4073-4079. (23) Page, J. S.; Rubakhin, S. S.; Sweedler, J. V. Analyst 2000, 125, 555-562. (24) Johnson, T.; Bergquist, J.; Ekman, R.; Nordhoff, E.; Schurenberg, M.; Kloppel, K.-D.; Muller, M.; Lehrach, H.; Gobom, J. Anal. Chem. 2001, 73, 1670-1675.
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complete mass profile of the sample is gained by reducing analyte suppression.23,24 There are a variety of examples where CE has been combined with MALDI MS. These methods range from continuous deposition of the CE eluent onto a membrane26,27 to the collection of fractions in small vials or directly onto a MS target.23,24,28,29 Here, nanoliter-volume fractions are collected into spots (bins) on a target, each representing a variable (e.g., 15 s) elution window, which allows easier interfacing to a variety of detection modes. This reduces some of the high separation efficiency of CE in order for easier access to the sample, preservation of the sample after analysis, fraction re-injection, and the ability to perform on-target chemical analysis of selected analytes.23 Combining CE with MALDI MS provides easier data analysis by reducing the complexity in the mass spectra through the separation of the analytes and through the elimination of salt adducts by eluting them in a MALDI-friendly buffer. CE-purified analytes can also be subjected to chemical tests performed directly on the MALDI target, gaining more information on selected peptides.23 Perhaps the biggest disadvantages of MALDI MS are related to sensitivity and quantitation, especially for peptides in complex samples such as cells when standards are not available. Calibration and even semiquantitation in MALDI requires the peptides of interest be synthesized. While radionuclide detection does not provide the molecular information of MALDI MS, it allows for quantitative measurements, as well as identical response for different peptides (assuming the same radionuclide tag). Hence, we have also investigated the use of CE with radionuclide detection.30,31 As common tags such as 3H and 35S lack sufficient energy to penetrate the capillary wall, on-column approaches are not effective for compounds containing these tags,32-34 so that an off-column approach is called for. Using a postcolumn deposition method, we have achieved detection sensitivities of 130 zmol for 35S-labeled peptides.30,31 As a combination of CE with MALDI MS and radionuclide detection has not yet been demonstrated, we have modified the deposition approach to make it compatible with these two detection modes. The goals of this study are to further expand the information content of single-cell analysis by incorporating these complementary planar detection techniques with CE separations. We demonstrate the effectiveness of combining CE with both MALDI MS and radionuclide detection for the assay of a single ∼40-µm neuron from the bag cell cluster from Aplysia californica. This represents (25) Hsieh, S.; Dreisewerd, K.; van der Schors, R. C.; Jimenez, C. R.; StahlZeng, J.; Hillenkamp, F.; Jorgenson, J. W.; Garaerts, W. P. M.; Li, K. W. Anal. Chem. 1998, 70, 1847-1852. (26) Zhang, H.; Caprioli, R. M. J. Mass Spectrom. 1996, 31, 1039-1046. (27) Preisler, J.; Hu, P.; Rejtar, T.; Karger, B. L. Anal. Chem. 2000, 72, 47854795. (28) Walker, K. L.; Chiu, R. K.; Monnig, C. A.; Wilkens, C. L. Anal. Chem. 1995, 67, 4197-4204. (29) Murray, K. K. Mass Spectrom. Rev. 1997, 16, 283-299. (30) Tracht, S. E.; Cruz, L.; Stobba-Wiley, C. M.; Sweedler, J. V. Anal. Chem. 1996, 68, 3922-3927. (31) Tracht, S.; Toma, V.; Sweedler, J. V. Anal. Chem. 1994, 66, 2382-2389. (32) Pentoney, S. L.; Zare, R. N.; Quint, J. F. Anal. Chem. 1989, 61, 16421647. (33) Pentoney, S. L.; Zare, R. N.; Quint, J. F. In Analytical Biotechnology: Capillary Electrophoresis and Chromatography; Horvath, C., Nikelly, J. G., Eds.; ACS Symposium Series 434; American Chemical Society: Washington, DC, 1990; p 60-89. (34) Gordon, J. S.; Vasile, S.; Hazlett, T.; Squillante, M. IEEE Trans. Nucl. Sci. 1993, 40, 1162-1164.
