Anal. Chem. 2003, 75, 3010-3018
Quantitative Determination of Noncovalent Binding Interactions Using Automated Nanoelectrospray Mass Spectrometry Sheng Zhang,*,† Colleen K. Van Pelt,† and David B. Wilson‡
Advion BioSciences, Inc., 15 Catherwood Road, Ithaca, New York 14850, and Department of Molecular Biology and Genetics, Cornell University, New York 14853
Electrospray ionization mass spectrometry (ESI-MS) has proven to be an extremely powerful tool for studying biomolecular structures and noncovalent interactions. Here we report a method using a fully automated, chipbased nanoESI-MS system to determine the dissociation constants (Kd) for the complexes of two different proteins with their ligands. The automated nanoelectrospray system, consisting of the NanoMate and ESI chip, serves functionally as a combination of autosampler and nanoelectrospray ionization source. This system provides all the advantages of conventional nanoelectrospray plus automated, high-throughput analyses without carryover. The automated nanoESI system was used to investigate quantitative noncovalent interactions between ribonuclease A (RNase A) and cytidylic acid ligands (2′-CMP, CTP), a well-characterized model protein-ligand complex, and between an inactive endocellulase mutant (Thermobifida fusca Cel6A D117Acd) and four oligosaccharide ligands (cellotriose, cellotetraose, cellopentaose, cellohexaose). Both titration and competitive binding approaches were performed prior to automated nanoESIMS analysis with a Q-TOF mass spectrometer. Dissociation constants for each complex were calculated from the sum of ion peak areas of free and complexed proteins during the titration and competition experiments. The measured Kd values for the RNase A-CMP and Cel6A D117AcdG3 complexes were found to be in excellent agreement with the available published values obtained by standard spectroscopic titration techniques. To our knowledge, this is the first report of using an ESI-MS approach to study the interactions between a cellulase and oligosaccharides. The results provide new insights for understanding the nature of cellulase-cellulose interactions. It is well known that many important biological functions are mediated through noncovalent interactions between biopolymers and other components existing in the cell.1,2 Traditionally, proteins were targeted as they possess structures for specific molecular * To whom correspondence should be addressed. Phone: 607-266-0665 ×223. Fax: 607-266-0749. E-mail:
[email protected]. † Advion BioSciences, Inc. ‡ Cornell University. (1) Pramanik, B. N.; Bartner, P. L.; Mirza, U. A.; Liu, Y. H.; Ganguly, A. K. J. Mass Spectrom. 1998, 33, 911-920.
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recognition by ligands. With the advance of combinatorial chemistry and the establishment of large drug candidate libraries, developments in rapid-screening technologies for identifying potential therapeutic targets in complex mixtures are of considerable interest and have been essential to the pharmaceutical industry. Electrospray ionization mass spectrometry (ESI-MS) has proven to be a useful tool for studying biomolecular structures and noncovalent interactions.3,4 Electrospray is a soft ionization technique that introduces the weakly bound complexes formed in solution into the gas phase where they can be analyzed by mass spectrometry. Since the first reports in 1991 by Ganem and Henion,3,4 the use of conventional ESI-MS for studying noncovalent complexes in solution has been successfully demonstrated and reviewed.1,2,5-10 There have been many studies using this technique to determine the stoichiometry and dissociation constants for a variety of biological noncovalent complexes.11-18 Some of the authors have shown that equilibrium binding constants can be determined by titration experiments11-13,18 while others have used competition experiments.18-20 Dissociation constants mea(2) Daniel, J. M.; Friess, S. D.; Rajagopalan, S.; Wendt, S.; Zenobi, R. Int. J. Mass Spectrom. 2002, 216, 1-27. (3) Ganem, B.; Li, T.; Henion, J. D. J. Am. Chem. Soc. 1991, 113, 6294-6296. (4) Ganem, B.; Li, T.; Henion, J. D. J. Am. Chem. Soc. 1991, 113, 7818-7820. (5) Ganem, B.; Henion, J. D. Bioorg. Med. Chem. 2003, 11, 311-314. (6) Winston, R. L.; Fitzgerald, M. C. Mass Spectrom. Rev. 1997, 16, 165-179. (7) Smith, R. D.; Bruce, J. E.; Wu, Q.; Lei, Q. P. Chem. Soc. Rev. 1997, 26, 191-202. (8) Loo, J. A. Mass Spectrom. Rev. 1997, 16, 1-23. (9) Veenstra, T. D. Biophys. Chem. 1999, 79, 63-79. (10) Schalley, C. A. Mass Spectrom. Rev. 2001, 20, 253-309. (11) Gao, J.; Cheng, X.; Chen, R.; Sigal, G. B.; Bruce, J. E.; Schwartz, B. L.; Hofstadler, S. A.; Anderson, G. A.; Smith, R. D.; Whitesides, G. M. J. Med. Chem. 1996, 39, 1949-1955. (12) Jorgensen, T. J. D.; Roepstorff, P.; Heck, A. J. Anal. Chem. 1998, 70, 44274432. (13) Lim, H. K.; Hsieh, Y. L.; Ganem, B.; Henion, J. J. Mass Spectrom. 