Anal. Chem. 2005, 77, 5036-5040
Combination Detergent/MALDI Matrix: Functional Cleavable Detergents for Mass Spectrometry Jeremy L. Norris, Ned A. Porter, and Richard M. Caprioli*
Department of Chemistry and Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232-8575
This study reports the synthesis of the first functional cleavable detergent designed specifically for applications in mass spectrometry. Upon cleavage, two inert compounds and the MALDI matrix are formed, eliminating sources of potential interference originating from traditional cleavable detergents. Analysis of peptides demonstrates that MALDI matrix generated in situ results in MALDI spectra equivalent to those prepared using established protocols. Analysis of the membrane protein diacylglycerol kinase was accomplished using the combination detergent/MALDI matrix. Applications of the functional cleavable detergents to the profiling of whole cell lysates results in increased signal-to-noise ratios of many ions and the detection of additional proteins previously not observed. The use of detergents to enhance the solubility of otherwise insoluble compounds has been part of general biochemical protocols for decades. Detergents provide a means by which these molecules can be extracted and isolated from other components of the cell for further study.1 Unfortunately, many of the detergents used today also hinder analytical characterization of biomolecules because they aggregate to form noncovalent, nonspecific complexes with the desired analyte. One specific example of this phenomenon is the suppression of ions that occurs during mass spectrometry of proteins that are detergent solubilized. The difficulties associated with detergents in mass spectrometry are well documented and continue to impede efforts to develop analytical techniques to study hydrophobic proteins using mass spectrometry.2-7 There have been a number of studies that suggest there are no intrinsic properties of membrane proteins that would prohibit analysis using mass spectrometry provided * To whom correspondence should be addressed: (e-mail) R.Caprioli@ vanderbilt.edu. (1) Stryer, L. Biochemistry, 4th ed.; W. H. Freeman: New York, 1995. (2) Schnaible, V.; Michels, J.; Zeth, K.; Freinang, J.; Welte, W.; Bu ¨ hler, S.; Glocker, M. O.; Przybylski, M. Int. J. Mass Spectrom. Ion Processes 1997, 169/170, 165-177. (3) Rosinke, B.; Strupat, K.; Hillenkamp, F.; Rosenbusch, J.; Dencher, N.; Kru ¨ger, U.; Galla, H.-J. J. Mass Spectrom. 1995, 30, 1462-1468. (4) Bo ¨rnsen, K. O.; Gass, M. A. S.; Bruin, G. J. M.; Adrichem, J. H. M. v.; Biro, M. C.; Kresbach, G. M.; Ehrat, M. Rapid Commun, Mass Spectrom, 1997, 11, 603-609. (5) Jeannot, M. A.; Zheng, J.; Li, L. J, Am, Soc, Mass Spectrom, 1999, 10, 512520. (6) Zhang, N.; Li, L. Anal. Chem. 2002, 74, 1729-1736. (7) Puchades, M.; Westman, A.; Blennow, K.; Davidsson, P. Rapid Commun, Mass Spectrom, 1999, 13, 344-349.
