Anal. Chem. 2003, 75, 6642-6647
Mass Spectrometry of Intracellular and Membrane Proteins Using Cleavable Detergents Jeremy L. Norris, Ned A. Porter, and Richard M. Caprioli*
Mass Spectrometry Research Center, Departments of Chemistry and Biochemistry, Vanderbilt University, 465 21st Avenue South, Medical Research Building 3, Room 9160, Nashville, Tennessee 37232-8575
Detergents have been used to enhance the solubility of hydrophobic biomolecules for decades. Despite the widespread use of detergents in biochemistry, the presence of these molecules often complicates further analysis by mass spectrometry. This study presents a solution to this problem by outlining a method utilizing a novel cleavable detergent, 3-[3-(1,1-bisalkyloxyethyl)pyridin-1-yl]propane1-sulfonate (PPS). This detergent can be used to extract protein contained within the interior of the cell by disrupting cell membranes. Once the proteins are free from the cell, PPS also assists in protein solubilization by shielding the hydrophobic regions of the newly extracted protein from unfavorable interactions with water. The added advantage of PPS over conventional detergents such as sodium dodecyl sulfate or n-octylglucoside is that the detergent properties that interfere with MALDI mass spectrometry can be eliminated prior to analysis. PPS was found to improve sensitivity in MALDI analyses of both soluble proteins and membrane proteins without degrading spectral quality. The virtues of this strategy were applied to whole cell extracts. Analysis of these extracts resulted in an overall increase in both the number of peaks observed and overall signal intensity. Rapid and comprehensive analysis of the proteome is critical for the advancement of biomedical science. The ability to determine the cellular state of individualized cells will lead to exciting new discoveries about how diseases manifest themselves through protein expression and localization.1-5 Accompanying the emerging field of proteomics in its development are a number of innovations in the way scientists analyze proteins.6,7 Among these innovations, mass spectrometry occupies a position of critical importance.4,5,8-11 For example, matrix-assisted laser desorption * To whom correspondence should be addressed. E-mail: R.Caprioli@ vanderbilt.edu. (1) Petricoin, E. F.; Zoon, K. C.; Kohn, E. C.; Barrett, J. C.; Liotta, L. A. Nature Rev. Drug Discovery 2002, 1, 683-695. (2) Adam, B.-L.; Vlahou, A.; Semmes, O. J.; George, L.; Wright, J. Proteomics 2001, 1, 1264-1270. (3) Rarekh, R. Nat. Biotechnol. 1999, 17, BV 19-20. (4) Pandey, A.; Mann, M. Nature 2000, 405, 837-846. (5) Aebersold, R.; Goodlett, D. R. Chem. Rev. 2001, 101, 269-295. (6) Lee, K. H. Trends Biotechnol. 2001, 19, 217-222. (7) Dutt, M. J.; Lee, K. H. Curr. Opin. Biotechnol. 2000, 11, 176-179. (8) Yates, J. R., III. Trends Genet. 2000, 16, 5-8. (9) Shevchenko, A.; Jensen, O. N.; Podtelejnikov, A. V.; Sagliocco, F.; Wilm, M.; Vorm, O.; Mortensen, P.; Shevchenko, A.; Boucherie, H.; Mann, M. Proc. Nat. Acad. Sci. U.S.A. 1996, 93, 14440-14445.