1995, 30, 708-714. (14) Ayed, A.; Krutchinsky, A. N.; Ens, W.; Standing, K. G.; Duckworth, H. W. Rapid Commun. Mass Spectrom. 1998, 12, 339-344. (15) Griffey, R. H.; Hofstadler, S. A.; Sannes-Lowery, K. A.; Ecker, D. J.; Crooke, S. T. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 10129-10133. (16) Pocsfalvi, G.; Ritieni, A.; Randazzo, G.; Dobo, A.; Malorni, A. J. Agric. Food Chem. 2000, 48, 5795-5801. (17) Beck, J. L.; Colgrave, M. L.; Ralph, S. F.; Sheil, M. M. Mass Spectrom. Rev. 2001, 20, 61-87. (18) Sannes-Lowery, K. A.; Griffey, R. H.; Hofstadler, S. A. Anal. Biochem. 2000, 280, 264-271. (19) Kempen, E. C.; Brodbelt, J. S.; Bartsch, R. A.; Jang, Y.; Kim, J. S. Anal. Chem. 1999, 71, 5493-5500. 10.1021/ac034089d CCC: $25.00
© 2003 American Chemical Society Published on Web 06/03/2003
sured by both approaches have been found to correlate well with those measured by solution-based techniques. The advantages of ESI-MS over other techniques for noncovalent binding studies include high sensitivity, speed of analysis, capability of obtaining stoichiometric information, and ability to identify unknown molecules.1,2 In addition, using ESI coupled to Fourier transform ion cyclotron resonance mass spectrometry, interactions between multiple targets and ligands can be simultaneously monitored, which greatly increase screening throughput.21,22 However, one concern for screening complexes by ESI-MS is the consumption of both ligand and target macromolecule. The need for relatively large amounts of purified target is a significant practical challenge for conventional ESI-MS analysis, particularly when low expression or poor purification yield limits the availability of a biological target. Thus, the application of nanoelectrospray MS for studying noncovalent binding complexes is highly desirable. In addition to the lower sample consumption and higher sensitivity offered by nanoelectrospray as compared to conventional ESI, nanoelectrospray with its nanoliter per minute flow rates is also believed to be softer or more gentle than conventional ESI-MS during the transfer of complexes from solution into the gas phase23,24 and can therefore be used to monitor the dissociation of protein complexes.25 However, the disadvantages of nanoelectrospray are its low sample throughput due to tedious single tip alignment procedures, potential sample carryover, and poor reproducibility of the relative intensities of the complexes due to the variable shape of the spray tip and the capillary-cone distance for each repeat analysis.26 Due to these issues, relatively little information on dissociation constants for noncovalent complexes in drug-screening applications has been reported using the nanoelectrospray approach.27 To overcome these disadvantages, we have used a fully automated nanoelectrospray system (NanoMate with ESI chip), developed and characterized recently in our laboratory,28,29 for nanoelectrospray analyses of noncovalent interactions. The ESI chip is a microchip device that consists of a 10 × 10 array of nozzles. The ESI chip is manipulated by a robot achieving automated sample delivery to each nozzle for MS analysis. The system removes the manual, tedious alignment processes of nanoelectrospray while offering a one-time spray optimization and enhances spray stability and reproducibility. In this paper, we report and evaluate the automated nanoESI system for noncovalent complex detection and determination of dissociation constants. Two protein-ligand systems were used (20) Leize, E.; Jaffrezic, A.; Van Dorsselaer, A. J. Mass Spectrom. 1996, 31, 537544. (21) Wigger, M.; Eyler, J. R.; Benner, S. A.; Li, W.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 2002, 13, 1162-1169. (22) Sannes-Lowery, K. A.; Drader, J. J.; Griffey, R. H.; Hofstadler, S. A. Trends Anal. Chem. 2000, 19, 481-491. (23) Rostom, A. A.; Robinson, C. V. Curr. Opin. Struct. Biol. 1999, 9, 135-141. (24) Chung, E. W.; Henriques, D. A.; Renzoni, D.; Morton, C. J.; Mulhern, T. D.; Pitkeathly, M. C.; Ladbury, J. E.; Robinson, C. V. Protein Sci. 1999, 8, 1962-1970. (25) Vis, H.; Dobson, C. M.; Robinson, C. V. Protein Sci. 1999, 8, 1368-1370. (26) Gabelica, V.; Vreuls, C.; Filee, P.; Duval, V.; Joris, B.; Pauw, E. D. Rapid Commun. Mass Spectrom. 2002, 16, 1723-1728. (27) Benkestock, K.; Van Pelt, C. K.; Åkerud, T.; Sterling, A.; Edlund, P. O.; Roeraade, J. J. Biomol. Screening 2003, 8, 247-256. (28) Schultz, G. A.; Corso, T. N.; Prosser, S. J.; Zhang, S. Anal. Chem. 2000, 72, 4058-4063. (29) Van Pelt, C. K.; Zhang, S.; Henion, J. D. J. Biomol. Tech. 2002, 13, 72-84.
