- Email: [email protected]
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are readily obtained after column characterization. In most cases, the Kd values that can be determined range from 0.1 to 300 M for 5-cm columns; however, weaker binders can be evaluated with longer columns with a larger column capacity. Only small quantities of ligand samples are required, typically a few milliliters of 1 M solutions. However, nonspecific binding of compounds to the column can be problematic; this is estimated by running compound mixtures through blank biotin-blocked columns. FAC=MS also requires a volatile buffer such as ammonium acetate, causing limitations in buffer additives that might be essential for ligand binding or protein stabilization. Acknowledgments M.M.P. and O.H. acknowledge funding from the Natural Sciences and Engineering Research Council of Canada (NSERC). B.Z. was supported by a postdoctoral fellowship from the Alberta Heritage Foundation for Medical Research (AHFMR), X.Q. by an Alberta Research Council carbohydrate graduate scholarship, and B.R. by summer studentships from NSERC and AHFMR.
[27] Determination of Protein–Oligosaccharide Binding by Nanoelectrospray Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry By Weijie Wang, Elena N. Kitova, and John S. Klassen Introduction
Diverse biological functions, including cellular growth and adhesion, bacterial and viral infections, inflammation, and the immune response, depend on the recognition of specific carbohydrates by proteins. A detailed understanding of carbohydrate recognition requires analytical methods that can provide information about the specificity and affinity of these interactions. There are a number of established analytical techniques suitable for the study of protein–carbohydrate binding. High-field nuclear magnetic resonance (NMR) and X-ray analysis have been used to characterize the three-dimensional structure of a number of protein– carbohydrate complexes. The binding affinity can be evaluated by semiquantitative methods such as inhibition of hemagglutination, precipitation assays, and enzyme-linked immunosorbent assays (ELISAs), or by quantitative methods such as isothermal titration microcalorimetry (ITC) and
METHODS IN ENZYMOLOGY, VOL. 362
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surface plasmon resonance or Biacore assays. Each of these techniques has advantages and disadvantages.1 ITC is the best method for determining the association thermochemistry for protein–carbohydrate complexes and has found wide application; however, it fails to distinguish binding to different protein quaternary structures and, in certain cases, to provide direct information about binding stoichiometry. It also requires milligram quantities of protein and ligand for each analysis. Mass spectrometry (MS), with its speed, sensitivity, specificity, and ability to directly determine binding stoichiometry, is a powerful tool for studying noncovalent biomolecular complexes. Although a number of ionization techniques, such as matrix-assisted laser desorption=ionization and laser-induced liquid beam ionization=desorption, have been shown to be amenable to the production of specific noncovalent complexes in the gas phase,2 electrospray (ES) and its low-flow variant, nanoflow electrospray (nanoES), are the predominant ionization techniques. An important feature of the ES (and nanoES) technique is the ability to maintain the solution close to physiological conditions, at neutral pH and ambient temperature, which is important for preserving the native protein structure and equilibrium between bound and unbound species in solution, up to the formation of the gas-phase ions. NanoES, which generally operates at solution flow rates of 1–10 nl=min, is particularly well suited for the study of weakly bound complexes3 and has the added advantage of consuming only picomoles of analyte. Beyond the detection of noncovalent complexes, ES-MS is increasingly being used to evaluate binding affinity and stoichiometry. A number of quantitative binding studies have appeared, dealing with protein–protein and protein–small molecule complexes,4 protein–oligonucleotide complexes,5 and peptide and RNA-binding antibiotics6 and small molecule– RNA complexes.7 Daniel and co-workers have described these quantitative 1
J. J. Lundquist and E. J. Toone, Chem. Rev. 102, 555 (2002); D. R. Bundle, Methods Enzymol. 247, 288–305 (1994); D. R. Bundle, in ‘‘Carbohydrates’’ (S. Hecht, ed.), p. 370. Oxford University Press, Oxford, 1998. 2 J. M. Daniel, S. D. Friess, S. Rajagopalan, S. Wendt, and R. Zenobi, Int. J. Mass Spectrom. 216, 1 (2002). 3 M. S. Wilm and M. Mann, Anal. Chem. 68, 1 (1996). 4 A. Ayed, A. N. Krutchinsky, W. Ens, K. G. Standing, and H. W. Duckworth, Rapid Commun. Mass Spectrom. 12, 339 (1998). 5 M. J. Greig, H. Gaus, L. L. Cummins, H. Sasmor, and H. R. Griffey, J. Am. Chem. Soc. 117, 10765 (1995). 6 T. J. D. Jorgensen, P. Roepstorff, and A. J. R. Heck, Anal. Chem. 70, 4427 (1998); K. A. Sannes-Lowery, R. H. Griffey, and S. A. Hofstadler, Anal Biochem. 280, 264 (2000). 7 R. H. Griffey, K. A. Sannes-Lowery, J. J. Drader, V. Mohan, E. E. Swayze, and S. A. Hofstadler, J. Am. Chem. Soc. 122, 9933 (2000).
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studies in a review.2 Although far from being an established method, the aforementioned studies indicate that ES-MS can, in certain cases, provide quantitative binding information. The first direct observation of a protein–oligosaccharide complex, hen egg white lysozyme and a hexasaccharide of N-acetylglucosamine, by ESMS was reported in 1991.8 More recently, van Dongent and Heck described a study of carbohydrate ligands with the lectin apo-concanavalin (Con A),9 which exists in both dimer–tetramer forms in solution. From the ES-MS data, the authors determined the binding stoichiometry of the different quaternary Con A complexes and demonstrated that dimer and tetramer forms exhibit similar affinities for the carbohydrates investigated. This binding information could not be obtained by any other analytical technique. Despite the success of these earlier studies, ES-MS has not found wide application in the area of protein–carbohydrate binding because the detection of protein– carbohydrate complexes is generally more challenging than other protein–ligand complexes. A major cause of this is the low binding affinity, generally in the range of 103 to 105 M1, characteristic of protein–carbohydrate complexes.10 With few exceptions, ES-MS studies of protein–ligand complexes have been restricted to moderately or strongly bound complexes, with association constants >105 M1. The detection of low-affinity complexes requires the use of high ligand concentrations, which tend to suppress the formation of gas-phase protein and protein–ligand ions and lead to the formation of nonspecific complexes. Further, many carbohydrate-binding proteins are heterogeneous in structure and composition, resulting in a distribution of ions with similar mass-to-charge ratios (m=z). This, combined with the high m=z typical of protein and complex ions produced from solutions at neutral pH (m=z > 3000), and the low molecular weight of many model carbohydrate ligands (mono- to tetrasaccharides), requires the use of mass analyzers with high m=z and high-resolution capabilities. In this article, we describe the application of nanoES and Fouriertransformion cyclotron resonance (FT-ICR) MS to evaluate the binding affinity and stoichiometry for two carbohydrate-binding proteins. The first part of this work focuses on results obtained in our laboratory for the binding of a single-chain variable fragment (scFv) of a monoclonal antibody, Se155–4,11 with its native trisaccharide ligand. The influence of several 8
B. Ganem, Y.-T. Li, and J. D. Henion. J. Am. Chem. Soc. 113, 7818 (1991). W. D. van Dongent and A. J. R. Heck, Analyst 125, 583 (2000). 10 E. J. Toone, Curr. Opin. Struct. Biol. 4, 719 (1994); T. K. Dam, R. Roy, S. K. Das, S. Oscarson, and C. F. Brewer, J. Biol. Chem. 275, 14223 (2000). 11 A. Zdanov, Y. Li, D. R. Bundle, S.-J. Deng, C. R. MacKenzie, S. A. Narang, N. M. Young, and M. Cygler, Proc. Natl. Acad. Sci. USA 91, 6423 (1994). 9