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that the solubility problems can be overcome.2,3,8,9 Reagents that provide for the extraction and solubilization of hydrophobic proteins, while allowing subsequent analysis of the isolated protein using mass spectrometry, will be of great value. Cleavable detergents are reagents that have properties similar in all ways to conventional detergents.10-12 However, cleavable detergents have the added benefit that when they are no longer necessary, they can be selectively degraded to yield products that have reduced or no surface activity. These innovative reagents have found a number of applications in industrial processes and chemical syntheses because they can be easily and completely removed when they are no longer needed. The development of cleavable detergents is an established field that has experienced significant resurgence in the past few years. The reason for such renewed interest is due in large part to novel applications of these reagents to problems in the analysis of biologically significant molecules. Various types of cleavable detergents have been synthesized that are suitable for the analysis of proteins using two-dimensional gel electrophoresis.13-17 These techniques were not successfully reduced to practice until the recent introduction of an acid-labile surfactant15-17 based on a structure first synthesized by Jaeger et al.18 These compounds allow two-dimensional gel analyses of a quality comparable to that of traditional SDSPAGE analyses while retaining the ability to identify the separated proteins using chromatography and mass spectrometry. Acidcleavable detergents are also useful for mass spectrometric analysis of intact membrane proteins and analyses requiring the direct profiling of cells and tissue with matrix-assisted laser (8) Ghaim, J. B.; Tsatsos, P. H.; Katsonouri, A.; Mitchell, D. M.; SalcedoHernandez, R.; Gennis, R. B. Biochim. Biophys. Acta 1997, 1330, 113120. (9) Cadene, M.; Chait, B. T. Anal. Chem. 2000, 72, 5655-5658. (10) Hellberg, P.-E.; Bergstrm, K.; Holmberg, K. J. Surfactants, Deterg. 2000, 3, 81-91. (11) Holmberg, K. In Novel surfactants: preparation, applications, and biodegradability; Holmberg, K., Ed.; Marcel Dekker: New York, 1998; Vol. 74, pp 333-359. (12) Jaeger, D. A. Supramol. Chem. 1995, 5, 27-30. (13) Epstein, W. W.; Jones, D. S.; Bruenger, E.; Rilling, H. C. Anal. Biochem. 1982, 21, 304-312. (14) Dunkin, I. R.; Gittinger, A.; Sherrington, D. C.; Whittaker, P. J. Chem. Soc., Chem. Commun. 1994, 2245-2246. (15) Meng, F.; Cargile, B. J.; Patrie, S. M.; Johnson, J. R.; McLoughlin, S. M.; Kelleher, N. L. Anal. Chem. 2002, 74, 2923-2929. (16) Ko ¨nig, S.; Schmidt, O.; Rose, K.; Thanos, S.; Besselmann, M.; Zeller, M. Electrophoresis 2003, 24, 751-756. (17) Ross, A. R. S.; Lee, P. J.; Smith, D. L.; Landridge, J. I.; Whetton, A. D.; Gaskell, S. J. Proteomics 2002, 2, 928-936. (18) Jaeger, D. A.; Jamrozik, J.; Golich, T. G.; Clennan, M. W.; Mohebalian, J. J. Am. Chem. Soc. 1989, 111, 3001-3006. 10.1021/ac050460g CCC: $30.25
© 2005 American Chemical Society Published on Web 06/16/2005
Scheme 1. Synthesis of the Functional Cleavable Detergent, 2-Cyano-3-(4-dodecyloxymethyloxyphenyl)acrylic Acida
Figure 1. Combination detergents/MALDI matrixes. Compound 1 is a derivative of the MALDI matrix, HCCA. Compound 2 is a derivative of the matrix, 3,5-dimethoxy-4-hydroxy-cinnamic acid (sinapinic acid).