for quantitative determination of noncovalent binding interactions. One is ribonuclease A (RNase A) complexed with cytidine 2′monophosphate, a well-characterized model system30-33 with abundant published dissociation constant information. This model system is used to demonstrate the utility of the automated nanoelectrospray system and to validate both titration and competition experiments. The other example is an endocellulase complexed with four oligosaccharide ligands including cellotriose (G3), cellotetraose (G4), cellopentaose (G5), and cellohexaose (G6). Cellulase is a well-known hydrolyase, which degrades cellulose into cellobiose and glucose, and has potential commercial value.34 Fully understanding the interactions of cellulase with cellulose and making the native cellulose more accessible to cellulase are thought to be the keys to increasing cellulase activity on native cellulose.35 Measurement of cellulase binding has been traditionally performed by fluorescence titration using 4-methylumbelliferyl-modified β-cellotrioside (MUG3) and β-cellobiose (MUG2) as substrates. The binding constant for a native oligosaccharide (G3) to cellulase was experimentally determined by a displacement titration using MUG2 as an indicator ligand.36,37 Other G4, G5, and G6 ligands were bound too tightly to be determined using the displacement titration approach. Additional methods for measuring the binding affinities of G4, G5, and G6 are needed in order to understand the relationship between binding and activity for cellulose. To determine the dissociation constants for G3, G4, G5, and G6, one of the most intensively characterized cellulases, an inactive endocellulase mutant (Cel6A D117Acd) from the bacterium Thermobifida fusca, was selected for the study.36-39 This inactive mutant has lost nearly all activity but retains normal substrate binding.37 Thus, the use of this mutant prevents any hydrolysis during the noncovalent binding experiments. The three-dimensional structure of its wild type (Cel6Acd) is available40 and is shown in Figure 1B; also shown in Figure 1B are the structures of the four oligosaccharide ligands used in this study. Both titration and competitive binding were measured using automated nanoESI-MS analysis. The Kd value for G3 determined by nanoESI-MS was compared with published data by fluorescence titration.36 The values of the dissociation constants of Cel6A D117Acd to four oligosaccharides were compared, and the binding interactions and mechanism are discussed. This is the first report, using an ESI-MS approach, to study the interactions of a cellulase and oligosaccharides. EXPERIMENTAL SECTION Materials. Bovine pancreatic RNase A (MWav )13 682), cytidine 2′-monophosphate (2′-CMP, MWav ) 323.2), cytidine (30) Jones, C. L.; Fish, F.; Muccio, D. D. Anal. Biochem. 2002, 302, 184-190. (31) Brandts, J. F.; Lin, L. N. Biochemistry 1990, 29, 6927-6940. (32) Wiseman, T.; Williston, S.; Brandts, J. F.; Lin, L. N. Anal. Biochem. 1989, 179, 131-137. (33) Straume, M.; Freire, E. Anal. Biochem. 1992, 203, 259-268. (34) Sun, Y.; Cheng, J. Bioresour. Technol. 2002, 83, 1-11. (35) Mosier, N. S.; Hall, P.; Ladisch, C. M.; Ladisch, M. R. Adv. Biochem. Eng. Biotechnol. 1999, 65, 23-40. (36) Barr, B. K.; Wolfgang, D. E.; Piens, K.; Claeyssens, M.; Wilson, D. B. Biochemistry 1998, 37, 9220-9229. (37) Wolfgang, D. E.; Wilson, D. B. Biochemistry 1999, 38, 9746-9751. (38) Zhang, S.; Barr, B. K.; Wilson, D. B. Eur. J. Biochem. 2000, 267, 244-252. (39) Zhang, S.; Wilson, D. B. J. Biotechnol. 1997, 57, 101-113. (40) Spezio, M.; Wilson, D. B.; Karplus, P. A. Biochemistry 1993, 32, 99069916.