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experimental parameters on the mass spectrometry-derived binding constants is discussed. Second, we describe a binding study of the multivalent B5 homopentamer of the Shiga-like toxin 1 [SLT-1(B5)] with the Pk trisaccharide.12 The excellent agreement between the mass spectrometry and ITC-derived association constants obtained in these two studies demonstrates that, under appropriate experimental conditions, nanoES-MS can provide quantitative information about solution binding for protein– oligosaccharide complexes. Experimental Methods
Materials The carbohydrate-binding antibody single-chain fragment11 (molecular mass, 26,539 Da) and the Shiga-like toxin type 1 homopentamer13 (molecular mass, 38,440 Da) are produced by recombinant technology. Sodium dodecyl sulfate–polyacrylamide slab gel electrophoresis (SDS–PAGE) in a 12% gel with reducing agent (dithiothreitol, DTT) is performed to confirm the purity of the proteins after they are isolated by affinity chromatography.13,14 Both proteins are concentrated and dialyzed against deionized water, using MicroSep microconcentrators (molecular mass cutoff, 10,000 Da; Sin-Can, Calgary, Alberta), and lyophilized before MS analysis. The trisaccharide ligands are synthesized at the University of Alberta (Edmonton, Canada) in the laboratory of D. R. Bundle. Sample Preparation Proteins are stored at 20 as lyophilized samples or as concentrated aqueous solutions (>0.1 mM) in 50–100 mM ammonium acetate buffer. The protein sample is weighed immediately after removing it from the lyophilizer and dissolved in a known volume of aqueous buffer solution. When lyophilizing the protein is undesirable, the concentration of dissolved protein can be determined from the absorbance measured at 280 nm and an extinction coefficient calculated from the amino acid sequence of the protein. Because oligosaccharides are highly hygroscopic, they may contain adsorbed water if stored as dry samples. This water can be removed in a ‘‘drying pistol’’, wherein the sample is gently dried in a vacuum chamber maintained at 5 torr and 56 . 12
E. N. Kitova, P. I. Kitov, D. R. Bundle, and J. S. Klassen, Glycobiology 11, 605 (2001). G. Mulvey, R. Vanmaele, M. Mrazek, M. Cahill, and G. D. Armstrong, J. Microbiol. Methods 32, 247 (1998). 14 D. R. Bundle, E. Eichler, M. A. J. Gidney, M. Meldal, A. Ragauskas, B. W. Sigurskjold, B. Sinnot, D. C. Watson, M. Yaguchi, and N. M. Young, Biochemistry 33, 5172 (1994). 13
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The pH of the nanoES solutions is maintained near pH 7 with 50 mM ammonium bicarbonate (pH 7.2) or ammonium acetate (pH 6.8) buffer. The actual pH is confirmed by measurements performed with a pH meter with microelectrodes (710Aplus pH=ISE meter and microcombination pH electrodes; Thermo Orion, Beverly, MA) which is suitable for solution volumes of a few microliters. Mass Spectrometry All experiments are performed with an Apex 47e Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometer (Bruker-Daltonics, Billerica, MA) equipped with an external nanoelectrospray source (Analytica of Branford, Branford, CT). A simplified illustration of the instrument is shown in Fig. 1. NanoES tips (3-m o.d. and 1.0-m i.d.) are pulled from aluminosilicate tubes (1-mm o.d., 0.68-mm i.d.), using a P-97 or P-2000 laser puller (Sutter Instrument, Novato, CA). A platinum wire, inserted into the nanoES tip, is used to establish electrical contact with the analyte solution. The tip is positioned <1.0 mm from a stainless steel sampling capillary, using a microelectrode holder. A potential of 800 to 1100 V is applied to the Pt wire in the nanotip in order to spray the solution. Typically, a stable electrospray ion current of 0.1 A is obtained. The solution flow rate ranges from 1 to 10 nl=min depending on the diameter of the nanotip, the electrospray voltage, and the composition of the solution. The droplets and gaseous ions produced by nanoES are introduced into the vacuum chamber of the mass spectrometer through a heated stainless steel sampling capillary (0.43-mm i.d.), maintained at a temperature of 150 . The gaseous ions sampled by the capillary (52 V) are transmitted through a skimmer (4 V) and accumulated in a trapping hexapole. Unless otherwise noted, the ions are accumulated in the hexapole for 1.5 s. After
Fig. 1. Schematic illustration of the Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer with nanoelectrospray ion source.
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accumulation, ions are ejected and accelerated (about 2700 V) through the fringing field of a 4.7-T superconducting magnet, decelerated, and introduced into the ion cell. The typical pressure for the instrument is 5 1010 mbar. Data acquisition is performed with Bruker Daltonics (Billerica, MA) XMASS software (version 5.0). The time-domain spectra consist of the sum of 30 transients containing 128,000 data points, per transient. Calculating Kassoc The equilibrium constant, Kassoc, for the association reaction involving a protein (P) and a ligand (L) [Eq. (1)] is given by the following expression: PþLÐPL
ð1Þ
Kassoc ¼ ½PL equil =½P equil ½L equil
ð2Þ
The equilibrium concentrations, [PL]equil, [P]equil, and [L]equil, can be deduced from the initial concentration of protein and ligand in solution, [P]0 and [L]0, and the relative abundance of the bound and unbound protein ions, P Lnþ and Pnþ, measured in the mass spectrum. Assuming that the ionization and detection efficiencies for the P Lnþ and Pnþ ions are similar, which is reasonable when the molecular weight of ligand is small compared with the protein,2 the ratio of the ion intensities (I) of the bound and unbound protein ions, determined from the mass spectrum, should be equivalent to the equilibrium concentrations in solution, that is, ½IðP Lnþ Þ/ IðPnþ Þ ¼ ½PL equil /½P equil . Ideally, this ratio (R) should be independent of charge state; however, this is sometimes not the case. The reason for this is not understood, but may reflect the statistical nature of the charging mechanism in the ES process. Consequently, it is advisable to average the ratios measured for all of the observed charge states (n). Because the ion signal in FT-ICR MS is proportional to the abundance of the ion as well as the charge state of the ion, the average value of R should be calculated using the charge-normalized ion intensities: P ½IðPLÞnþ =n ½PL equil n R¼ ð3Þ ¼ P ½P equil ½IðPÞnþ =n n
The equilibrium concentration, [PL]equil, can be determined from the measured R value and [P]0. ½PL equil ¼
R½P 0 1þR
ð4Þ
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Once [PL]equil has been calculated, equilibrium concentration [L]equil can be found from ½L equil ¼ ½L 0 ½PL equil
ð5Þ
The equilibrium concentration of free ligand in solution cannot be determined directly from the mass spectrum because the ionization and detection efficiencies for the protein and ligand ions are expected to be different because of the large difference in m=z and, potentially, other factors. Kassoc can then be solved using Eqs. (6a) and (6b): Kassoc ¼
½PL equil ½P equil ð½L 0 ½PL equil Þ
Kassoc ¼
R ½L 0
R½P 0 1þR
ð6aÞ
ð6bÞ
When the protein (or protein assembly) can bind to N ligands (where N > 1), there are N reactions to be considered: PþLÐPL
ð7aÞ
P L þ L Ð P L2
ð7bÞ
P L2 þ L Ð P L3
ð7cÞ
.. .
.. .
.. .
P LN1 þ L Ð P LN
.. . ð7NÞ
Here, we describe only the simplest case, in which all N binding sites are equivalent, with identical binding constants. The treatment of more complicated cases has been discussed elsewhere.15 The equilibrium concentrations, [PL], [PL2], , [PLN], can be determined from relative abundance of the corresponding ions observed in the mass spectrum and Eq. (8a). Then, using these values, the equilibrium concentration of L can be found from Eq. (8b): ½P þ ½PL þ ½PL2 þ þ ½PLN1 þ ½PLN ¼ ½P 0
15
ð8aÞ
C. Tanford, ‘‘Physical Chemistry of Macromolecules.’’ John Wiley & Sons, New York, 1961.
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½L þ ½PL þ 2½PL2 þ þ ðN 1Þ½PLN1 þ ðNÞ½PLN ¼ ½L 0
ð8bÞ
Kassoc can be determined from any of the following equations, which are based on the general expression Ki ¼ Kassoc (N i þ 1)=i, where i is the number of occupied binding sites15: ½PL ¼ ðNÞKassoc ½P ½L
ð9aÞ
½PL2 ðN 1ÞKassoc ¼ ½PL ½L 2 .. . ½PLN Kassoc ¼ ½PLN1 ½L N
.. .