desorption/ionization mass spectrometry (MALDI-MS).19 These applications demonstrate that the increased solubility induced by the presence of cleavable detergents results in increased signalto-noise ratios and greater numbers of proteins observed in a given MALDI-MS profile. The benefit of increased solubility is realized without the corresponding loss in mass spectral quality normally associated with the addition of conventional detergents. Although the cleavable detergents previously described provide a novel and effective approach for proteomics analyses, ion suppression effects may still interfere with the analysis. Cleavage of these reagents removes the surface-active properties from the solution, but the cleavage products may inhibit crystallization of the matrix and give nonoptimal performance. One solution to this problem is to incorporate a functional chemical species into the detergent designed to enhance crystal formation that will be released upon cleavage of the detergent. For MALDI-MS, an obvious choice for the functional component of the reagent is one of the known MALDI matrixes. This study outlines the design and synthesis of a combination detergent/MALDI matrix and the application of this new class of cleavable reagents for MALDIMS of peptide and protein mixtures. These functional cleavable detergents, shown in Figure 1, are based on the MALDI matrixes, R-cyano-4-hydroxycinnamic acid (HCCA) and 3,5-dihydroxycinnamic acid (sinapinic acid). Results demonstrate the utility of these compounds to increase the number of signals observed in a single mass spectrum, yielding a more complete protein profile. EXPERIMENTAL SECTION Synthesis. Synthesis of cleavable detergents described in this paper is specifically detailed in the Supporting Information. Peptide Analysis. Three peptides (neurotensin, substance P, angiotensin II) were solubilized in a 9 mM solution of 2-cyano-3(4-dodecyloxymethoxyphenyl)acrylic acid (1) in 10% acetonitrile at a concentration of 1 µM each. A 1-µL aliquot of the mixture was deposited onto the MALDI target. A 0.1-µL volume of 1% HCl was added to the droplet to initiate cleavage of the detergent. The sample was allowed to dry under ambient conditions. The samples were analyzed using an Applied Biosystems Voyager-STR (Framingham, MA) operating in linear mode and equipped with a nitrogen laser (337 nm) operating at 2 Hz. The instrument operating parameters were optimized for best resolution at m/z 1600. Preparation of Cell Lysates. The human colorectal cell line, RKO, was grown in DMEM Hi Glucose (Gibco BRL, Gaithers(19) Norris, J. L.; Porter, N. A.; Caprioli, R. M. Anal. Chem. 2003, 75, 66426647.
a Conditions: (a) TMS-Cl, (CH O) , 2 h; (b) NaOH, DMSO, 2 n room temperature, 12 h.
burg, MD) medium supplemented with 10% fetal calf serum (Gemini Bio-Products, Inc., Calabasas, CA) and 1% penicillin/ streptomycin (Sigma, St. Louis, MO). Three cell extracts containing each cleavable detergent were prepared along with a separate extract containing commercial detergents. Control cell extracts were lysed in RIPA lysis buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 1% (v/v) Nonidet P-40, 0.1% (v/v) SDS, 0.5% (w/v) sodium deoxycholate, 50 mM NaF, 0.1 mM NaV, 1 mM dithiothreitol, and the protease inhibitors antipain (10 µg/mL), leupeptin (10 µg/mL), pepstatin A (10 µg/mL), chymostatin (10 µg/mL), phenylmethanesulfoyl fluoride (50 µg/mL) (Sigma), and 4-(2aminoethyl)benzenesulfonyl fluoride (200 µg/mL) (CalbiochemNovabiochem Corp.)]. Cell extracts containing cleavable detergents were prepared using a modified version of RIPA buffer that excludes the detergent components SDS, nonidet P-40, and sodium deoxycholate. The combination detergent/MALDI matrix (2) solutions contained 0.1, 1, and 10 mM cleavable detergent. Additional controls were also prepared excluding all detergent components. Cells were incubated for 1 h in the presence of lysis buffer followed by homogenization. Cell debris was removed by centrifugation at 3000g for 30 min. Membrane Protein Preparation. The membrane protein HisDAGK, expressed in Escherichia coli, was purified from whole cell lysate using a Ni(II) resin bathed in 0.5% dodecylmaltoside (DM). DM was removed from a volume of 400 µL of resin containing 1 mg/mL DAGK. The resin was reconstituted in 700 µL of a 0.5% solution of cleavable detergent 2. The resin was incubated at 4 °C for a period of 10 min. The purified, resin-bound protein was eluted from the resin using 250 mM imidazole in 0.5% combination detergent/MALDI matrix (2). Control experiments were performed in the absence of cleavable detergents. Samples were prepared for MALDI-MS analysis using the procedures outlined below. Sample Preparation of MALDI Analysis. Solutions of membrane protein and RKO cell lystates solubilized in combination detergent/MALDI matrix were prepared using the following method. A volume of 0.5 µL of each detergent-solubilized protein was deposited on the MALDI target along with 0.5 µL of sinapinic acid. The sinapinic acid was prepared at a concentration of 20 mg/mL in 50% acetonitrile. This matrix solution is mixed in a 5:1 ratio with 1% HCl prior to use. Cleavage of the detergent is Analytical Chemistry, Vol. 77, No. 15, August 1, 2005
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Scheme 2. Synthesis of Functional Acid-Cleavable Detergent, 3-(4-Dodecyl oxymethyloxy-3,5-dimethyloxyphenyl)acrylic Acida
a Conditions: (a) DMAP, pyridine, CH Cl , 2 h; (b) Ph P, EtAc, 24 h; (c) triethylamine, benzene, 16 h; (d) TMS-Cl, (CH O) , 2 h; (e) 2 2 3 2 n triethylamine, benzene, 12 h; (f) TBAF, THF, 1 h.