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ionization mass spectrometry (MALDI-MS) is one of the premier tools for the analysis of complex mixtures of biomolecules. This tool has been exploited in a number of ways allowing researchers to directly analyze cells and tissue with minimal sample pretreatment while preserving the spatial information intrinsic to the sample.12-15 In addition, other ionization techniques, e.g., electrospray ionization, also allow molecular measurements to be obtained from complex biological samples, making mass spectrometry an ideal tool for rapid and direct protein analysis of biological samples. The general experimental protocol for MALDI-MS involves first mixing with or extracting the analyte into a solution containing an organic matrix molecule. Cocrystallization of the matrix and the analyte follows. The cocrystals formed are then irradiated using a laser having the appropriate wavelength to affect desorption of the matrix. During this desorption process, ions of the analyte are produced in the gas phase and subsequently analyzed.16 Although sample preparation steps for MALDI analysis are relatively simple, there are some significant shortcomings of the technique. The requirement to form cocrystals of matrix and analyte presupposes that various proteins in the sample must be soluble under the same solvent conditions. Thus, differences in protein solubility can bias the analysis in favor of soluble proteins and against hydrophobic, e.g., membrane, proteins. On the other hand, several published papers show that hydrophobic membrane proteins can be successfully analyzed by MALDI mass spectrometry by overcoming the problem of membrane protein solubility.17-20 The problem of membrane protein solubility is not new, and traditionally, biochemists have employed detergents to increase protein solubility.21 With a variety of detergents commercially (10) Mo, W.; Karger, B. L. Curr. Opin. Chem. Biol. 2002, 6, 666-675. (11) Gygi, S. P.; Aebersold, R. Curr. Opin. Chem. Biol. 2000, 4, 489-494. (12) Stoeckli, M.; Chaurand, P.; Hallahan, D. E.; Caprioli, R. M. Nat. Med. 2001, 7, 493-496. (13) Chaurand, P.; Stoeckli, M.; Caprioli, R. M. Anal. Chem. 1999, 71, 52635270. (14) Lay, J. O., Jr. Mass Spectrom. Rev. 2001, 20, 172-194. (15) Li, L.; Garden, R. W.; Sweedler, J. V. Trends Biotechnol. 2000, 18, 151160. (16) Zenobi, R.; Knochenmuss, R. Mass Spectrom. Rev. 1998, 17, 337-366. (17) Schnaible, V.; Michels, J.; Zeth, K.; Freinang, J.; Welte, W.; Bu ¨ hler, S.; Glocker, M. O.; Przybylski, M. Int. J. Mass Spectrom. 1997, 169/170, 165177. (18) Ghaim, J. B.; Tsatsos, P. H.; Katsonouri, A.; Mitchell, D. M.; SalcedoHernandez, R.; Gennis, R. B. Biochim. Biophys. Acta 1997, 1330, 113120. (19) Rosinke, B.; Strupat, K.; Hillenkamp, F.; Rosenbusch, J.; Dencher, N.; Kru ¨ger, U.; Galla, H.-J. J. Mass Spectrom. 1995, 30, 1462-1468. (20) Cadene, M.; Chait, B. T. Anal. Chem. 2000, 72, 5655-5658. 10.1021/ac034802z CCC: $25.00
© 2003 American Chemical Society Published on Web 10/29/2003
Scheme 1. Synthesis of PPSa
Figure 1. 3-[3-(1,1-Bisalkyloxyethyl)pyridin-1-yl]propane-1-sulfonate (PPS).