ð9bÞ .. . ð9NÞ
An average Kassoc can be determined from the binding constant determined for each of the binding reactions, Eqs. (9a)–(9N). Results and Discussion
I. Binding of scFv and Gal[Abe]Man Using nanoES-FT-ICR=MS, we have investigated the binding affinity of the single-chain variable domain fragment (scFv), based on the carbohydrate-binding antibody Se155-4,11 for the trisaccharide ligand -dGal(1!2)[-d-Abe(1!3)] -d-Manp!OMe, where Gal is galactose, Abe is abequose, and Man is mannose. We refer to this ligand as Gal[Abe]Man. A Kassoc of (1.6 0.2) 105 M1 for this interaction in 50 mM Tris and 150 mM NaCl at pH 8.0 and 298 K has been previously determined by ITC.14 A typical nanoES mass spectrum obtained from an aqueous buffered solution of scFv and Gal[Abe]Man is shown in Fig. 2. The dominant ions observed in the mass spectrum correspond to the protonated, unbound scFv ion, (scFv þ nH)nþ, and the protonated complex, (scFv L þ nH)nþ, with charge states of n ¼ 9–12. As described in Experimental Methods, Kassoc can be deduced from the ion intensity ratio, R ¼ I(scFvL)nþ= I(scFv)nþ, determined from the mass spectrum and the initial analyte concentrations. However, the value R, and, consequently, Kassoc, is sensitive to experimental parameters=conditions, such as analyte concentration, number and duration of MS measurements, and source conditions. We have investigated the influence of these experimental parameters on the MS-derived binding affinity and have identified optimal conditions for
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Fig. 2. NanoES mass spectrum of an aqueous solution (pH 6.8) containing scFv (12 M) and its native ligand, Gal[Abe]Man L (6 M).
the determination of Kassoc. The results from this study are described below. Influence of experimental conditions on MS-derived Kassoc Requirement for Multiple Measurements. For a given nanoES tip, the R values obtained from sequential measurements performed under identical conditions were found to fluctuate, significantly in some cases. Shown in Fig. 3a are the R values obtained from five nanoES tips pulled under identical conditions. For each tip, R was determined from six sequential measurements. The duration of each measurement was approximately 1 min. It can be seen in Fig. 3a that the value of R obtained from a given tip fluctuates by as much as 15%, compared with the average value (see Fig. 3b). The reason for this fluctuation is not understood but likely reflects changes in the spray characteristics of the nanoES tip with time. The difference in behavior observed for the different tips may reflect small differences in tip geometry. Shown in Fig. 3b are the average values of R, determined from the six measurements, for each tip. Despite fluctuations in the single
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Fig. 3. (A) Distribution of R obtained from five different nanoES tips and six individual measurements. (B) Comparison of the averaged value of R for each nanoES tip (dashed line indicating the average value of R calculated for all five tips), and the expected R value derived from the reported Kassoc (solid column).
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measurements, the average R values were found to be similar, ranging from 0.63 to 0.68. An R value of 0.66 was obtained as the average for all five tips; this is close to the expected value of 0.64, which can be calculated from the ITC-derived Kassoc and the initial protein and ligand concentrations. The average R, value, for all five tips, corresponds to a Kassoc of 1.8 105 M1, which is in excellent agreement with ITC-derived value of 1.6 105 M1. These results indicate that binding constants determined from single MS measurements might be unreliable; the use of multiple measurements carried out with different tips is recommended. However, on the basis of results discussed in the proceeding section, the use of sequential measurements, lasting more than several minutes with a single tip, is not advisable when the stability of the protein–carbohydrate complex is sensitive to the solution pH. pH Changes in NanoES Solution. It is well established that electrochemical reactions, which occur at the electrode in the nanoES tip, can alter the pH of the nanoES solution. For aqueous solutions in positive ion mode, the dominant electrochemical reaction at the platinum electrode used in the present work is the oxidation of H2O, leading to the production of H3Oþ [Eq. (10)] and a decrease in pH:16 2H2 O Ð O2 þ 4Hþ þ 4e
ð10Þ
In the present experiments, spraying for approximately 30 min resulted in a decrease in the bulk pH of the nanoES solution from 6.8 to 6.0. Van Berkel and co-workers have shown that the end of the nanoES tip, where the droplets are formed, experiences a more significant drop in pH compared with the bulk solution.17 Therefore, the actual pH of the nanoES droplets produced after 30 min of spray is expected to be substantially less than 6.0. In Fig. 4a, the R values determined from a single tip are plotted versus spray time. The magnitude of R decreases with increasing spray duration, from a value of 0.62 to 0.45, corresponding to a decrease of 45% in Kassoc. This behavior is consistent with a reduction in the stability of the scFv-Gal[Abe]Man complex, which decreases significantly below pH 5.5, resulting from a decrease in solution pH.14 The influence of spray duration on the binding of scFv to the structural analog, Tal[Abe]Man, for which a binding constant of 1.17 105 M1 has been determined by ITC,18 was also investigated. The bulk pH of the solution decreased from pH 6.8 to 5.6 after 35 min of spraying and R decreased 16
G. J. Van Berkel, F. Zhou, and J. T. Aronson, Int. J. Mass Spectrom. 162, 55 (1997). G. J. Van Berkel, K. G. Asano, and P. D. Schnier, J. Am. Soc. Mass Spectrom. 12, 853 (2001). 18 D. R. Bundle, unpublished results (1996). 17
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Fig. 4. Value of R plotted versus the spray duration for scFv and (A) its native ligand, Gal[Abe]Man (d); (B) Tal[Abe]Man (m).
from 0.82 to 0.45 (see Fig. 4b), corresponding to a decrease of 50% in the Kassoc. Influence of Ligand Concentration. In general, MS-derived binding constants are determined, not from measurements at a single set of analyte concentrations, but from titration experiments in which the concentration
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of one analyte is fixed and the concentration of the other is varied.5,19,20 In the present study, binding constants were determined over a range of ligand concentrations, 6 106 to 1.9 105 M, with the protein concentration fixed at 1.81 105 M. In practice, an R of 0.1 is the smallest value that can be reliably determined from the MS data; this places a lower limit on the ligand concentration. For the given protein concentration, this lower limit corresponds to a ligand concentration of 3 106 M. The observation of scFv(Gal[Abe]Man)2nþ ions at ligand concentrations exceeding that of the protein by more than a factor of 2 establishes the upper limit for the ligand concentration; see Fig. 5. Because the scFv has only one carbohydrate-binding site, these ions must be artifacts of the nanoES process. The observation of nonspecific complexes that result from the formation of random intermolecular interactions between analyte molecules as the nanoES droplets shrink due to evaporation of solvent is not uncommon, particularly when working at high analyte concentrations.9,21 The appearance of nonspecific complexes obscures the true solution composition, making the reliable determination of Kassoc all but impossible. As expected, increasing the ligand concentration results in an increase in the relative abundance of scFv(Gal[Abe]Man)nþ ions (Fig. 5). Shown in Fig. 6 are the values of R measured at different ligand concentrations. The predicted values, based on the ITC-derived Kassoc, are also shown in Fig. 6 and are found to be in good agreement with the MS values. The MSderived Kassoc values determined at the different ligand concentrations are summarized in Table I. In-Source Dissociation. The influence of several source parameters, such as inlet capillary temperature, source voltages, and hexapole accumulation times, on the relative abundance of scFvLnþ and scFvnþ ions was also examined. Of these, only the accumulation time in the hexapole was found to have a significant effect on R. Shown in Fig. 7 are mass spectra, obtained from a single nanoES tip, recorded with accumulation times of 1.0, 3.0, and 6.0 s. The fraction of scFvLnþ ions, relative to scFvnþ ions, decreases significantly with increasing accumulation time. The R values, determined over a range of accumulation times, are shown in Fig. 8. Under our experimental conditions, R decreased by 59% on increasing the 19
K. Hirose, J. Inclusion Phenomena Macrocyclic Chem. 39, 193 (2001). J. A. Loo, P. Hu, P. McConnel, W. T. Mueller, T. K. Sawyer, and V. Thanabal, J. Am. Soc. Mass Spectrom. 8, 234 (1997); H.-K. Lim, Y. L. Hsieh, B. Ganem, and J. Henion, J. Mass Spectrom. 30, 708 (1995); R. H. Griffey, S. A. Hofstadler, K. A. Sannes-Lowery, D. J. Ecker, and S. T. Crooke, Proc. Natl. Acad. Sci. USA 96, 10129 (1999). 21 C. V. Robinson, E. W. Chung, B. B. Kragelund, J. Knudsen, R. T. Aplin, F. M. Poulsen, and C. M. Dobson, J. Am. Chem. Soc. 118, 8646 (1996); V. Gabelica, E. De Pauw, and F. Rosu, J. Mass Spectrom. 34, 1328 (1999). 20
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Fig. 5. NanoES mass spectra obtained for aqueous solutions containing scFv (18.1 M) and Gal[Abe]Man (L) at increasing concentrations: (A) 10.8 M; (B) 35.6 M; and (C) 45.1 M.
390
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Fig. 6. Dependence of R on the concentration of ligand (L). ( ) MS-derived value, () value predicted from the ITC-derived Kassoc.