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Figure 1. Structures of proteins and ligands used in this study. (A) Structures of 2′-CMP and CTP and a ribbon diagram of RNase A in a complex with 2′-CMP adapted from 1ROB.pdb;45 (B) Structures of G3, G4, G5, G6, and Cel6Acd which shows the ribbon diagram of Cel6Acd with G4 modeled into the active site, adapted from 1TML.pdb.40 Both diagrams were created using the RasMol program.46
triphosphate (CTP, MWav ) 483.1), and ammonium acetate were purchased from Sigma (St. Louis, MO). Cellotriose (G3, MWav ) 504.4), cellotetraose (G4, MWav ) 666.6), cellopentaose (G5, MWav ) 828.7), and cellohexaose (G6, MWav ) 990.9) were purchased from Seikagaku Corp. (Tokyo, Japan), and all other chemical reagents, unless otherwise noted, were obtained from Aldrich (Milwaukee, WI). Cel6A D117Acd protein (MWav ) 30363) was overexpressed and purified from Escherichia coli BL21(DE3) after its gene was cloned into pET26b(+) (J. H. Kim, unpublished data) from the parent pDW4 plasmid containing the Cel6A D117A gene.37 The purified protein was dialyzed against 5 mM ammonium acetate, pH 6.8. Protein concentrations were determined from the OD280 nm using extinction coefficients ) 57 580 M-1 cm-1 for Cel6A D117Acd and ) 8160 M-1 cm-1 for RNase A. Sample Preparation for Noncovalent Interaction Assays. For RNase A-2′-CMP interactions, RNase A protein was kept constant at 10 µM (10 pmol/µL) in a final solution containing 10 mM ammonium acetate, pH 6.8. In titration experiments, RNase A was mixed with various concentrations (from 1 to 20 µM) of 3012
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2′-CMP and allowed to equilibrate at room temperature for at least 10 min prior to nanoESI-MS analysis. For RNase A-CTP reactions, RNase A was constant at 4 µM containing 10 mM ammonium acetate and titrated with CTP from 1 to 8 µM. In competitive experiments, equimolar 2′-CMP and CTP (4 µM) were mixed with 4 µM RNase A in 10 mM ammonium acetate, pH 6.8. The mixture was then incubated at room temperature for 10 min followed by MS analysis. The analysis of the titration samples was performed from the lowest concentration of ligands to the highest. For the Cel6A D117Acd-G3 titration, protein (50 µM) was mixed with 15-100 µM G3 in water at room temperature for 15 min. An equal amount of 0.1% acetic acid was added to each reaction prior to nanoES/MS analysis. In the G4 titration experiments, Cel6A D117Acd (24 µM) was mixed with 6-96 µM G4 in water, and in the G5 titration experiments, the protein (6 µM) was mixed with 1-15 µM G5. For both the G4 and G5 titrations, the samples were incubated at room temperature for 15 min before an equal amount of 0.1% acetic acid was added to each reaction prior to analysis. For competitive binding experiments, 36 µM G3
and G4 were mixed with 36 µM protein, while for the G4-G5 and G5-G6 competitions, 10 and 2 µM concentrations of each ligand were used with 10 and 2 µM protein, respectively. All samples were incubated at room temperature for 15 min, and an equal amount of 0.1% acetic acid was added before nanoESI-MS analysis. All titration and competition samples were prepared in triplicate and analyzed by automated nanoESI-MS using the NanoMate and Q-TOF-MS. Automated NanoESI-MS Analysis. All analyses were performed on a Q-TOF micro mass spectrometer (Micromass, Beverly MA) equipped with an automated nanoelectrospray system, the NanoMate 100 (Advion BioSciences, Inc. Ithaca, NY).29 The NanoMate holds a 96-well sample plate, a rack of 96 disposable, conductive pipet tips, and an ESI chip that was positioned ∼5 mm from the sampling cone of the Q-TOF micro. The NanoMate, controlled by ChipSoft software, sequentially picked up a new pipet tip, aspirated 3 µL of sample from the 96well plate where 5 µL of sample was placed in each well, and delivered the sample to the inlet side of the ESI chip. The ESI chip consists of a 10 × 10 array of nozzles etched from the planar surface of a silicon wafer. A channel extends from the nozzle through the chip to an inlet etched on the opposite planar surface.28 It is the disposable pipet tip, containing the sample to be analyzed, that seals against the inlet channel of the chip. Nanoelectrospray was initiated by applying a 1.55-kV spray voltage and a 0.3 psi nitrogen head pressure to the sample in the pipet tip. The estimated flow rate emitting from the nozzles of the ESI chip was 100 nL/min. Following sample infusion and mass spectrometric analysis, the used pipet tip was ejected. The typical operating conditions for the Q-TOF micro mass spectrometer and NanoMate were as follows: spray voltage, 1.55 kV; sample pressure, 0.3 psi; sample cone voltage, 30 V; source temperature, 45 °C. The collision gas pressure was activated. The Q-TOF was operated in TOF-MS positive ion mode with the fullscan mass spectra recorded in profile mode. Data for each sample was acquired for 2 min in the mass range between m/z 1000 and 3500. A sample list containing up to 96 sample entries was created in MassLynx, and the instrument was programmed to wait for a contact closure signal. The NanoMate sent a contact closure to the Q-TOF micro at the start of each infusion experiment, triggering the Q-TOF micro to begin its 2-min acquisition. Upon the completion of the run, the Q-TOF micro had acquired files, each with their respective infusion-derived full-scan mass analysis. Each spectrum was integrated from the average of 58 scans with a scan rate of 1 scan/2 s. All data were processed using MassLynx software version 3.5 from Micromass. Calculation of Dissociation Constants. The calculation of Kd and the application of competitive binding experiments by ESIMS analysis were modified based on a previous report by Loo and co-workers.41 Using the ESI-MS approach, the stoichiometry and relative abundance of both free protein and the protein-ligand complex can be simultaneously determined, as the number of binding sites for a given protein-ligand complex can be determined from the spectrum based on the mass difference between the free target and its complex. Only one binding site was found (41) Loo, J. A.; Hu, P.; McConnell, P.; Mueller, W. T.; Sawyer, T. K.; Thanabal, V. J Am. Soc. Mass Spectrom. 1997, 8, 234-243.