available, conditions can be established that allow most biochemical experiments to be performed. Unfortunately, in a sample that is to be analyzed by mass spectrometry, the presence of detergents causes significant difficulties.17,19,22-25 It is therefore important to address this issue with new reagents and methods. This study describes a new methodology that employs cleavable detergents to enhance the solubility of proteins to be analyzed by MALDI mass spectrometry. Cleavable detergents are surfactants that have an easily cleavable bond connecting the hydrophilic head to the hydrophobic tail of the surfactant. Introduction of this chemical group into the surfactant provides a mechanism by which unwanted surfactant properties, e.g., foaming and aggregation, can be eliminated in a controlled way following protein solubilization. The products of detergent degradation exhibit significantly reduced surface activity, eliminating these detergent associated problems. Cleavable detergents have been used in a variety of ways in the chemical industry with applications ranging from environmentally friendly, biodegradable detergents to chemical synthesis.26-28 In contrast, only a few applications have focused on biological applications of these surfactants and their use as replacements for common biological detergents.29-32 These applications focus on two-dimensional gel technology attempting to make a substitute for sodium dodecyl sulfate that can be used in an electrophoresis platform. An additional application of cleavable detergents in the field of mass spectrometry is to use these agents as a way of enhancing the results of direct protein and peptide profiling of cells and tissue by MALDI-MS.33,34 This paper details this approach using a novel class of cleavable detergents, 3-[3(1,1-bisalkyloxyethyl)pyridin-1-yl]propane-1-sulfonate (PPS) (Figure 1), which are useful for protein solubilization prior to mass (21) Stryer, L. Biochemistry, 4th ed.; W. H. Freeman: New York, 1995. (22) 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. (23) Jeannot, M. A.; Zheng, J.; Li, L. J. Am. Soc. Mass Spectrom. 1999, 10, 512520. (24) Zhang, N.; Li, L. Anal. Chem. 2002, 74, 1729-1736. (25) Puchades, M.; Westman, A.; Blennow, K.; Davidsson, P. Rapid Commun. Mass Spectrom. 1999, 13, 344-349. (26) Hellberg, P.-E.; Bergstrom, K.; Holmberg, K. J. Surf. Deterg. 2000, 3, 8191. (27) Jaeger, D. A. Supramol. Chem. 1995, 5, 27-30. (28) Holmberg, K. In Novel surfactants: preparation, applications, and biodegradability; Holmberg, K., Ed.; Marcel Dekker: New York, 1998; Vol. 74, pp 333-359. (29) Ko ¨nig, S.; Schmidt, O.; Rose, K.; Thanos, S.; Besselmann, M.; Zeller, M. Electrophoresis 2003, 24, 751-756. (30) Meng, F.; Cargile, B. J.; Patrie, S. M.; Johnson, J. R.; McLoughlin, S. M.; Kelleher, N. L. Anal. Chem. 2002, 74, 2923-2929. (31) Epstein, W. W.; Jones, D. S.; Bruenger, E.; Rilling, H. C. Anal. Biochem. 1982, 119, 304-312. (32) Ross, A. J.; Lee, P. J.; Smith, D. L.; Landridge, J. I.; Whetton, A. D.; Gaskell, S. J. Proteomics 2002, 2, 928-936. (33) Norris, J. L.; Porter, N. A.; Caprioli, R. M. Proceedings of the 49th ASMS Conference on Mass Spectrometry and Allied Topics, Chicago, IL, 2001. (34) Norris, J. L.; Porter, N. A.; Caprioli, R. M. Proceedings of the 50th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, FL, 2002.