TABLE I R Values and Association Constants for scFv and Gal[Abe]Mana Concentration of Gal[Abe]Man (M)
MS
ITCb
6.6 8.6 9.9 10.8 12.1 14.3 16.5 19.0
0.36 0.45 0.56 0.66 0.69 0.85 1.25 1.38
0.33 0.47 0.55 0.64 0.72 0.91 1.12 1.36
a
R
Kassoc 105 (M1)(MS)
Differencec (%)
1.98 1.50 1.62 1.84 1.47 1.42 1.95 1.62
+23.8 6.3 +1.3 +5.7 8.1 11.3 +21.9 +1.3
Determined by nanoES-FT-ICR=MS (MS) at different ligand concentrations. R values calculated from the ITC-derived Kassoc of (1.6 0.2) 105M1 from Ref. 14. c Values correspond to {[Kassoc (MS) Kassoc (ITC)]=Kassoc (ITC)} 100% b
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Fig. 7. NanoES mass spectra of an aqueous solution of scFv (18.1 M) and Gal[Abe]Man (L) (10.8 M) obtained at different hexapole accumulation times: (A) 1 s; (B) 3 s; (C) 6 s.
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Fig. 8. A plot of R as a function of hexapole accumulation time.
accumulation time from 1 s (R ¼ 0.63) to 6 s (R ¼ 0.26), leading to a decrease of 77% in Kassoc. This effect is attributed to the dissociation of the scFvLnþ ions while stored in the hexapole. The pressure in the hexapole is not uniform but ranges from 103 to 105 torr. Acceleration of the trapped ions by the high radio-frequency (rf) field (1 kV) applied to the hexapole rods results in some of the complex ions being collisionally heated and subsequently dissociating. These results clearly indicate that shorter accumulation times, which minimize the extent of collision-induced dissociation, will lead to more reliable binding constants. However, spectra acquired with short accumulation times, less than 1 s, suffer from poor signal-to-noise(S=N) ratios. Therefore, an accumulation time of 1.5 s was chosen as a compromise between enhancing the S=N ratio of the spectra and minimizing in-source dissociation. II. Binding of SLT-1(B5) and Pk Trisaccharide Our laboratory has also applied nanoES-FT-ICR MS to investigate the binding of the Pk trisaccharide to the multivalent protein complex
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SLT-1 (B5).12 SLT-1(B5) is a homopentamer consisting of five subunits, each with a molecular mass of 7688 Da. From solution NMR studies,22 it is known that each subunit has one dominant Pk-binding site (referred to as ‘‘site 2’’), with two additional binding sites (sites 1 and 3), per subunit, suggested from the crystal structure.23 The crystal structure of the SLT1(B5) complex and the location of binding site 2 are shown in Fig. 9. From ITC measurements, it has been shown that binding between SLT-1(B5) and Pk at site 2 is noncooperative with a Kassoc of (1.5 0.5) 103 M1.24 The Kassoc values for sites 1 and 3 have not been determined but are believed to be significantly smaller than for site 2. A nanoES spectrum obtained for a solution of SLT-1(B5) at pH 7.2 is shown in Fig. 10. The dominant ions correspond to the intact pentamer (i.e., B5nþ) with charge states of 11 to 13. The absence of monomer or smaller oligomer ions indicates that the pentamer is stable in the nanoES solution and that its quaternary structure is preserved throughout the nanoES process. The long accumulation times (in the hexapole), up to 15 s, which were necessary to obtain adequate S=N ratios for the B5nþ ions, were not found to promote dissociation of the complex. To establish the binding constant for Pk, nanoES-MS measurements were performed on aqueous solutions (pH 7.2) containing 5.0 M SLT1(B5) and three different concentrations of Pk (45, 154, and 310 M). Despite the low binding constant, complexes of SLT-1(B5) and Pk, (B5Pik)nþ, were readily observed (Fig. 11a–c) and, as expected, the degree of complexation increased with the concentration of the ligand. The number of bound ligands and even nature of the charging agents are easily identified from the FT-ICR mass spectra. At the highest concentration investigated (310 M) complexes with up to five bound Pk ligands were observed (Fig. 11c). In contrast to the behavior observed for the scFv-Gal[Abe]Man system, nonspecific complexes of SLT 1(B5) and Pk were not observed in the mass spectra. The reason for this difference in behavior is not known. It may be that the nonspecific complexes, if present, dissociate before detection. On the basis of the relative abundance of the protonated (B5Pik)nþ ions and the initial concentration of protein and ligand, the equilibrium concentration of B5 and the B5Pik complexes [Eqs. (11a–11e)] could be
22
H. Shimizu, R. A. Field, S. W. Homans, and A. Donohue-Rolfe, Biochemistry 37, 11078 (1998). 23 H. Ling, A. Boodhoo, B. Hazes, M. D. Cummings, G. D. Armstrong, J. L. Brunton, and R. J. Read, Biochemistry 37, 1777 (1998); D. J. Bast, L. Banerjee, C. Clark, R. J. Read, and J. L. Brunton, Mol. Microbiol. 32, 953 (1999). 24 P. M. St. Hilaire, M. K. Boyd, and E. J. Toone, Biochemistry 33, 14452 (1994).
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Fig. 9. Crystal structure of the complex SLT-1(B5)(Pk)5.
calculated. This in turn allowed for the determination of Kassoc for the five noncooperative binding sites of SLT-1(B5): B5 þ Pk Ð B5 Pk
ð11aÞ
B5 Pk þ Pk Ð B5 Pk2
ð11bÞ
B5 Pk2 þ Pk Ð B5 Pk3
ð11cÞ
B5 Pk3 þ Pk Ð B5 Pk4
ð11dÞ
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Fig. 10. NanoES mass spectrum of an aqueous solution (pH 7.2) of 5.0 M SLT-1(B5).
B5 Pk4 þ Pk Ð B5 Pk5
ð11eÞ
Reliable Kassoc values could be determined only when the relative ion abundance was determined from MS data with high S=N (S=N > 3). For example, at the highest ligand concentration (310 M), complexes with up to five Pk ligands were observed in the mass spectrum, allowing for the determination of Kassoc for i ¼ 1–5. However, because of the poor S=N for the B5P5k ion, its relative abundance and, consequently, Kassoc for i ¼ 5, could not be accurately determined. The MS-derived Kassoc values determined for the þ12 and þ13 charge states are listed in Table II. The values in parentheses were calculated from MS data with low S=N and were not used to calculate the average association constant. The individual binding constants reported in Table II are similar, ranging from 1 103 to 4 103 M1, and independent of the ligand concentration and charge state. The overall Kassoc, corresponding to the average of the binding constants obtained for the þ12 and þ13 charge states and the three ligand concentrations, (2.5 0.8) 103 M1, is in excellent agreement with the ITC-derived value of (1.5 0.5) 103 M1.24 In the present study, the number of binding sites and the value of Kassoc were known in advance. To confirm by MS that there were only five dominant binding sites, nanospectra acquired at significantly higher ligand concentrations, so that the five binding sites become saturated, are necessary. However, a dramatic decrease in the S=N for the (B5Pi k)nþ ions was
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Fig. 11. NanoES mass spectra of aqueous solutions (pH 7.2) containing 5.0 M SLT-1(B5) and three different concentrations of Pk: (A) 45 M, (B) 154 M, and (C) 310 M.
observed at ligand concentrations of >500 M and the relative ion abundance could not be accurately determined. This study represents the first direct determination by nanoES-MS of the binding stoichiometry and affinity for a protein–ligand complex with an association constant in the 103 M1 range. On the basis of the spectrometric data obtained for solutions of SLT-1(B5) and Pk, it was confirmed
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TABLE II Association Constants for Binding of SLT1(B5) and Pka Concentration of Pk (M)
Number of bound Pk
Kassoc 103 (M1) Charge state +12
45
154
310
a
1 2 3 1 2 3 4 1 2 3 4 5
2.0 3.5 (4.4) 1.6 2.2 3.1 1.8 1.7 2.3 1.9 4.6 (21.4)
Charge state +13 2.8 3.8 (5.0) 1.8 2.0 2.7 2.5 1.6 1.7 3.2 3.2 (9.9) Average = 2.5
0.8
Determined by nanoES-FT-ICR=MS.
that the five dominant Pk binding sites (i.e., site 2) of SLT-1(B5) operate in a noncooperative manner. Further, from the relative abundance of the (B5Pi k)nþ ions observed in the mass spectra, an association constant, in excellent agreement with the ITC-derived value, was determined. Summary
The two studies presented here demonstrate that nanoES-FT-ICR MS is a powerful method for studying the association of oligosaccharide ligands with monomeric and multimeric proteins. It permits the facile identification of the occupancy of binding sites, information that is not readily available by other techniques. Its high-resolution capability is ideally suited to the observation of interactions between a large protein receptor and a relatively small oligosaccharide ligand. The sensitive and rapid determination of association constants for protein carbohydrate complexes is expected to find wide application.