for both RNase A and Cel6A D117Acd (see Figures 2 and 5). Dissociation constants were then calculated from the following equation for a single ligand binding:
Kd )
[R][L] [R]([Li] - [RL]) ) [RL] [RL]
(1)
The sum of absolute peak areas (intensities) of respective charge states was substituted for concentrations, where [R] represents the peak area for all charge states of free protein, [RL] indicates the sum of all charge state peak areas of protein-ligand complex, and [L] is the concentration of the unbound ligand after the reaction reached equilibrium. The conversion of [RL] from peak area to concentration units is based on the fact that the total protein concentration [Rtotal] used in the study is known. For one binding site, [Rtotal] is equal to the sum of both [R] and [RL] peak areas. Thus, the peak areas of [RL] and [R] can be converted to concentration units. Substituting the [L] with [Li] - [RL], where [Li] represents the initial concentration of the ligand gives the following equation:
[RL]/[R] ) 1/Kd([Li] - [RL])
(2)
Thus, plotting [RL]/[R] versus ([Li] - [RL]) produces a straight line with 1/Kd as the slope. In the competitive binding experiments where an equimolar concentration of R, L1, and L2 were used, the Kd was calculated based on the following equations:
KdRL1 ) [R]([R] + [RL2])/[RL1]
(3)
KdRL2 ) [R]([R] + [RL1])/[RL2]
(4)
The [L1], which is the equilibrium concentration of ligand 1, is equal to ([R] + [RL2]) under the initial equal molar case, where [Ri ] ) [R] + [RL1] + [RL2], and [L1i] ) [L1] + [RL1]. As [Ri] ) [L1i], so [L1] ) [R] + [RL2]. Meanwhile, [L2], which is the equilibrium concentration of ligand 2, is equal to ([R] + [RL1]) under the same conditions as described above for L1. RESULTS AND DISCUSSION The goal of this work was to investigate whether the automated nanoelectrospray platform (NanoMate and ESI chip) coupled to MS could be used for the reliable determination of noncovalent interactions as well as for the quantitative determination of absolute dissociation constants for protein target and ligands. Initial experiments demonstrated the feasibility and reproducibility of the automated nanoelectrospray platform using a wellcharacterized RNase A-2′CMP system as a “gold standard” (see Figure 1A). The results showed that, in three different titration experiments using different nozzles for each sample in each trial, the reproducibility of the relative peak abundance (areas) for free RNase A ions [R] and RNase A-2′CMP complex ions [RL] was satisfactory as shown in Table 1. The resulting Kd values were in good agreement with those obtained by other methods (see below), indicating that the automated nanoelectrospray system Analytical Chemistry, Vol. 75, No. 13, July 1, 2003
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Table 1. Titration Data with Equilibrium Concentrations Deduced from the Sum of Measured Peak Areas for an RNase A-CMP Complex [RL] and Unbound RNase A [R] trial 1a
trial 2
trial 3
2′-CMP (µM)
[R]
Kd [RL] (µM)
[R]
Kd [RL] (µM)
[R]
Kd [RL] (µM)
0 1 2 3 4 5 6 8 10 12 15
4471 6064 4573 4771 4755 4502 3768 2828 1865 1780 1349
0 529 955 1693 2395 3094 3354 3821 3832 4735 5113
4858 6233 5555 5343 4652 4535 3837 3835 2174 2086 1339
0 592 1210 1832 2492 2749 3284 4475 4286 4692 4978
6988 8045 4660 4184 5056 4853 4480 3554 2694 2683 1832
0 779 1016 1396 2607 2611 3377 3933 4692 5864 6076
av SD RSD a
2.27 1.30 1.07 1.29 1.35 1.45 1.67 1.59 1.78 1.87 1.56 0.26 16.59
1.40 0.97 1.30 0.96 2.02 1.62 2.24 1.71 2.26 1.92 1.67 0.50 29.97
1.21 0.96 1.49 1.16 2.79 2.26 2.48 2.09 2.35 2.21 1.90 0.63 32.92
RNase A concentration was maintained constant at 10 µM.