a (a) H SO , benzene, 80 °C; (b) 1,3-propane sultone, benzene, 2 4 40 °C.
spectrometric analysis. In contrast to other MALDI tolerant detergents such as n-octylglucoside, PPS is a strong detergent that can be used to aggressively solubilize hydrophobic proteins and lyse cells. The key advantage of the cleavable detergent PPS over conventional detergents is that, following cleavage, MALDI mass spectrometric sensitivity and spectral quality are virtually unaffected. EXPERIMENTAL SECTION Synthesis. Compounds were synthesized in two steps according to the procedure outlined in Scheme 1. All reagents were purchased from Sigma-Aldrich and used without purification unless otherwise noted. 3-(1,1-Bishexyloxyethyl)pyridine (4a). A volume of 0.22 mL (2 mmol) of 3-acetylpyridine was combined with 1.5 mL (12 mmol) of 1-hexanol in 5 mL of benzene. A catalytic amount of sulfuric acid was added to the reaction mixture. The reaction was carried out at 80 °C for 12 h with azeotropic removal of water from the reaction mixture. The reaction was quenched with NaHCO3(aq), and product 4a was extracted with CH2Cl2. The organic layer was dried over MgSO4. Excess 1-hexanol was removed under high vacuum (∼10-4 Torr) and recovered using a cold trap. Reaction proceeded with quantitative conversion to product 4a, a colorless oil. 1H NMR (300 MHz, CDCl3): δ ) 8.73 (dd, J ) 2.4, 0.6 Hz, 1H; ArH), δ ) 8.50 (dd, J ) 4.7, 1.8 Hz, 1H; ArH), δ ) 7.77 (ddd, J ) 6.3, 2.1, 1.8 Hz, 1H; ArH), δ ) 7.24 (m, 1H; ArH), δ ) 3.40 (dd, 2H; OCH2CH2), δ ) 3.23 (dd, 2H; OCH2CH2), δ ) 1.56 (m, 7H; OCH2CH2CH2, C(O)2Me), δ ) 1.33 (m, 12H), δ ) 0.87 (t, 6H; CH2Me). 13C NMR (300 MHz, CDCl3): δ ) 148.7, 148.4, 139.2, 122.9, 99.94, 61.27, 31.7, 29.8, 27.0, 26.0, 22.6, 14.0. HRMS (ESI) calcd for C19H33NO2 (M + Na)+, 330.2403; found, 330.2390. 3-(1,1-Bisoctyloxyethyl)pyridine (4b). A volume of 0.22 mL (2 mmol) of 3-acetylpyridine was combined with 1.9 mL (12 mmol) of 1-octanol in 5 mL of benzene. To the reaction mixture was added a catalytic amount of sulfuric acid. The reaction was carried out at 80 °C for 12 h with azeotropic removal of water from the reaction mixture. The reaction was quenched with NaHCO3(aq), and product 4b was extracted with CH2Cl2. The organic layer was dried over MgSO4. Excess 1-octanol was removed under high vacuum (∼10-4 Torr) and recovered using a cold trap. Reaction proceeded with 73.6% conversion to product 4b. 1H NMR (300 MHz, CDCl3): δ ) 8.78 (d, J ) 4.6 Hz, 1H; ArH), δ ) 8.49 (dd, J ) 4.5, 1.6 Hz, 1H; ArH), δ ) 7.77 (ddd, J ) 4.0, 2.0, 2.0 Hz, 1H; ArH), δ ) 7.23 (dd, J ) 7.7, 5.3 Hz, 1H; ArH), δ ) 3.38 (dd, 2H; OCH2CH2), δ ) 3.23 (dd, 2H; OCH2CH2), δ ) 1.53 (m, 7H; Analytical Chemistry, Vol. 75, No. 23, December 1, 2003