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are readily obtained after column characterization. In most cases, the Kd values that can be determined range from 0.1 to 300 M for 5-cm columns; however, weaker binders can be evaluated with longer columns with a larger column capacity. Only small quantities of ligand samples are required, typically a few milliliters of 1 M solutions. However, nonspecific binding of compounds to the column can be problematic; this is estimated by running compound mixtures through blank biotin-blocked columns. FAC=MS also requires a volatile buffer such as ammonium acetate, causing limitations in buffer additives that might be essential for ligand binding or protein stabilization. Acknowledgments M.M.P. and O.H. acknowledge funding from the Natural Sciences and Engineering Research Council of Canada (NSERC). B.Z. was supported by a postdoctoral fellowship from the Alberta Heritage Foundation for Medical Research (AHFMR), X.Q. by an Alberta Research Council carbohydrate graduate scholarship, and B.R. by summer studentships from NSERC and AHFMR.
[27] Determination of Protein–Oligosaccharide Binding by Nanoelectrospray Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry By Weijie Wang, Elena N. Kitova, and John S. Klassen Introduction
Diverse biological functions, including cellular growth and adhesion, bacterial and viral infections, inflammation, and the immune response, depend on the recognition of specific carbohydrates by proteins. A detailed understanding of carbohydrate recognition requires analytical methods that can provide information about the specificity and affinity of these interactions. There are a number of established analytical techniques suitable for the study of protein–carbohydrate binding. High-field nuclear magnetic resonance (NMR) and X-ray analysis have been used to characterize the three-dimensional structure of a number of protein– carbohydrate complexes. The binding affinity can be evaluated by semiquantitative methods such as inhibition of hemagglutination, precipitation assays, and enzyme-linked immunosorbent assays (ELISAs), or by quantitative methods such as isothermal titration microcalorimetry (ITC) and
METHODS IN ENZYMOLOGY, VOL. 362
Copyright 2003, Elsevier (USA). All rights reserved. 0076-6879/03 $35.00
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surface plasmon resonance or Biacore assays. Each of these techniques has advantages and disadvantages.1 ITC is the best method for determining the association thermochemistry for protein–carbohydrate complexes and has found wide application; however, it fails to distinguish binding to different protein quaternary structures and, in certain cases, to provide direct information about binding stoichiometry. It also requires milligram quantities of protein and ligand for each analysis. Mass spectrometry (MS), with its speed, sensitivity, specificity, and ability to directly determine binding stoichiometry, is a powerful tool for studying noncovalent biomolecular complexes. Although a number of ionization techniques, such as matrix-assisted laser desorption=ionization and laser-induced liquid beam ionization=desorption, have been shown to be amenable to the production of specific noncovalent complexes in the gas phase,2 electrospray (ES) and its low-flow variant, nanoflow electrospray (nanoES), are the predominant ionization techniques. An important feature of the ES (and nanoES) technique is the ability to maintain the solution close to physiological conditions, at neutral pH and ambient temperature, which is important for preserving the native protein structure and equilibrium between bound and unbound species in solution, up to the formation of the gas-phase ions. NanoES, which generally operates at solution flow rates of 1–10 nl=min, is particularly well suited for the study of weakly bound complexes3 and has the added advantage of consuming only picomoles of analyte. Beyond the detection of noncovalent complexes, ES-MS is increasingly being used to evaluate binding affinity and stoichiometry. A number of quantitative binding studies have appeared, dealing with protein–protein and protein–small molecule complexes,4 protein–oligonucleotide complexes,5 and peptide and RNA-binding antibiotics6 and small molecule– RNA complexes.7 Daniel and co-workers have described these quantitative 1
J. J. Lundquist and E. J. Toone, Chem. Rev. 102, 555 (2002); D. R. Bundle, Methods Enzymol. 247, 288–305 (1994); D. R. Bundle, in ‘‘Carbohydrates’’ (S. Hecht, ed.), p. 370. Oxford University Press, Oxford, 1998. 2 J. M. Daniel, S. D. Friess, S. Rajagopalan, S. Wendt, and R. Zenobi, Int. J. Mass Spectrom. 216, 1 (2002). 3 M. S. Wilm and M. Mann, Anal. Chem. 68, 1 (1996). 4 A. Ayed, A. N. Krutchinsky, W. Ens, K. G. Standing, and H. W. Duckworth, Rapid Commun. Mass Spectrom. 12, 339 (1998). 5 M. J. Greig, H. Gaus, L. L. Cummins, H. Sasmor, and H. R. Griffey, J. Am. Chem. Soc. 117, 10765 (1995). 6 T. J. D. Jorgensen, P. Roepstorff, and A. J. R. Heck, Anal. Chem. 70, 4427 (1998); K. A. Sannes-Lowery, R. H. Griffey, and S. A. Hofstadler, Anal Biochem. 280, 264 (2000). 7 R. H. Griffey, K. A. Sannes-Lowery, J. J. Drader, V. Mohan, E. E. Swayze, and S. A. Hofstadler, J. Am. Chem. Soc. 122, 9933 (2000).
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studies in a review.2 Although far from being an established method, the aforementioned studies indicate that ES-MS can, in certain cases, provide quantitative binding information. The first direct observation of a protein–oligosaccharide complex, hen egg white lysozyme and a hexasaccharide of N-acetylglucosamine, by ESMS was reported in 1991.8 More recently, van Dongent and Heck described a study of carbohydrate ligands with the lectin apo-concanavalin (Con A),9 which exists in both dimer–tetramer forms in solution. From the ES-MS data, the authors determined the binding stoichiometry of the different quaternary Con A complexes and demonstrated that dimer and tetramer forms exhibit similar affinities for the carbohydrates investigated. This binding information could not be obtained by any other analytical technique. Despite the success of these earlier studies, ES-MS has not found wide application in the area of protein–carbohydrate binding because the detection of protein– carbohydrate complexes is generally more challenging than other protein–ligand complexes. A major cause of this is the low binding affinity, generally in the range of 103 to 105 M1, characteristic of protein–carbohydrate complexes.10 With few exceptions, ES-MS studies of protein–ligand complexes have been restricted to moderately or strongly bound complexes, with association constants >105 M1. The detection of low-affinity complexes requires the use of high ligand concentrations, which tend to suppress the formation of gas-phase protein and protein–ligand ions and lead to the formation of nonspecific complexes. Further, many carbohydrate-binding proteins are heterogeneous in structure and composition, resulting in a distribution of ions with similar mass-to-charge ratios (m=z). This, combined with the high m=z typical of protein and complex ions produced from solutions at neutral pH (m=z > 3000), and the low molecular weight of many model carbohydrate ligands (mono- to tetrasaccharides), requires the use of mass analyzers with high m=z and high-resolution capabilities. In this article, we describe the application of nanoES and Fouriertransformion cyclotron resonance (FT-ICR) MS to evaluate the binding affinity and stoichiometry for two carbohydrate-binding proteins. The first part of this work focuses on results obtained in our laboratory for the binding of a single-chain variable fragment (scFv) of a monoclonal antibody, Se155–4,11 with its native trisaccharide ligand. The influence of several 8
B. Ganem, Y.-T. Li, and J. D. Henion. J. Am. Chem. Soc. 113, 7818 (1991). W. D. van Dongent and A. J. R. Heck, Analyst 125, 583 (2000). 10 E. J. Toone, Curr. Opin. Struct. Biol. 4, 719 (1994); T. K. Dam, R. Roy, S. K. Das, S. Oscarson, and C. F. Brewer, J. Biol. Chem. 275, 14223 (2000). 11 A. Zdanov, Y. Li, D. R. Bundle, S.-J. Deng, C. R. MacKenzie, S. A. Narang, N. M. Young, and M. Cygler, Proc. Natl. Acad. Sci. USA 91, 6423 (1994). 9
