Anal. Chem. 2006, 78, 5296-5301
Articles
Decomposition of ESR Spectra Using MALDI-TOF Mass Spectrometry Werner L. Vos, Louic S. Vermeer, Cor J. A. M. Wolfs, Ruud B. Spruijt, and Marcus A. Hemminga*
Laboratory of Biophysics, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands
ESR (or EPR) spectroscopy on spin-labeled site-directed cysteine mutants is ideally suited for structural studies of membrane proteins due to its high sensitivity and its low demands with respect to sample purity and preparation. Many features can be inferred from the spectral line shape of an ESR spectrum, but the analysis of ESR spectra is complicated when multiple sites with different line shapes are present. Here, we present a method to decompose the spectrum of a doubly labeled peptide that is composed of a singly labeled, noninteracting component and a doubly labeled, dipolar-broadened component using a combination of optical and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. The effect on the interspin distance calculation based on the dipolar broadening is quantified and discussed. A continuing challenge in structural biology is the unraveling of the structure and function of membrane proteins. For the structure calculation of water-soluble proteins, X-ray diffraction and high-field NMR are routinely applied nowadays, but for membrane proteins, there is no well-defined strategy for obtaining a protein structure.1 Therefore techniques based on site-directed labeling are becoming increasingly important as alternative tools for structure determination of membrane proteins.2-6 Recently, using fluorescence7,8 and ESR techniques,9 we have shown already how such a site-directed labeling approach leads to detailed structural information on membrane proteins. * Corresponding author. Telephone: +31-317-482044. Fax: +31-317-482725. E-mail:
[email protected]. (1) Torres, J.; Stevens, T. J.; Samso, M. Trends Biochem. Sci. 2003, 28, 137144. (2) Steinhoff, H.-J.; Radzwill, N.; Thevis, W.; Lenz, V.; Brandenburg, D.; Antson, A.; Dodson, G.; Wollmer, A. Biophys. J. 1997, 73, 3287-3298. (3) Xiao, W.; Poirier, M. A.; Benett, M. K.; Shin, Y.-K. Nat. Struct. Biol. 2001, 8, 308-311. (4) Lakshmikanth, G. S.; Sridevi, K.; Krishnamoorthy, G.; Udgaonkar, J. B. Nat. Struct. Biol. 2001, 8, 799-804. (5) Brown, L. J.; Sale, K. L.; Hills, R.; Rouviere, C.; Song, L.; Zhang, X.; Fajer, P. G. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 12765-12770. (6) Cuello, L. G.; Cortes, D. M.; Perozo, E. Science 2004, 306, 491-495. (7) Vos, W. L.; Koehorst, R. B. M.; Spruijt, R. B.; Hemminga, M. A. J. Biol. Chem. 2005, 280, 38522-38527. (8) Koehorst, R. B. M.; Spruijt, R. B.; Vergeldt, F. J.; Hemminga, M. A. Biophys. J. 2004, 87, 1445-1455. (9) Sˇ trancar, J.; Koklic, T.; Arsov, Z.; Filipic, B.; Stopar, D.; Hemminga, M. A. J. Chem. Inf. Model. 2005, 45, 394-406.