can be used for quantitative studies of high-throughput noncovalent binding interactions with good reproducibility. Determination of RNase A Target and Complex Concentrations. For the RNase A model system in 100% aqueous conditions (10 mM ammonium acetate, pH 6.8), three charge states from +8 to +6 were observed over the mass range of m/z 1000-3000 for both free RNase A and the RNase A-CMP and the RNase A-CTP complexes (Figure 2). This charge-state distribution is identical to those from a conventional ESI source with a 5 µL/min flow injection of similar samples.42 In the RNase A samples, the low abundant phosphate adduct ions were observed for each charge state as shown in Figure 2. Upon deconvolution of the spectra for all three samples, average masses of 13 682.8, 14 005.8, and 14 166.0 Da were obtained (Figure 3), which is in good agreement with the theoretical average masses for RNase A, RNase A-CMP, and RNase A-CTP, respectively. The RNase A-CMP and RNase A-CTP complexes increased in mass by 323 and 483 Da, respectively, as compared to free RNase A. Panels A-D of Figure 2 show that ∼90% of the RNase A signal comes from the +7 charge state at m/z 1955.65. The RNase A complexes with both 2′-CMP at m/z 2001.97 and CTP at m/z 2024.59 do not change the observed charge-state distribution. However, it is surprising that the ratio of free RNase A to RNase A-CMP/CTP complex ion intensity increases as the charge state decreases from +8 to +6 (see RNase A-2′-CMP example in panel E of Figure 2). For example, at the +8 charge state, the ratio of free RNase A ion intensity to RNase A-2′-CMP complex ion intensity was found to be 0.65, which is lower than those, 0.73 and 1.1, at the +7 and +6 charge states, respectively. This result suggests that either the binding of CMP/CTP to RNase A or the presence of ligand in the analyzed sample creates a slight change of the charge-state distribution for the complex, although it did not change the fact that ∼90% of the complex signal is in the +7 charge state. It is unlikely that the presence of free ligand in the (42) Benkestock, K.; Edlund, P. O.; Roeraade, J. Rapid Commun. Mass Spectrom. 2002, 16, 2054-2059.
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Figure 2. Representative nanoelectrospray mass spectra of the noncovalent interactions of RNase A with 2′-CMP and CTP obtained in 10 mM ammonium acetate, pH 6.8. Arrows indicate the mass shift at each charge state for complexes formed. In panels A-D are the mass spectra of (A) 10 µM RNase A, (B) 10 µM RNase A plus 10 µM 2′-CMP, (C) 10 µM RNase A plus 10 µM CTP, and (D) 10 µM RNase A plus 10 µM 2′-CMP and 10 µM CTP. In the RNase A samples, the low-abundant phosphate adduct ions were observed for each charge state. All spectra are the sum of 58 scans. Panel E is the same as panel B, but the peaks at both the +8 and +6 charge states were magnified 6- and 10-fold, respectively. The ratios of free RNase A ion intensity to RNase A-2′-CMP complex ion intensity at the +8, +7, and +6 charge states were found to be 0.65, 0.73, and 1.1, respectively.
Figure 3. Deconvoluted mass spectra of RNase A and both the RNase A-2′-CMP and RNase A-CTP complexes obtained in 10 mM ammonium acetate, pH 6.8. In panels A-C are shown the mass spectra of (A) 10 µM RNase A, (B) 10 µM RNase A plus 10 µM 2′CMP, and (C) 10 µM RNase A plus 10 µM CTP. The low-abundant phosphate adduct ions (+ 98 Da) were observed in RNase A samples and are indicated by * in panel A. These adducts are also observed in panels B and C but are not indicated. The deconvoluted spectra were generated using the MaxEnt1 deconvolution program with the resolution at 0.1 Da/channel and the minimum intensity ratios at L66% and R66%. The deconvoluted mass spectra of both the RNase A-2′CMP and RNase A-CTP complexes increased in mass by 323 and 483 Da, respectively, as compared to free RNase A.
sample would affect the charge-state distribution differently for the free RNase A and the RNase A-ligand complexes. One possible explanation for the slight differences in charge-state distribution between RNase A and its complexes is that the RNase
Table 2. Dissociation Constants (Kd) of Ribonuclease A with Cytidylic Acid Ligands Obtained by Automated NanoESI-MS Analysis Kd (µM)a titration experiment
ligand cytidine 2′-monophosphate (2′-CMP) cytidine triphosphate (CTP) a
average of individual points
slope of plot
competitive binding expt
1.71 ( 0.33
2.00 ( 0.43
2.3 ( 0.4
0.80 ( 0.2
0.74 ( 0.3
0.75 ( 0.4
The values represent the mean ( SD of three measurements.