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experimental parameters on the mass spectrometry-derived binding constants is discussed. Second, we describe a binding study of the multivalent B5 homopentamer of the Shiga-like toxin 1 [SLT-1(B5)] with the Pk trisaccharide.12 The excellent agreement between the mass spectrometry and ITC-derived association constants obtained in these two studies demonstrates that, under appropriate experimental conditions, nanoES-MS can provide quantitative information about solution binding for protein– oligosaccharide complexes. Experimental Methods
Materials The carbohydrate-binding antibody single-chain fragment11 (molecular mass, 26,539 Da) and the Shiga-like toxin type 1 homopentamer13 (molecular mass, 38,440 Da) are produced by recombinant technology. Sodium dodecyl sulfate–polyacrylamide slab gel electrophoresis (SDS–PAGE) in a 12% gel with reducing agent (dithiothreitol, DTT) is performed to confirm the purity of the proteins after they are isolated by affinity chromatography.13,14 Both proteins are concentrated and dialyzed against deionized water, using MicroSep microconcentrators (molecular mass cutoff, 10,000 Da; Sin-Can, Calgary, Alberta), and lyophilized before MS analysis. The trisaccharide ligands are synthesized at the University of Alberta (Edmonton, Canada) in the laboratory of D. R. Bundle. Sample Preparation Proteins are stored at 20 as lyophilized samples or as concentrated aqueous solutions (>0.1 mM) in 50–100 mM ammonium acetate buffer. The protein sample is weighed immediately after removing it from the lyophilizer and dissolved in a known volume of aqueous buffer solution. When lyophilizing the protein is undesirable, the concentration of dissolved protein can be determined from the absorbance measured at 280 nm and an extinction coefficient calculated from the amino acid sequence of the protein. Because oligosaccharides are highly hygroscopic, they may contain adsorbed water if stored as dry samples. This water can be removed in a ‘‘drying pistol’’, wherein the sample is gently dried in a vacuum chamber maintained at 5 torr and 56 . 12
E. N. Kitova, P. I. Kitov, D. R. Bundle, and J. S. Klassen, Glycobiology 11, 605 (2001). G. Mulvey, R. Vanmaele, M. Mrazek, M. Cahill, and G. D. Armstrong, J. Microbiol. Methods 32, 247 (1998). 14 D. R. Bundle, E. Eichler, M. A. J. Gidney, M. Meldal, A. Ragauskas, B. W. Sigurskjold, B. Sinnot, D. C. Watson, M. Yaguchi, and N. M. Young, Biochemistry 33, 5172 (1994). 13
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The pH of the nanoES solutions is maintained near pH 7 with 50 mM ammonium bicarbonate (pH 7.2) or ammonium acetate (pH 6.8) buffer. The actual pH is confirmed by measurements performed with a pH meter with microelectrodes (710Aplus pH=ISE meter and microcombination pH electrodes; Thermo Orion, Beverly, MA) which is suitable for solution volumes of a few microliters. Mass Spectrometry All experiments are performed with an Apex 47e Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometer (Bruker-Daltonics, Billerica, MA) equipped with an external nanoelectrospray source (Analytica of Branford, Branford, CT). A simplified illustration of the instrument is shown in Fig. 1. NanoES tips (3-m o.d. and 1.0-m i.d.) are pulled from aluminosilicate tubes (1-mm o.d., 0.68-mm i.d.), using a P-97 or P-2000 laser puller (Sutter Instrument, Novato, CA). A platinum wire, inserted into the nanoES tip, is used to establish electrical contact with the analyte solution. The tip is positioned <1.0 mm from a stainless steel sampling capillary, using a microelectrode holder. A potential of 800 to 1100 V is applied to the Pt wire in the nanotip in order to spray the solution. Typically, a stable electrospray ion current of 0.1 A is obtained. The solution flow rate ranges from 1 to 10 nl=min depending on the diameter of the nanotip, the electrospray voltage, and the composition of the solution. The droplets and gaseous ions produced by nanoES are introduced into the vacuum chamber of the mass spectrometer through a heated stainless steel sampling capillary (0.43-mm i.d.), maintained at a temperature of 150 . The gaseous ions sampled by the capillary (52 V) are transmitted through a skimmer (4 V) and accumulated in a trapping hexapole. Unless otherwise noted, the ions are accumulated in the hexapole for 1.5 s. After
Fig. 1. Schematic illustration of the Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer with nanoelectrospray ion source.
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accumulation, ions are ejected and accelerated (about 2700 V) through the fringing field of a 4.7-T superconducting magnet, decelerated, and introduced into the ion cell. The typical pressure for the instrument is 5 1010 mbar. Data acquisition is performed with Bruker Daltonics (Billerica, MA) XMASS software (version 5.0). The time-domain spectra consist of the sum of 30 transients containing 128,000 data points, per transient. Calculating Kassoc The equilibrium constant, Kassoc, for the association reaction involving a protein (P) and a ligand (L) [Eq. (1)] is given by the following expression: PþLÐPL
ð1Þ
Kassoc ¼ ½PL equil =½P equil ½L equil
ð2Þ
The equilibrium concentrations, [PL]equil, [P]equil, and [L]equil, can be deduced from the initial concentration of protein and ligand in solution, [P]0 and [L]0, and the relative abundance of the bound and unbound protein ions, P Lnþ and Pnþ, measured in the mass spectrum. Assuming that the ionization and detection efficiencies for the P Lnþ and Pnþ ions are similar, which is reasonable when the molecular weight of ligand is small compared with the protein,2 the ratio of the ion intensities (I) of the bound and unbound protein ions, determined from the mass spectrum, should be equivalent to the equilibrium concentrations in solution, that is, ½IðP Lnþ Þ/ IðPnþ Þ ¼ ½PL equil /½P equil . Ideally, this ratio (R) should be independent of charge state; however, this is sometimes not the case. The reason for this is not understood, but may reflect the statistical nature of the charging mechanism in the ES process. Consequently, it is advisable to average the ratios measured for all of the observed charge states (n). Because the ion signal in FT-ICR MS is proportional to the abundance of the ion as well as the charge state of the ion, the average value of R should be calculated using the charge-normalized ion intensities: P ½IðPLÞnþ =n ½PL equil n R¼ ð3Þ ¼ P ½P equil ½IðPÞnþ =n n
The equilibrium concentration, [PL]equil, can be determined from the measured R value and [P]0. ½PL equil ¼
R½P 0 1þR
ð4Þ
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Once [PL]equil has been calculated, equilibrium concentration [L]equil can be found from ½L equil ¼ ½L 0 ½PL equil
ð5Þ
The equilibrium concentration of free ligand in solution cannot be determined directly from the mass spectrum because the ionization and detection efficiencies for the protein and ligand ions are expected to be different because of the large difference in m=z and, potentially, other factors. Kassoc can then be solved using Eqs. (6a) and (6b): Kassoc ¼
½PL equil ½P equil ð½L 0 ½PL equil Þ
Kassoc ¼
R ½L 0
R½P 0 1þR
ð6aÞ
ð6bÞ
When the protein (or protein assembly) can bind to N ligands (where N > 1), there are N reactions to be considered: PþLÐPL
ð7aÞ
P L þ L Ð P L2
ð7bÞ
P L2 þ L Ð P L3
ð7cÞ
.. .
.. .
.. .
P LN1 þ L Ð P LN
.. . ð7NÞ
Here, we describe only the simplest case, in which all N binding sites are equivalent, with identical binding constants. The treatment of more complicated cases has been discussed elsewhere.15 The equilibrium concentrations, [PL], [PL2], , [PLN], can be determined from relative abundance of the corresponding ions observed in the mass spectrum and Eq. (8a). Then, using these values, the equilibrium concentration of L can be found from Eq. (8b): ½P þ ½PL þ ½PL2 þ þ ½PLN1 þ ½PLN ¼ ½P 0
15
ð8aÞ
C. Tanford, ‘‘Physical Chemistry of Macromolecules.’’ John Wiley & Sons, New York, 1961.
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protein–carbohydrate binding studied by nanoes-ms
½L þ ½PL þ 2½PL2 þ þ ðN 1Þ½PLN1 þ ðNÞ½PLN ¼ ½L 0
ð8bÞ
Kassoc can be determined from any of the following equations, which are based on the general expression Ki ¼ Kassoc (N i þ 1)=i, where i is the number of occupied binding sites15: ½PL ¼ ðNÞKassoc ½P ½L
ð9aÞ
½PL2 ðN 1ÞKassoc ¼ ½PL ½L 2 .. . ½PLN Kassoc ¼ ½PLN1 ½L N
.. .