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OCH2CH2CH2, C(O)2Me), δ ) 1.24 (m, 20H), δ ) 0.85 (t, 6H; CH2Me). 13C NMR (300 MHz, CDCl3): δ ) 148.7, 148.4, 139.2, 134.0, 122.8, 99.9, 61.3, 31.8, 29.8, 29.5, 29.3, 27.0, 26.4, 22.6, 14.4. HRMS (ESI) calcd for C23H41NO2 (M + Na)+, 386.3029; found, 386.3028. 3-[3-(1,1-Bishexyloxyethyl)pyridin-1-yl]propane-1-sulfonate (1a). A mass of 0.407 g (3.33 mmol) of 1,3-propane sultone and 5 mL of dry benzene were added to 0.600 g (1.95 mmol) of 4a. The reaction proceeded for 24 h at 40 °C at which time 0.220 mL (2.7 mmol) of pyridine was added to the reaction. The reaction proceeded for an additional 24 h. The pyridine reacts with the excess 1,3-propane sultone to form a water soluble zwitterionic compound which was extracted using NaHCO3(aq). The aqueous layer was back extracted three times with chloroform. The combined organic layer was dried over MgSO4. A mass of 0.48 g (65.9% yield) of the product was isolated as a white solid. 1H NMR (300 MHz, DMSO): δ ) 9.12 (m, 2H; ArH), δ ) 8.61 (d, J ) 8.4 Hz, 1H; ArH), δ ) 8.22 (dd, J ) 8.1, 5.8 Hz, 1H; ArH), δ ) 4.84 (t, 2H; CH2SO3-), δ ) 3.42 (m, 4H; OCH2CH2), δ ) 2.50 (t, 2H; CH2CH2SO3-), δ ) 2.29 (t, 2H; CH2N+), δ ) 1.63 (m, 7H; OCH2CH2CH2, C(O)2Me), δ ) 1.33 (m, 12H), δ ) 0.93 (t, 6H; CH2Me). 13C NMR (300 MHz, DMSO): δ ) 144.4, 143.65, 142.95, 142.8, 127.91, 99.0, 61.2, 59.9, 47.0, 31.1, 29.1, 27.6, 25.9, 25.4, 22.1, 13.9. HRMS (ESI) calcd for C22H39NO5S (M + Na)+, 452.2441; found, 452.2428. 3-[3-(1,1-Bisoctyloxyethyl)pyridin-1-yl]propane-1-sulfonate (1b). A mass of 0.230 g (1.89 mmol) of 1,3-propane sultone and 2 mL of dry toluene were added to 0.341 g (0.938 mmol) of 4b. The reaction proceeded for 24 h at 40 °C at which time 0.100 mL (1.24 mmol) of pyridine was added to the reaction. The reaction proceeded for an additional 24 h. The product was washed using NaHCO3(aq), and the aqueous layer is back extracted three times with chloroform. The combined organic layer was dried over MgSO4. A mass of 0.405 g (88.9% yield) of the product was isolated as a white solid. 1H NMR (300 MHz, CDCl3): δ ) 9.65 (d, J ) 6.0 Hz, 1H; ArH), δ ) 8.68 (s, 1H; ArH), δ ) 8.36 (d, J ) 8.1 Hz, 1H; ArH), δ ) 8.15 (dd, J ) 8.1, 5.7 Hz, 1H; ArH), δ ) 5.07 (t, 2H; CH2SO3-), δ ) 3.42 (dd, 2H; OCH2CH2), δ ) 3.15 (dd, 2H; OCH2CH2), δ ) 2.94 (t, 2H; CH2CH2SO3-), δ ) 2.56 (t, 2H; N+CH2), δ ) 1.56 (m, 7H; OCH2CH2CH2, C(O)2Me), δ ) 1.27 (m, 20H), δ ) 0.92 (t, 6H; CH2Me). 13C NMR (300 MHz, CDCl3): δ ) 146.5, 145.5, 142.6, 141.6, 128.6, 99.1, 62.2, 61.0, 47.0, 31.8, 29.6, 29.4, 29.2, 28.1, 26.8, 26.3, 22.6, 14.1. HRMS (ESI) calcd for C26H47NO5S (M+Na)+, 508.3067; found, 508.3063. CMC Measurement. Critical micelle concentrations (CMCs) were determined by the DuNo¨y ring method using a manual tensiometer. CMC measurements were made in 50 mM TRIS‚ HCl (pH 7.4).35 Analysis of Standard Proteins. The standard protein, porcine insulin, was purchased from Sigma-Aldrich (St. Louis, MO) and used without further purification. Monitoring the signal-to-noise ratio as a function of insulin concentration tested the sensitivity of MALDI analysis in the presence of cleavable detergent. Control experiments without detergent and with 0.5% n-octylglucoside were performed for comparison. (35) Although these values will deviate from results obtained in pure water, rigorous control of pH prevents hydrolysis from occurring during the experiment.