A-ligand complex possesses an induced conformation change upon ligand binding. The finding of a relatively higher protonated portion of RNase A-ligand complex than free RNase A suggests that the conformation change of the complex is slightly less compacted. To minimize these differences between free RNase A and its complexes, the contributions to the signal from all three charge states were summed for this work. For each integrated spectrum, the peak areas for free RNase A and the RNase A complexes were calculated using the MassLynx software. Based on the observed results and other reports, the binding of the ligand to the protein target did not change the overall ionization efficiency of the complex.7,8,18 As a result, it was assumed for all calculations that the free protein and protein-ligand complexes share similar ionization efficiencies, and the measured peak areas for both free protein and its complexes correlate with their solution equilibrium concentrations. To verify the titration results, a competitive binding experiment with equal molar CMP and CTP to the target protein was carried out as shown in panel D of Figure 2. The peak areas of all three ions (one free RNase and two complexes) were summed from three charge states, and the Kd from both experiments was compared as described below. Kd Results of RNase A-2′-CMP and RNase A-CTP. Using automated nanoESI-MS, the Kd of RNase A-2′-CMP and RNase A-CTP were measured by the titration approach using a constant RNase A concentration and titrating the ligands. Increasing the amount of ligand in the mixture results in the formation of more complex, and the change in the ratio of free RNase A to bound RNase A can be used to calculate the Kd. For each initial ligand concentration used in the titration, the Kd can be directly calculated based on eq 1 and a final Kd can be averaged from all points for the different ligand concentrations. The results are shown in Table 2, where the Kd for RNase A-2′-CMP and RNase A-CTP were 1.71 ( 0.33 and 0.8 ( 0.2 µM, respectively. Alternatively, the plot of [RL]/[R] versus [Li] - [RL] for a titration was also used to determine the Kd. The plots for one measurement of both the RNase A-CMP and RNase A-CTP titrations are shown in Figure 4. The Kds were calculated to be 2.0 ( 0.43 and 0.74 ( 0.3 µM, respectively. Competitive binding assays for both 2′-CMP and CTP produced consistent values with 2.3 ( 0.4 µM for the CMP complex and 0.75 ( 0.4 µM for the CTP complex (Table 2), suggesting that nonspecific binding in this assay was minimized and that the automated nanoESI-MS analysis method used in this study is quite reproducible as both the titration and competition approaches gave comparable results.
Figure 4. Plots of [RL]/[R] versus [Li] - [RL] for the titration of RNase A with cytidylic acid ligands. (A) The Kd for RNase A and 2′CMP was determined by keeping the RNase A concentration at 10 µM and titrating 2′-CMP from 1 to 15 µM. A linear least-squares fit gives a correlation coefficient of 0.98 with a Kd ) 1.77 µM from the inverse of the slope. (B) The Kd for RNase A and CTP was determined by keeping RNase A concentration at 4 µM and titrating CTP from 0.5 to 8 µM. A linear least-squares fit gives a correlation coefficient of 0.985 with a Kd ) 0.72 µM from the inverse of the slope. Table 3. Comparison of Dissociation Constants of RNase A-2′-CMP Complex Measured by Different Methods methods
Kd (µM) (ITC)a
isothermal titration calorimetry differential scanning calorimetry (DSC)b circular dichroism (CD)c automated NanoESI-MS (this work)
1.0 ( 0.8 0.5 ( 0.8 1.6 ( 0.4 2.0 ( 0.4
a Straume and Freire.33 In 15 mM potassium acetate (KOAc) buffer, pH 5.5 at 25 °C. b Wiseman et al.32 In 200 mM KOAc, 200 mM KCl buffer, pH 5.5 at 28 °C. c Jones et al.30 In 50 mM KOAc buffer, pH 5.5 at 25 °C.
Comparison of the Kd values with previously published data measured by alternative spectroscopic methods for the same RNase A-2′-CMP complex further demonstrates the reliability of the described nanoelectrospray system. Table 3 summarizes the Kd values of RNase A-2′-CMP obtained by isothermal titration calorimetry,33 differential scanning calorimetry,31,33 and circular dichroism.30 The results clearly show that the Kd value derived from the present approach is in good agreement with the reported values, so that solution-phase binding affinity is reflected in the nanoESI mass spectra. In addition, our results from both the titration and competitive assays show that CTP has a 2-3-fold higher binding affinity than 2′-CMP to RNase A. The active sites of the structures of RNase A and RNase A-ligand complexes provide insight into the higher binding affinity for CTP. This difference could be attributed to the additional diphosphate in CTP and the different attachment position of the phosphate group to ribose (5′ versus 2′). To obtain accurate data in a titration experiment, the target protein concentration should generally be below the expected Kd while the ligand is titrated through the expected Kd.18 However, Analytical Chemistry, Vol. 75, No. 13, July 1, 2003
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Figure 5. Representative nanoelectrospray mass spectra of the noncovalent interactions for Cel6A D117Acd with G3, G4, G5, and G6. Arrows indicate the mass shift at each charge state for complexes formed. In panels A-E are shown the mass spectra of (A) 3 µM Cel6A D117Acd, (B) 3 µM Cel6A D117Acd plus 15 µM G3, (C) 3 µM Cel6A D117Acd plus 5 µM G4, (D) 3 µM Cel6A D117Acd plus 3 µM G5, and (E) 3 µM Cel6A D117Acd plus 3 µM G6. All spectra are the sum of 58 scans.