monitored using MALDI-MS. If required, the sample is washed on target using 0.1% TFA. Protein Mass Spectrometry. Samples were analyzed using an Applied Biosystems Voyager-STR equipped with a nitrogen laser (337 nm) operating at 20 Hz. Data were acquired using automatic acquisition mode. Twenty shots were averaged per spectrum, and these spectra were accumulated for a total of 300 shots per spectrum. The instrument operating parameters were optimized for best resolution at m/z 12 000. RESULTS AND DISCUSSION Detergent Synthesis and Degradation. Synthesis of the cleavable detergents was carried out according to the reactions in Schemes 1 and 2. The details of the synthesis of all compounds are provided as Supporting Information. The synthesis of the acidcleavable detergent based on the MALDI matrix HCCA is shown in Scheme 1. Compound 1 is synthesized by alkylating HCCA using chloromethyl dodecyl ether (4) in the presence of base. The product, 1, was isolated in 65% yield after two steps. The detergent derived from sinapinic acid, 3-(4-dodecyloxymethyoxy3,5-dimethoxyphenyl)acrylic acid (2), is synthesized according to the procedure in Scheme 2. To prevent alkylation of the acid and the phenol of sinapinic acid by 4, a protected sinapinic acid, 3-(4hydroxy-3,5-dimethoxyphenyl)acrylic acid 2-trimethylsilanyl ethyl ester (11), was synthesized. This allows for selective alkylation of the phenol followed by deprotection to create the final product 2. Trimethylsilylethanol (7) was reacted with bromoacetyl bromide (6) to generate bromoacetic acid 2-trimethylsilanyl ethyl ester (8), which is then reacted with triphenylphosphine to produce the phosphonium salt, (2′-trimethylsilyl)ethoxycarbonylmethylen-triphenylphosphonium bromide (9). Compound 9 was used in a Wittig reaction with 3,5-dimethoxy-4-hydroxybenzaldehyde (syringealdehyde) (10) to synthesize the protected matrix 11. Compound 11 was alkylated using chloromethyl dodecyl ether (4) followed by deprotection of 12 using tetrabutylammonium fluoride. The overall yield of the product (2) was 47%. Combination detergents/MALDI matrixes are a class of cleavable detergent designed specifically for use in MALDI-MS. To 5038
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reduce the number of interfering compounds present during the mass spectrometric analysis, one of the MALDI matrixes was incorporated as the head of the detergent. Because these reagents are functional surfactants, cleavage of the surfactant releases a reagent that is now useful for another purpose. In this case, cleavage of combination detergent/MALDI matrixes generates one of the known MALDI matrixes, making sample preparation fast and efficient. Fast and efficient cleavage makes it possible to prepare the samples directly on the MALDI target. Combination detergents/MALDI matrixes are acetals and are cleaved via the same mechanism as the acid-cleavable detergents described in previous studies.15-17,19 Cleavage of the sinapinic acidderived functional detergent 2 proceeds according to the scheme in Scheme 3. The products of the cleavage reaction are the MALDI matrix sinapinic acid, formaldehyde, and 1-dodecanol. To provide fast and complete cleavage of the detergent on target, a highly acidic matrix solution (pH