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Preparation of Membrane Protein. The membrane protein poly-His Rv0011c, expressed in Mycobacterium tuberculosis, was purified from whole cell lysate using a Ni(II) resin bathed in 0.5% dodecyl maltoside (DM). DM was removed from a volume of 400 µL of resin containing 1 mg/mL of Rv0011c. The resin was reconstituted in 700 µL of a 0.5% PPS solution. The resin was incubated at 4 °C for a period of 10 min. The purified, resin-bound proteins were eluted from the resin using 250 mM imidazole in 0.5% PPS. Control experiments were done in the absence of PPS. Concentration of solubilized Rv0011c was estimated using gel electrophoresis and by comparing the intensity of the PPS solubilized Rv0011c with that of standards on a silver stained PAGE gel. Samples were prepared for MALDI-MS analysis using the procedure outlined in following paragraphs. Preparation of Cell Lysates. The human colorectal cell line, RKO, was grown in DMEM Hi glucose (Gibco BRL, Gaithersburg, MD) medium supplemented with 10% fetal calf serum (Gemini Bio-Products, Inc., Calabasas, CA) and 1% penicillin/streptomycin (Sigma, St. Louis, MO). Cell extracts containing PPS were prepared along with separate extracts 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), phenylmethylsulfoyl fluoride (50 µg/mL) (Sigma), and 4-(2-aminoethyl)benzenesulfonylfluoride (200 µg/ mL) (Calbiochem-Novabiochem Corp.)]. Cell extracts containing PPS were prepared using a modified version of RIPA buffer that excludes the detergent components SDS, nonidet P-40, and sodium deoxycholate. Cells were incubated for 1 h in the presence of lysis buffer followed by homogenization. Cell debris was removed by centrifugation at 3000 × g for 30 min. MALDI Sample Preparation/Detergent Cleavage. All samples were prepared for analysis by dialyzing 20 µL of each sample against 3:2:1 water/2-propanol/formic acid (pH 1.4). The dialysis membranes used have MWCO of 3500, and the dialysis was done for 2 h. The low pH of this solution will begin to degrade the detergent, 6-PPS, while removing the cleavage products and other nonprotein impurities from the solution. Dialyzing against 33% organic solvent helps to maintain the stability of hydrophobic proteins in solution. Samples were mixed on target in a 1:1 ratio with sinapinic acid (20 mg/mL in 50% acetonitrile in 0.1%TFA). Mass Spectrometry. Samples were analyzed using an Applied Biosystems Voyager DE STR (Framingham, MA) equipped with a nitrogen laser (λ ) 337 nm). RESULTS AND DISCUSSION Detergent Synthesis. The cleavable detergent, PPS, was synthesized using the method outlined in Scheme 1. A number of considerations must be addressed in order to design a molecule that functions well when used with MALDI mass spectrometry. One prerequisite is that the conditions required for cleavage not chemically alter the protein or introduce reagents that have a deleterious effect on the MALDI process. There are many different paradigms for cleavable surfactant design one could consider.26-28 Due to the nature of the MALDI experiment, an acid cleavable group was chosen. Prior to analysis, the pH of the analyte is lowered to approximately pH 2 with little or no modification to
Scheme 2. Cleavage of PPS
protein primary structure. The acidic conditions will initiate cleavage of an acid sensitive group such as a ketal or acetal. This reaction has been used in a variety of reagents in combination with analysis by mass spectrometry.29,30,32 Another design consideration is the tolerance of the MALDI process toward the products generated by cleavage of the detergent. This is of particular importance in applications that require the direct analysis of cells and tissue. Scheme 2 outlines the cleavage reaction of PPS. The hydrophobic tail of PPS was designed having a double chain configuration to reduce the length of the required hydrophobic chains to eight carbons or less individually. Upon cleavage, two molecules of a volatile alcohol are generated. This is preferable to a single long chain because high molecular weight alcohols are not volatile and would remain in the sample, interfering with crystallization. Likewise, the zwitterionic head of the surfactant must not interfere with cocrystallization of the analyte in the matrix, cause ion suppression of the protein, or otherwise react with the analyte. Subsequent data will demonstrate