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ESR spectroscopy has become an invaluable tool for structural studies of proteins, in addition to techniques such as NMR and X-ray diffraction. The advantage of ESR spectroscopy is that it can be applied relatively easily in a variety of environments such as lipid vesicles or even biological systems, making the technique particularly useful for studying membrane proteins (for a review, see ref 10). Strategies that use ESR spectroscopy to study membrane proteins usually involve extrinsic probes (spin labels) that are covalently linked to cysteine residues, which can be introduced in a protein using genetic engineering. Based on the ESR spectral line shape, many features are inferred about the labeled sites, including dynamics, immersion depth of the spin label into the membrane, or, in the case of doubly labeled proteins, the interspin distance. However, a line shape analysis can be complicated when multiple labeled sites are present, and the ESR spectrum is a composition of different spectra, especially when the spectra are similar in shape. For doubly spin-labeled proteins, the presence of singly labeled proteins due to incomplete labeling can strongly affect the spectral line shape, ultimately introducing an uncertainty in the calculated distance.11,12 Commonly used methods to calculate the distance between the spin labels from the dipolar broadened ESR spectrum include least-squares fitting of spectral line shapes 13 and analysis of the spectral broadening via the second moment.11 Here we present a chemical method for the quantitative decomposition of the ESR spectrum of a spin-labeled peptide with two labeling sites into a singly labeled, noninteracting component and a doubly labeled, dipolar-broadened spectrum based on matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry. This approach is applied to accurately determine the interspin distance in a doubly labeled peptide from the dipolar-broadened spectrum, by using a second moment analysis. This opens up the possibility to fully exploit the possibilities offered by ESR spectroscopy in combination with site-directed mutagenesis for the structural study of membrane proteins. (10) Borbat, P. P.; Costa-Filho, A. J.; Earle, K. A.; Moscicki, J. K.; Freed, J. H. Science 2001, 291, 266-269. (11) Radzwill, N.; Gerwert, K.; Steinhoff, H.-J. Biophys. J. 2001, 80, 2856-2866. (12) Persson, M.; Harbridge, J. R.; Hammarstro ¨m, P.; Mitri, R.; Mårtensson, L.G.; Carlsson, U.; Eaton, G. R.; Eaton, S. S. Biophys. J. 2001, 80, 28862897. (13) Farahbakhsh, Z. T.; Huang, Q.-L.; Ding, L.-L.; Altenbach, C.; Steinhoff, H.J.; Horwitz, J.; Hubbell, W. L. Biochemistry 1995, 34, 509-516. 10.1021/ac0521448 CCC: $33.50
© 2006 American Chemical Society Published on Web 06/30/2006
Figure 1. One-letter codes of the peptides that were used in this study. The original amino acid numbering in the yeast V-ATPase is depicted for peptide I. The peptides span the shaded area, the cysteine residues are indicated in dark gray.
MATERIALS AND METHODS Peptide Synthesis. The peptides I, II, and III (see Figure 1) were produced on solid support using continuous-flow chemistry by Pepceuticals Ltd. (Leicester, UK). Peptides were >90% pure, as determined by high-performance liquid chromatography and mass spectrometry. Peptide Design. Subunit a, product of the Vph1 gene in yeast, plays a crucial role in the proton pumping mechanism of vacuolar H+-ATPase.14 The subunit is composed of several transmembrane R-helices, which contain a number of buried charged residues. Some of these residues are important for protein activity, i.e., K593, H729, R735, H743, E789, and R799.14 One of these residues, R735, was shown to be absolutely required for proton translocation.15 The peptide that was studied in this work was designed to include the essential arginine R735, as well as the two nearby histidines H729 and H743. In addition, the peptide was selected to contain two nearby cysteines C723 and C725, which are suitable targets for site-directed spin labeling. A combination of these prerequisites resulted in peptide I, as depicted in Figure 1, spanning