ð9bÞ .. . ð9NÞ
An average Kassoc can be determined from the binding constant determined for each of the binding reactions, Eqs. (9a)–(9N). Results and Discussion
I. Binding of scFv and Gal[Abe]Man Using nanoES-FT-ICR=MS, we have investigated the binding affinity of the single-chain variable domain fragment (scFv), based on the carbohydrate-binding antibody Se155-4,11 for the trisaccharide ligand -dGal(1!2)[-d-Abe(1!3)] -d-Manp!OMe, where Gal is galactose, Abe is abequose, and Man is mannose. We refer to this ligand as Gal[Abe]Man. A Kassoc of (1.6 0.2) 105 M1 for this interaction in 50 mM Tris and 150 mM NaCl at pH 8.0 and 298 K has been previously determined by ITC.14 A typical nanoES mass spectrum obtained from an aqueous buffered solution of scFv and Gal[Abe]Man is shown in Fig. 2. The dominant ions observed in the mass spectrum correspond to the protonated, unbound scFv ion, (scFv þ nH)nþ, and the protonated complex, (scFv L þ nH)nþ, with charge states of n ¼ 9–12. As described in Experimental Methods, Kassoc can be deduced from the ion intensity ratio, R ¼ I(scFvL)nþ= I(scFv)nþ, determined from the mass spectrum and the initial analyte concentrations. However, the value R, and, consequently, Kassoc, is sensitive to experimental parameters=conditions, such as analyte concentration, number and duration of MS measurements, and source conditions. We have investigated the influence of these experimental parameters on the MS-derived binding affinity and have identified optimal conditions for
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Fig. 2. NanoES mass spectrum of an aqueous solution (pH 6.8) containing scFv (12 M) and its native ligand, Gal[Abe]Man L (6 M).
the determination of Kassoc. The results from this study are described below. Influence of experimental conditions on MS-derived Kassoc Requirement for Multiple Measurements. For a given nanoES tip, the R values obtained from sequential measurements performed under identical conditions were found to fluctuate, significantly in some cases. Shown in Fig. 3a are the R values obtained from five nanoES tips pulled under identical conditions. For each tip, R was determined from six sequential measurements. The duration of each measurement was approximately 1 min. It can be seen in Fig. 3a that the value of R obtained from a given tip fluctuates by as much as 15%, compared with the average value (see Fig. 3b). The reason for this fluctuation is not understood but likely reflects changes in the spray characteristics of the nanoES tip with time. The difference in behavior observed for the different tips may reflect small differences in tip geometry. Shown in Fig. 3b are the average values of R, determined from the six measurements, for each tip. Despite fluctuations in the single
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Fig. 3. (A) Distribution of R obtained from five different nanoES tips and six individual measurements. (B) Comparison of the averaged value of R for each nanoES tip (dashed line indicating the average value of R calculated for all five tips), and the expected R value derived from the reported Kassoc (solid column).
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measurements, the average R values were found to be similar, ranging from 0.63 to 0.68. An R value of 0.66 was obtained as the average for all five tips; this is close to the expected value of 0.64, which can be calculated from the ITC-derived Kassoc and the initial protein and ligand concentrations. The average R, value, for all five tips, corresponds to a Kassoc of 1.8 105 M1, which is in excellent agreement with ITC-derived value of 1.6 105 M1. These results indicate that binding constants determined from single MS measurements might be unreliable; the use of multiple measurements carried out with different tips is recommended. However, on the basis of results discussed in the proceeding section, the use of sequential measurements, lasting more than several minutes with a single tip, is not advisable when the stability of the protein–carbohydrate complex is sensitive to the solution pH. pH Changes in NanoES Solution. It is well established that electrochemical reactions, which occur at the electrode in the nanoES tip, can alter the pH of the nanoES solution. For aqueous solutions in positive ion mode, the dominant electrochemical reaction at the platinum electrode used in the present work is the oxidation of H2O, leading to the production of H3Oþ [Eq. (10)] and a decrease in pH:16 2H2 O Ð O2 þ 4Hþ þ 4e
ð10Þ
In the present experiments, spraying for approximately 30 min resulted in a decrease in the bulk pH of the nanoES solution from 6.8 to 6.0. Van Berkel and co-workers have shown that the end of the nanoES tip, where the droplets are formed, experiences a more significant drop in pH compared with the bulk solution.17 Therefore, the actual pH of the nanoES droplets produced after 30 min of spray is expected to be substantially less than 6.0. In Fig. 4a, the R values determined from a single tip are plotted versus spray time. The magnitude of R decreases with increasing spray duration, from a value of 0.62 to 0.45, corresponding to a decrease of 45% in Kassoc. This behavior is consistent with a reduction in the stability of the scFv-Gal[Abe]Man complex, which decreases significantly below pH 5.5, resulting from a decrease in solution pH.14 The influence of spray duration on the binding of scFv to the structural analog, Tal[Abe]Man, for which a binding constant of 1.17 105 M1 has been determined by ITC,18 was also investigated. The bulk pH of the solution decreased from pH 6.8 to 5.6 after 35 min of spraying and R decreased 16
G. J. Van Berkel, F. Zhou, and J. T. Aronson, Int. J. Mass Spectrom. 162, 55 (1997). G. J. Van Berkel, K. G. Asano, and P. D. Schnier, J. Am. Soc. Mass Spectrom. 12, 853 (2001). 18 D. R. Bundle, unpublished results (1996). 17
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Fig. 4. Value of R plotted versus the spray duration for scFv and (A) its native ligand, Gal[Abe]Man (d); (B) Tal[Abe]Man (m).
from 0.82 to 0.45 (see Fig. 4b), corresponding to a decrease of 50% in the Kassoc. Influence of Ligand Concentration. In general, MS-derived binding constants are determined, not from measurements at a single set of analyte concentrations, but from titration experiments in which the concentration
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of one analyte is fixed and the concentration of the other is varied.5,19,20 In the present study, binding constants were determined over a range of ligand concentrations, 6 106 to 1.9 105 M, with the protein concentration fixed at 1.81 105 M. In practice, an R of 0.1 is the smallest value that can be reliably determined from the MS data; this places a lower limit on the ligand concentration. For the given protein concentration, this lower limit corresponds to a ligand concentration of 3 106 M. The observation of scFv(Gal[Abe]Man)2nþ ions at ligand concentrations exceeding that of the protein by more than a factor of 2 establishes the upper limit for the ligand concentration; see Fig. 5. Because the scFv has only one carbohydrate-binding site, these ions must be artifacts of the nanoES process. The observation of nonspecific complexes that result from the formation of random intermolecular interactions between analyte molecules as the nanoES droplets shrink due to evaporation of solvent is not uncommon, particularly when working at high analyte concentrations.9,21 The appearance of nonspecific complexes obscures the true solution composition, making the reliable determination of Kassoc all but impossible. As expected, increasing the ligand concentration results in an increase in the relative abundance of scFv(Gal[Abe]Man)nþ ions (Fig. 5). Shown in Fig. 6 are the values of R measured at different ligand concentrations. The predicted values, based on the ITC-derived Kassoc, are also shown in Fig. 6 and are found to be in good agreement with the MS values. The MSderived Kassoc values determined at the different ligand concentrations are summarized in Table I. In-Source Dissociation. The influence of several source parameters, such as inlet capillary temperature, source voltages, and hexapole accumulation times, on the relative abundance of scFvLnþ and scFvnþ ions was also examined. Of these, only the accumulation time in the hexapole was found to have a significant effect on R. Shown in Fig. 7 are mass spectra, obtained from a single nanoES tip, recorded with accumulation times of 1.0, 3.0, and 6.0 s. The fraction of scFvLnþ ions, relative to scFvnþ ions, decreases significantly with increasing accumulation time. The R values, determined over a range of accumulation times, are shown in Fig. 8. Under our experimental conditions, R decreased by 59% on increasing the 19
K. Hirose, J. Inclusion Phenomena Macrocyclic Chem. 39, 193 (2001). J. A. Loo, P. Hu, P. McConnel, W. T. Mueller, T. K. Sawyer, and V. Thanabal, J. Am. Soc. Mass Spectrom. 8, 234 (1997); H.-K. Lim, Y. L. Hsieh, B. Ganem, and J. Henion, J. Mass Spectrom. 30, 708 (1995); R. H. Griffey, S. A. Hofstadler, K. A. Sannes-Lowery, D. J. Ecker, and S. T. Crooke, Proc. Natl. Acad. Sci. USA 96, 10129 (1999). 21 C. V. Robinson, E. W. Chung, B. B. Kragelund, J. Knudsen, R. T. Aplin, F. M. Poulsen, and C. M. Dobson, J. Am. Chem. Soc. 118, 8646 (1996); V. Gabelica, E. De Pauw, and F. Rosu, J. Mass Spectrom. 34, 1328 (1999). 20
[27]
protein–carbohydrate binding studied by nanoes-ms
389
Fig. 5. NanoES mass spectra obtained for aqueous solutions containing scFv (18.1 M) and Gal[Abe]Man (L) at increasing concentrations: (A) 10.8 M; (B) 35.6 M; and (C) 45.1 M.
390
[27]
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Fig. 6. Dependence of R on the concentration of ligand (L). ( ) MS-derived value, () value predicted from the ITC-derived Kassoc.