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the first example of CE/MALDI MS analysis of the processing of a prohormone in a single neuron along with the first example of the combination of CE with MALDI MS and radionuclide detection. The use of nanovial injection and nanoliter fraction collection is critical for such mass-limited samples to increase the percentage of the cellular matrix introduced into the capillary and to decrease dilution upon deposition of the fractions. The peptides and proteins in the CE fractions are subsequently mass profiled by MALDI MS. The radioactivity emanating from the radiolabeled peptides is then detected by a phosphor imager. This complements the elution time and molecular weight information with highly sensitive, quantitative information. EXPERIMENTAL SECTION Reagents. The CE buffer consisted of a 80 mM phosphate solution (pH 2.5) made from monobasic ammonium phosphate and phosphoric acid, both purchased from Fisher Scientific (Pittsburgh, PA). The water used for the CE buffer and throughout the experiment came from a Milli-Q purifying system (Millipore, Boston, MA). Acetonitrile was purchased from Fisher Scientific. The MALDI matrix was a 15 mg/mL solution of 2,5-dihydroxybenzoic acid (DHB) (ICN Biomedicals, Aurora, OH) in purified water with 1 vol % trifluoroacetic acid (TFA) (Aldrich, Milwaukee, WI). The medium used for cellular culture was Leibovitz’s L-15 (Life Technologies, Gaithersburg, MD), and the protease was a type IX Bacterial from Sigma (St. Louis, MO). For radiolabeling, the 35S-methionine was purchased from Amersham (Arlington Heights, IL, Catalog No. S51015). Sample Preparation and Radiolabeling. A. californica (100250 g) were obtained from Aplysia Research Facility (University of Miami, Miami, FL) or collected in the wild off the coast of California and kept in an aquarium containing continuously circulating, aerated, and filtered artificial seawater (ASW) at a temperature of 14-15 °C until used. Molluscs were anesthetized by the injection of isotonic MgCl2 (∼30 to ∼50% of body weight) into the body cavity. The bag cell clusters were dissected and washed for ∼1 h in ASW at pH 7.7 consisting of (in mM) 460 NaCl, 10 KCl, 10 CaCl2, 22 MgCl2, 6 MgSO4, 10 HEPES, 100 units/ mL penicillin G, 100 µg/mL streptomycin, and 100 µg/mL gentamicine. This was followed by ∼24-h incubation of the clusters in 0.5 mCi 35S-methionine containing culture medium (50% supplemented with inorganic salts Leibovitz’s L-15 medium and 50% Aplysia hemolymph).35 The culture medium was then substituted with ASW-antibiotic solution before dissection of the bag cell cluster. The handling and disposal of 35S-Met and radionuclidelabeled culturing media requires safe-handling procedures as outlined in each institution’s division of environmental health and safety guidelines. Neuron Isolation. To isolate a neuron from the bag cell cluster, the ganglionic sheath was enzymatically digested by incubating the clusters in ASW-antibiotic solution containing 1% protease at 36 °C for 1-2.5 h depending on the size of the cluster. After the sheath removal, the cells were dissected using tungsten needles and transferred to individual glass nanovials. The nanovials containing the individual cells were then placed in a -15 °C freezer to preserve the sample until analysis and to lyse the cell within (35) Clark, G. A.; Hawkins, R. D.; Kandel, E. R. Learn. Mem. 1994, 1, 243257.
Figure 1. Diagram of the CE system highlighting the nanovial sampling system, the postcolumn fraction collector, and the complementary detection modes of mass spectrometry and radionuclide detection.
the vial. The nanovials were made from standard glass capillaries with a 0.5-mm i.d. and 1.0-mm o.d. (A-M Systems, Everett, WA).23 To make the vial, a silver-based epoxy plug (Epo-Tek, Billerica, MA) was introduced into the capillary by placing the tip of the capillary into the epoxy and allowing it to wick to a desired height. The epoxy was hardened in a 70 °C oven for several hours, and then the capillary was cut at a measured height above the plug leaving behind a shallow well of ∼150 nL in volume, which served as the sample vial. CE System/Nanovial Sampling. To inject the contents of the neuron into the CE system, a nanovial containing the cell was taken from a -15 °C freezer and reconstituted by filling with a 45:45:10 solution of acetonitrile, water, and CE buffer (v/v). The solution in the nanovial was allowed to sit and evaporate to