residues 721-745 of the yeast Vph1 subunit. Peptides II and III were synthesized as single cysteine mutants by replacing C723 and C725 with an alanine residue. Spin Labeling. Typically, an amount of 2.5 mg of peptide was dissolved in 750 µL of dimethyl sulfoxide (DMSO) and 250 µL of water in a plastic tube. Subsequently, 100 µL of 9.5 mM methanethiosulfonate spin label, purchased from Toronto Research Chemical Inc., stock in DMSO was added, and the tube was slowly vortexed for 3 h at room temperature. A small amount of sample (10 µL) was stored at -18 °C for MALDI-TOF measurements; the remaining part of the sample was immediately purified on a Superdex-75 gel column using 10 mM sodium dodecyl sulfate (SDS) as eluent, buffered at pH 7.0 with 10 mM Na2HPO4. The flow speed was 60 mL/min, and every 2 min a fraction was collected. Fractions containing peptide were detected by monitoring the fluorescence at 350 nm with a Fluor LC 304 by LINEAR, using an excitation wavelength of 280 nm. Only the fraction with the highest fluorescence intensity was used for further experiments, typically appearing after ∼52 min. ESR Measurements. A 1-mL aliquot of the column fraction with the highest fluorescence intensity was concentrated by freezing it in liquid nitrogen, freeze-drying overnight, and rehydrating with 200 µL of water. A 1-mL aliquot of elution buffer was also concentrated this way as a reference for optical and ESR measurements. For the ESR measurements, a 200-µL glass capillary was filled with 40 µL of rehydrated sample. To avoid
motional averaging of the dipolar interaction, the sample was quickly frozen in liquid nitrogen and subsequently placed in the ESR cavity at 150 K. First-derivative absorption spectra were measured on a X-band Bruker Elexsys E500 equipped with a super high sensitivity probe head in combination with a SuperX bridge. Temperature was controlled with a quartz variable-temperature dewar insert. Spectra were recorded at 20-mT scan widths with a low microwave power (0.2 mW) to avoid saturation. The effect of SDS concentration was checked by the addition of the appropriate amount of concentrated (0.3 M) SDS buffer to the sample (peptide: SDS ratio ) 1:200), giving peptide/SDS ratios of 1:900 and 1:1600. Experimental conditions, such as filling of the capillary and position in the cavity, were kept identical for the different samples. Interspin Distance Calculations. The distance between two spin labels, r, depends on the second moment via16,17
(14) Nishi, T.; Forgac, M. Nat. Rev. Mol. Cell Biol. 2002, 3, 94-103. (15) Kawasaki-Nishi, S.; Nishi, T.; Forgac, M. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 12397-12402.
(16) Kokorin, A. I.; Zamarayev, K. I.; Grigoryan, G. L.; Ivanov, V. P.; Rozantsev, E. G. Biofizika 1972, 17, 24-41. (17) Steinhoff, H.-J. Front. Biosci. 2002, 7, 97-110.
r ) 2.32/(∆M2)1/6
(1)
Here r is the distance between the paramagnetic centers (in nm) and ∆M2 is the difference in the second spectral moment between the doubly (MD) and singly (MS) labeled spectrum (in units 10-8 T2):
∆M2 ) MD - MS
(2)
with
MD )
∫(B - B ∫G
MS )
∫(B - B ) G (B) dB ∫G (B) dB
2 FD) GD(B)
dB (3)
D(B) dB
and 2
FS
S
(4)
S
Here GD(B) is the absorption spectrum of the doubly spin-labeled protein sample and GS(B) is the corresponding spectrum without spin-spin interaction. Both are obtained by numeric integration of the first-derivative absorption spectrum. BFD and BFS are the first spectral moments of doubly and singly-labeled peptide, respectively, and B is the magnetic field.17
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Figure 2. ESR spectra of peptide I before (A) and after (C) decomposition. Spectrum A is a composition of interacting and noninteracting species. Spectrum B is the spectrum of singly labeled, noninteracting species and spectrum C is the dipolar-broadened spectrum of interacting species without the contribution of noninteracting species. For the purpose of presentation, the spectra are scaled to the same height. The temperature is 150 K. In (B), the spectrum of spin-labeled peptide II is shown. This spectrum is identical to that of spin-labeled peptide III.