an RNase A concentration less than 3 µM was experimentally difficult due to the sensitivity under the MS conditions needed to detect noncovalent interactions. Thus, target protein concentrations used were above the expected Kd. When the titration experiments with 2′-CMP were performed using 20 µM RNase A concentration, the Kd was 2.15 ( 0.45 µM determined from the slope of the titration plot. The results showed that there was no difference in the dissociation constants determined with RNase A concentrations of 20 or 10 µM in 2′-CMP titration experiments, suggesting that the concentration of the protein target can be above the expected Kd for accurate measurement. Determination of Cel6A D117Acd Target and Complex Concentrations. The target protein Cel6A D117Acd was tested under a variety of solvent and ESI conditions. The spectrum obtained from the standard ESI-MS conditions, with a source temperature of 80 °C, cone voltage of 60 V, and 50% methanol in water and 0.1% TFA, showed a broad charge-state distribution with the base peak at a charge state of +23 for Cel6A D117Acd (data not shown). However, the spectrum obtained under 100% aqueous conditions (0.05% acetic acid in water, pH 5.0) displayed a significant change with a compact charge distribution and a shift of charge-state maximum from +23 to +13 (see panel A of Figure 5). This reduction of charge state has been recognized as a feature of spectra obtained from a solution where the protein remains in a more native conformation during introduction to the ESI source.43 The spectra of Cel6A D117Acd in both the denatured and native conformations showed that there are two extra small peaks for each charge state (panel A of Figure 5) at ∼8% of total Cel6A D117Acd abundance. Deconvolution analysis showed that these peaks correspond to Cel6A D117Acd plus an additional two residues (AA) and seven residues (FPSQAAA) miscleaved from the Cel6A signal peptide (protein ID, P26222) during protein expression. As the miscleaved forms contain less than 10% of the total and are expected to retain full binding activity, their effects on the binding results should be very small. (43) Vis, H.; Heinemann, U.; Dobson, C. M.; Robinson, C. V. J. Am. Chem. Soc. 1998, 120, 6427-6428.
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To minimize the potential of nonspecific binding due to electrostatic interactions,18 Cel6A D117Acd was tested in three different volatile salts from 10 to 100 mM ammonium formate, ammonium acetate, and ammonium bicarbonate at pH 3.5-9.5. Unfortunately, none of the solvents gave a detectable signal even at 10 µM protein. Consequently, all the noncovalent interactions were carried out in 0.05% acetic acid in water. The addition of 0.05% acetic acid significantly improved the nanoelectrospray stability for Cel6A D117Acd compared to 100% water. It was found that, under these conditions, nonspecific binding for Cel6A D117Acd-ligand interactions was negligible as long as the maximum ligand concentration was kept below 60 µM and the ratio of ligand to target was below 5. Additional evidence supporting the lack of nonspecific binding came from the competitive binding results, which agree well with the titration results (see below). Four charge states from +14 to +11 were observed over the mass range of m/z 1500-3500 for both the free target and protein-ligand complexes (Figure 5). The binding of Cel6A D117Acd to ligands does not change the observed charge state. Upon deconvolution of the spectra for all five samples, average massesof 30 365.1(Cel6AD117Acd),30 869.4(Cel6AD117Acd+G3), 31 031.7 (Cel6A D117Acd+G4), 31 193.4 (Cel6A D117Acd+G5), and 31 356.1 Da (Cel6A D117Acd+G6) were obtained as shown in Figure 6. These values were in good agreement with the theoretical average mass for all complexes (increases in mass of 504.3, 666.6, 828.3, and 991.0 Da for G3, G4, G5, and G6, respectively). The signals from all four charge states were summed for both free protein ions and complexed ions. For each integrated spectrum, the peak areas for free protein and the complexes were calculated using the MassLynx software. And again, it was assumed for all calculations that free protein and protein-ligand complexes have similar ionization efficiencies and the measured peak areas for both free protein and the complexes correlate with their solution equilibrium concentrations. Kd Determination for Cel6A D117Acd and Four Oligosaccharides. Using the same titration method described above, the
Figure 6. Deconvoluted mass spectra of the noncovalent interactions for Cel6A D117Acd with G3, G4, G5, and G6. In panels A-E are shown the mass spectra of (A) 3 µM Cel6A D117Acd, (B) 3 µM Cel6A D117Acd plus 15 µM G3, (C) 3 µM Cel6A D117Acd plus 5 µM G4, (D) 3 µM Cel6A D117Acd plus 3 µM G5, and (E) 3 µM Cel6A D117Acd plus 3 µM G6. In the Cel6A D117Acd samples, the lowabundant ions corresponding to Cel6A D117Acd plus two (30 357.8 Da) and seven residues (31 037.7 Da) miscleaved from its signal peptide are labeled by 1 and b, respectively, in panel A. The deconvoluted spectra were generated using the MaxEnt1 deconvolution program with the resolution at 0.1 Da/channel and the minimum intensity ratios at L66% and R66%. The deconvoluted mass spectra showed the complexes increased mass by 504.3, 666.6, 828.3, and 991.0 Da for G3, G4, G5, and G6, respectively, as compared to the free Cel6A D117Acd. Table 4. Dissociation Constants (Kd) of an Inactive Cel6A D117Acd Mutant with Four Oligosccharide Ligands Kd (µM)a titration experiment
ligand
average of individual points
slope of plot
competitive binding expt
cellotriose (G3)b cellotetraose (G4) cellopentaoase (G5) cellohexaose (G6)
77.5 ( 9.2 13.55 ( 1.19 0.854 ( 0.328 c
78.4 ( 3.5 14.43 ( 1.50 0.954 ( 0.05 c
76.9 ( 6.3 11.46 ( 1.2 0.80 ( 0.05