TABLE I R Values and Association Constants for scFv and Gal[Abe]Mana Concentration of Gal[Abe]Man (M)
MS
ITCb
6.6 8.6 9.9 10.8 12.1 14.3 16.5 19.0
0.36 0.45 0.56 0.66 0.69 0.85 1.25 1.38
0.33 0.47 0.55 0.64 0.72 0.91 1.12 1.36
a
R
Kassoc 105 (M1)(MS)
Differencec (%)
1.98 1.50 1.62 1.84 1.47 1.42 1.95 1.62
+23.8 6.3 +1.3 +5.7 8.1 11.3 +21.9 +1.3
Determined by nanoES-FT-ICR=MS (MS) at different ligand concentrations. R values calculated from the ITC-derived Kassoc of (1.6 0.2) 105M1 from Ref. 14. c Values correspond to {[Kassoc (MS) Kassoc (ITC)]=Kassoc (ITC)} 100% b
[27]
protein–carbohydrate binding studied by nanoes-ms
391
Fig. 7. NanoES mass spectra of an aqueous solution of scFv (18.1 M) and Gal[Abe]Man (L) (10.8 M) obtained at different hexapole accumulation times: (A) 1 s; (B) 3 s; (C) 6 s.
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Fig. 8. A plot of R as a function of hexapole accumulation time.
accumulation time from 1 s (R ¼ 0.63) to 6 s (R ¼ 0.26), leading to a decrease of 77% in Kassoc. This effect is attributed to the dissociation of the scFvLnþ ions while stored in the hexapole. The pressure in the hexapole is not uniform but ranges from 103 to 105 torr. Acceleration of the trapped ions by the high radio-frequency (rf) field (1 kV) applied to the hexapole rods results in some of the complex ions being collisionally heated and subsequently dissociating. These results clearly indicate that shorter accumulation times, which minimize the extent of collision-induced dissociation, will lead to more reliable binding constants. However, spectra acquired with short accumulation times, less than 1 s, suffer from poor signal-to-noise(S=N) ratios. Therefore, an accumulation time of 1.5 s was chosen as a compromise between enhancing the S=N ratio of the spectra and minimizing in-source dissociation. II. Binding of SLT-1(B5) and Pk Trisaccharide Our laboratory has also applied nanoES-FT-ICR MS to investigate the binding of the Pk trisaccharide to the multivalent protein complex
[27]
protein–carbohydrate binding studied by nanoes-ms
393
SLT-1 (B5).12 SLT-1(B5) is a homopentamer consisting of five subunits, each with a molecular mass of 7688 Da. From solution NMR studies,22 it is known that each subunit has one dominant Pk-binding site (referred to as ‘‘site 2’’), with two additional binding sites (sites 1 and 3), per subunit, suggested from the crystal structure.23 The crystal structure of the SLT1(B5) complex and the location of binding site 2 are shown in Fig. 9. From ITC measurements, it has been shown that binding between SLT-1(B5) and Pk at site 2 is noncooperative with a Kassoc of (1.5 0.5) 103 M1.24 The Kassoc values for sites 1 and 3 have not been determined but are believed to be significantly smaller than for site 2. A nanoES spectrum obtained for a solution of SLT-1(B5) at pH 7.2 is shown in Fig. 10. The dominant ions correspond to the intact pentamer (i.e., B5nþ) with charge states of 11 to 13. The absence of monomer or smaller oligomer ions indicates that the pentamer is stable in the nanoES solution and that its quaternary structure is preserved throughout the nanoES process. The long accumulation times (in the hexapole), up to 15 s, which were necessary to obtain adequate S=N ratios for the B5nþ ions, were not found to promote dissociation of the complex. To establish the binding constant for Pk, nanoES-MS measurements were performed on aqueous solutions (pH 7.2) containing 5.0 M SLT1(B5) and three different concentrations of Pk (45, 154, and 310 M). Despite the low binding constant, complexes of SLT-1(B5) and Pk, (B5Pik)nþ, were readily observed (Fig. 11a–c) and, as expected, the degree of complexation increased with the concentration of the ligand. The number of bound ligands and even nature of the charging agents are easily identified from the FT-ICR mass spectra. At the highest concentration investigated (310 M) complexes with up to five bound Pk ligands were observed (Fig. 11c). In contrast to the behavior observed for the scFv-Gal[Abe]Man system, nonspecific complexes of SLT 1(B5) and Pk were not observed in the mass spectra. The reason for this difference in behavior is not known. It may be that the nonspecific complexes, if present, dissociate before detection. On the basis of the relative abundance of the protonated (B5Pik)nþ ions and the initial concentration of protein and ligand, the equilibrium concentration of B5 and the B5Pik complexes [Eqs. (11a–11e)] could be
22
H. Shimizu, R. A. Field, S. W. Homans, and A. Donohue-Rolfe, Biochemistry 37, 11078 (1998). 23 H. Ling, A. Boodhoo, B. Hazes, M. D. Cummings, G. D. Armstrong, J. L. Brunton, and R. J. Read, Biochemistry 37, 1777 (1998); D. J. Bast, L. Banerjee, C. Clark, R. J. Read, and J. L. Brunton, Mol. Microbiol. 32, 953 (1999). 24 P. M. St. Hilaire, M. K. Boyd, and E. J. Toone, Biochemistry 33, 14452 (1994).
394
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[27]
Fig. 9. Crystal structure of the complex SLT-1(B5)(Pk)5.
calculated. This in turn allowed for the determination of Kassoc for the five noncooperative binding sites of SLT-1(B5): B5 þ Pk Ð B5 Pk
ð11aÞ
B5 Pk þ Pk Ð B5 Pk2
ð11bÞ
B5 Pk2 þ Pk Ð B5 Pk3
ð11cÞ
B5 Pk3 þ Pk Ð B5 Pk4
ð11dÞ
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protein–carbohydrate binding studied by nanoes-ms
395
Fig. 10. NanoES mass spectrum of an aqueous solution (pH 7.2) of 5.0 M SLT-1(B5).
B5 Pk4 þ Pk Ð B5 Pk5
ð11eÞ
Reliable Kassoc values could be determined only when the relative ion abundance was determined from MS data with high S=N (S=N > 3). For example, at the highest ligand concentration (310 M), complexes with up to five Pk ligands were observed in the mass spectrum, allowing for the determination of Kassoc for i ¼ 1–5. However, because of the poor S=N for the B5P5k ion, its relative abundance and, consequently, Kassoc for i ¼ 5, could not be accurately determined. The MS-derived Kassoc values determined for the þ12 and þ13 charge states are listed in Table II. The values in parentheses were calculated from MS data with low S=N and were not used to calculate the average association constant. The individual binding constants reported in Table II are similar, ranging from 1 103 to 4 103 M1, and independent of the ligand concentration and charge state. The overall Kassoc, corresponding to the average of the binding constants obtained for the þ12 and þ13 charge states and the three ligand concentrations, (2.5 0.8) 103 M1, is in excellent agreement with the ITC-derived value of (1.5 0.5) 103 M1.24 In the present study, the number of binding sites and the value of Kassoc were known in advance. To confirm by MS that there were only five dominant binding sites, nanospectra acquired at significantly higher ligand concentrations, so that the five binding sites become saturated, are necessary. However, a dramatic decrease in the S=N for the (B5Pi k)nþ ions was
396
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Fig. 11. NanoES mass spectra of aqueous solutions (pH 7.2) containing 5.0 M SLT-1(B5) and three different concentrations of Pk: (A) 45 M, (B) 154 M, and (C) 310 M.
observed at ligand concentrations of >500 M and the relative ion abundance could not be accurately determined. This study represents the first direct determination by nanoES-MS of the binding stoichiometry and affinity for a protein–ligand complex with an association constant in the 103 M1 range. On the basis of the spectrometric data obtained for solutions of SLT-1(B5) and Pk, it was confirmed
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protein–carbohydrate binding studied by nanoes-ms
397
TABLE II Association Constants for Binding of SLT1(B5) and Pka Concentration of Pk (M)
Number of bound Pk
Kassoc 103 (M1) Charge state +12
45
154
310
a
1 2 3 1 2 3 4 1 2 3 4 5
2.0 3.5 (4.4) 1.6 2.2 3.1 1.8 1.7 2.3 1.9 4.6 (21.4)
Charge state +13 2.8 3.8 (5.0) 1.8 2.0 2.7 2.5 1.6 1.7 3.2 3.2 (9.9) Average = 2.5
0.8
Determined by nanoES-FT-ICR=MS.
that the five dominant Pk binding sites (i.e., site 2) of SLT-1(B5) operate in a noncooperative manner. Further, from the relative abundance of the (B5Pi k)nþ ions observed in the mass spectra, an association constant, in excellent agreement with the ITC-derived value, was determined. Summary
The two studies presented here demonstrate that nanoES-FT-ICR MS is a powerful method for studying the association of oligosaccharide ligands with monomeric and multimeric proteins. It permits the facile identification of the occupancy of binding sites, information that is not readily available by other techniques. Its high-resolution capability is ideally suited to the observation of interactions between a large protein receptor and a relatively small oligosaccharide ligand. The sensitive and rapid determination of association constants for protein carbohydrate complexes is expected to find wide application.