Optical Measurements. UV absorption spectra were recorded in a 1-mm quartz cuvette with a Varian Cary 5E at room temperature. Peptide concentrations were determined based on the absorbance at 280 after correction for the SDS background via the extinction coefficients of tryptophan, tyrosine, and the nitroxide spin label. Effects of tryptophan oxidation and incomplete spin labeling were taken into account as described below. The final peptide concentrations are depicted in Table 3. MALDI-TOF Sample Preparation. A stainless steel MALDI target plate was prepared by the sandwich method. First a layer of matrix solution (10 µL of R-cyanohydroxycinnaminic acid in 400 µL of water, 500 µL of acetonitrile, and 100 µL of 3% trifluoroacetic acid solution) was added. Crystals appeared after evaporation of the solvent. The crystals were dissolved in 1 µL of the labeling solution previously stored at -18 °C, after which the target was put in a vacuum chamber overnight. This removed the DMSO, and crystals reappeared. Subsequently, another layer of matrix solution was added, and immediately after evaporation of the solvent and formation of crystals, mass spectra were recorded. MALDI-TOF mass spectra were recorded on a Bruker Ultraflex mass spectrometer, calibrated with a calibration mix of for peptides between 500 and 3500 u. RESULTS AND DISCUSSION ESR Spectroscopy. The ESR spectra of spin-labeled peptides I and II are depicted in Figure 2A and B, respectively. The spectrum of peptide I at 150 K is a powderlike ESR spectrum that is typical for randomly oriented, immobilized nitroxide spin labels. The spectrum shows a slight broadening resulting from dipolar interaction between the spin labels.2,18 The spectrum of singly labeled peptide II at 150 K (Figure 2B) is a powderlike spectrum with no observable dipolar broadening. MALDI-TOF Mass Spectrometry. In this work, experimental conditions for the MALDI experiments were carefully controlled (18) Rabenstein, M. D.; Shin, Y.-K. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 82398243.
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in order to minimize the effect of external factors, such as sample preparation, matrix solution, and matrix crystal morphology, which are known to affect the peak intensities of peptides.19 Moreover, intrinsic peptide properties such as charged side chains, the presence of aromatic amino acids, peptide hydrophobicity, and the potential to form stable secondary structure have been reported to affect peak intensities.20 In the present work, we are dealing with a limited number of molecular species that are similar in terms of net charge, surface charge distribution, hydrophobicity, and molecular mass; therefore, we believe that the relative intensities of the different peaks constitute a fair estimate of the fraction of each species in a particular condition. MALDI-TOF mass spectra of spin-labeled peptides I and II are depicted in Figure 3A and B, respectively. Peaks of the unlabeled species are indicated with I0, peaks of singly labeled species with IS, and peaks of doubly labeled species with ID. The assignments of the individual ions in the MALDI-TOF mass spectra of peptides I and II are summarized in Tables 1 and 2, respectively. The ions include both protonated molecules [M + H]+ and sodiated [M + Na]+ adduct ions. Both spectra are dominated by intense [M + H]+ ions. Peak heights of the most abundant isotopic species were used rather than integrated signals for the calculation of the fraction of unlabeled, singly labeled, and doubly labeled protein since this does not require a unique integration range and baseline selection for each system.21 The fraction of intact tryptophan residues was calculated likewise. It has been suggested that proton transfer for [M + H]+ and a gas-phase mechanism for formation of [M + Na]+ are the two competing mechanisms for the ionization step;22,23 therefore, the sum of the signal intensities of [M + H]+ and [M + Na]+ ions was assumed constant in our calculations. For both peptides, addition of one spin label leads to an increase in mass of 185.2 u (expected mass 184.1 u). The difference of 1 u is due to the addition of a proton (H•), suggesting the reduction of the nitroxide radical to hydroxylamine. This reaction can occur in photochemical reactions of nitroxides,24 probably during the ionization of the peptides in the mass spectrometer. Both spectra indicate oxidation of the unlabeled cysteine residues. This oxidation is apparent from the sulfinic acid peaks at +32 u of the unlabeled peptides I and II and at +32 u of singly labeled peptide I. Also, the tryptophan residues are partially oxidized, evident from the presence of doubly oxidized tryptophan or N-formylkynurenine 25 at +32 u of the fully labeled peptides I and II. Decomposition of the ESR Spectrum. The MALDI-TOF mass spectrum shows that a considerable fraction of singly labeled peptides is present in the case of spin-labeled peptide I (Figure 3). This implies that the ESR spectrum of spin-labeled peptide I (Figure 2A) is a superposition of singly labeled and doubly labeled (19) Cohen, S. L.; Chait, B. T. Anal. Chem. 1996, 68, 31-37. (20) Krause, E.; Wenschuh, H.; Jungblut, P. R. Anal. Chem. 1999, 71, 41604165. (21) Nelson, R. W.; McLean, M. A.; Hutchens, T. W. Anal. Chem. 1994, 66, 1408-1415. (22) Liao, P.-C.; Allison, J. J. Mass. Spectrom. 1995, 30, 408-423. (23) Ling, Y.-C.; Lin, L.; Chen, Y.-T. Rapid Commun. Mass Spectrom. 1998, 12, 317-327. (24) Gaffney, B. J. In Spin labeling: theory and applications; Berliner, L. J., Ed.; Academic Press: New York, 1976; Vol. I, pp 183-237. (25) Bienvenut, W. V.; De´on, C.; Pasquarello, C.; Campbell, J. M.; Sanchez, J.C.; Vestal, M. L.; Hochstrasser, D. F. Proteomics 2002, 2, 868-876.