that the pyridinium propyl sulfonate structure (5) chosen as the headgroup is easily removed from the sample and does not exhibit any of these properties at relevant concentrations. Detergent Properties. The critical micelle concentration of PPS was determined using surface tension measurements. Figure 2 shows a plot of the surface tension of TRIS buffered PPS (pH 7.4) as a function of PPS concentration. The observed CMCs of the different PPS analogues are 0.128 and 0.112 mM for compounds 1a and 1b, respectively. These values are used to determine the effective concentration range for PPS when solubilizing proteins. It is common practice to use a concentration equivalent to 1 × CMC;36 however, a number of higher concentrations were tested to empirically determine the optimum concentration for mass spectrometry. For each sample prepared, cleavage can be confirmed by acquiring a low mass MALDI spectrum (Figure 3). The characteristic molecular ion is [M + H]+ ) 430 for compound 1a and [M + H]+ ) 486 for compound 1b. Although the CMCs of both 1a and 1b are similar and both compounds are apparently useful for protein solubilization, cleavage of 1b is not as rapid. Exposure to an acidic environment of pH 1.4 results in cleavage of 1a within 2 h while 1b requires exposure overnight. In light of this fact and given that the effective concentrations of 1a and 1b are nearly identical, all following mass spectrometric data utilize compound 1a. Effect of Detergent on MALDI-MS. The typical problem associated with detergent additives in MALDI mass spectrometry is signal suppression. This is primarily caused through detergent interference with proper cocrystallization between the matrix and the analyte.17,19,22-25 The properties that allow detergents to solubilize hydrophobic molecules can also sequester the protein (36) Womack, M. D.; Kendall, D. A.; MacDonald, R. C. Biochim. Biophys. Acta 1983, 210, 210-215.
Figure 2. Critical micelle concentration of PPS.
Figure 3. MALDI mass spectra of cleavable detergent 1a (a) before and (b) after treatment with acid.
Figure 4. Effect of detergent cleavage on sinapinic acid crystallization.
preventing cocrystallization with matrix. Figure 4 demonstrates this effect as it applies to the cleavable detergent PPS. The four pictures depict the change in crystal morphology as a function of time when a solution of PPS is exposed to aqueous acid. A 0.128 mM (1 × CMC) aqueous solution of PPS was treated with 3:2:1 water/2-propanol/formic acid by solvent exchange through dialysis. An aliquot of this sample was removed at various time intervals to qualitatively determine the effect of cleavage on the MALDI process. Each aliquot was used to prepare a MALDI sample with sinapinic acid. The dried droplets show an increase in the density of sinapinic acid crystals as PPS degrades. At 30 min, PPS is still present resulting in an amorphous coating rather than a crystalline sample. After 140 min of treatment, crystals form which are suitable for MALDI analysis. No trace of PPS can be observed in the MALDI spectra of such samples. A significant effect on the quality of MALDI mass spectra of proteins is observed when the detergent is cleaved as shown in Figure 5. Even at relatively high concentrations of PPS, 0.5% (w/v) or approximately 90 × CMC, the effect on MALDI Analytical Chemistry, Vol. 75, No. 23, December 1, 2003
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Figure 7. MALDI-MS analysis of Rv0011c from Mycobacterium tuberculosis using PPS.
Figure 5. Effect of detergent cleavage on sensitivity.
Figure 6. Overall sensitivity compared to other methods.
sensitivity is minimal. Cleavage of the detergent causes a marked increase in sensitivity shown here by the increased signal-to-noise of insulin. These effects can be attributed to the presence and absence of detergent properties in the protein preparation and an increase in soluble insulin available for analysis. The sensitivity of the cleavable detergent method also compares well to other commercial detergents. The most popular and effective conventional detergent used for MALDI analysis is n-octylglucoside (NOG).20,37 Figure 6 compares the sensitivity of the MALDI-MS analysis of insulin in 0.5% NOG with that of PPS. Using this cleavable detergent enhances the S/N of insulin relative to NOG and control experiments in the absence of detergent. At low concentrations (