Figure 3. MALDI-TOF mass spectra of spin-labeled peptides I (A) and II (B). Peak assignments for peptides I and II are given in Tables 1 and 2, respectively. Peaks indicated with I0, IS, and ID are of unlabeled, singly labeled, and doubly labeled peptides, respectively. Table 1. Peak Assignments of the MALDI-TOF Mass Spectrum of Spin-labeled Peptide I mass (m/z)
assignment
2835.2 2867.2 2897.2
peptide I, unlabeled (1), one cysteine side chain oxidized to sulfinic acid (2), sodiated and (1), both cysteine side chains oxidized to sulfinic acid (1), both cysteine side chains oxidized to sulfinic acid, sodiated (6), ammonia loss Peptide I, singly labeled (6), tryptophan oxidized to hydroxytryptophan (6), tryptophan oxidized to 3-hydroxykynurenine (6), one cysteine side chain oxidized to sulfinic acid and (6), tryptophan doubly oxidized to 3-hydroxylkynurenine (9), sodiated (12), ammonia loss peptide I, doubly labeled (12), tryptophan oxidized to hydroxytryptophan (12), tryptophan oxidized to 3-hydroxykynurenine (12), tryptophan doubly oxidized to 3-hydroxykynurenine
2922.2 3003.4 3020.4 3036.4 3043.4 3052.4 3075.2 3188.7 3205.7 3221.7 3227.7 3237.7
theoretical mass 2834.4 2866.4 2889.4 and 2898.4 2921.4 3001.5 3018.5 3034.5 3038.5 3050.5 3073.5 3185.6 3202.6 3218.6 3222.6 3234.6
Table 2. Peak Assignments of the MALDI-TOF Mass Spectrum of Spin-Labeled Peptide II mass (m/z)
assignment
theoretical mass
2803.2 2835.2 2858.2 2971.4 2988.4 3004.4 3010.4 3020.5
peptide II, unlabeled (1), cysteine side chain oxidized to sulfinic acid (2), sodiated (5), loss of ammonia peptide II, singly labeled (5), tryptophan oxidized to hydroxytryptophan (5), tryptophan oxidized to 3-hydroxykynurenine (5), tryptophan oxidized to N-formylkynurenine
2802.4 2834.4 2857.4 2969.5 2986.5 3002.5 3006.5 3018.5
species. Since the spectrum of the doubly labeled peptide is required for further analysis of the ESR spectrum, the spectrum is decomposed. The decomposition is complicated by the fact that the singly labeled component is a mixture of two different species, i.e., peptide I with the spin label attached to the cysteine at
positions 3 and 6. The spectrum therefore should be decomposed by subtraction of the ESR spectra of peptides II and III, corrected for the concentration. However, since the ESR spectra of peptides II and III are identical at 150 K (spectra not shown), only the spectrum of peptide II, instead of a combination of peptides II Analytical Chemistry, Vol. 78, No. 15, August 1, 2006
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Table 3. Parameters Used for Spectral Decomposition of the Spin-Labeled Peptide I
peptide I peptide II a
fintact
fS
fD
P/103 M-1 × cm-1
[P]/10-5 M
[S]/10-5 M
0.85 0.81
0.35 0.67
0.37
6.4 ( 0.17 × 103a 6.0 ( 0.15 × 103a
4.7 ( 0.13 1.6 ( 0.04
1.6 ( 0.04 1.1 ( 0.03
Error estimated based on an uncertainty of 100 in the extinction coefficients of tryptophan, tyrosine, and the nitroxide spin label.
gdec ) gI - ([S]I/[S]II)gII
(5)
and III, is used for the decomposition. The decomposed spectrum of doubly labeled peptide is obtained by subtracting the ESR spectrum of peptide II (Figure 2B) of the ESR spectrum of peptide I (Figure 2A), corrected for the concentration: where gdec is the decomposed spectrum, gI and gII are the spectra of peptides I and II, respectively, and [S]I and [S]II are the concentrations of singly labeled peptide I and singly labeled peptide II (in a reference sample), respectively. The reference sample containing spin-labeled peptide II was recorded using identical conditions, such as filling and size of the capillary, positioning of the sample, and identical settings of the ESR apparatus. To calculate the concentration of peptide, the total concentration of peptide, [P], is calculated first, using Lambert-Beer’s law:
A280 ) p[P]l
(6)
Here, p is the extinction coefficient of the peptide, A280 is the absorption at 280 nm, and l is the path length of the cuvette. The extinction coefficient of the peptide, p, is calculated from the extinction coefficients of tryptophan (Tryp) tyrosine (Tyr) and the spin label (nit) at 280 nm. The value of Tryp was taken from the spectrum of N-acetyl-DL-tryptophanamide in phosphate buffer at pH 6.5 (Tryp ) 5690 M-1 cm-1).26 The value of Tyr was taken from the spectrum of glycyl-L-tyrosylglycine (Tyr ) 1280 M-1 cm-1).26 The value of nit was also taken from the literature (nit ) 220 M-1 cm-1).27 However, the MALDI-TOF mass spectra of peptides I and II (Figure 3, Tables 1 and 2) show that part of the tryptophan residues is oxidized to N-formylkynurenine, which does not absorb appreciably at 280 nm.28 Moreover, the peptides are not fully labeled. Therefore the extinction coefficient of the peptides at 280 nm is given by
p ) (f
intactTryp
+ (f S + 2f D)nit + Tyr)
(7)
Here, f intact is the fraction of peptide with intact tryptophan residues, f S is the fraction singly labeled peptide and f D is the fraction doubly labeled peptide. f intact, f S, and f D are given by
f intact ) Iintact/(I0 + IS + ID)
(8a)
f S ) IS/(I0 + IS + ID)
(8b)
f D ) ID/(I0 + IS + ID)
(8c)
Here, Iintact is the sum of the intensities of species with intact tryptophan residues in the MALDI-TOF mass spectrum, I0 is the sum of the intensities of the unlabeled species in the mass spec5300
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trum, IS is the sum of the intensities of the singly labeled species in the mass spectrum, and ID is the sum of the intensities of the doubly labeled species in the mass spectrum. The calculation of the fraction of peptide with intact tryptophan residues is slightly complicated by the fact that, in the case of peptide I, the peak of N-formylkynurenine for the singly labeled peptide overlaps with the sulfinic acid peak. Therefore, the ratio of N-formylkynurenine and its reaction intermediate 3-hydroxykynurenine of the doubly labeled peptide I was used to estimate the intensity of the N-formylkynurenine peak in the case of the singly labeled species. After calculation of the total concentration peptide, [P], the concentration singly labeled peptide [S], is readily calculated using eqs 6-8:
[S] ) f S[P]
(9)
The values for f intact, f S, f D, P, [P], and [S] are shown in Table 3. The values for f intact are comparable in both samples, indicating similar tryptophan oxidation for both samples. The value for f S is lower for peptide I, since the singly labeled peptide progressively reacts to doubly labeled species. Not all singly labeled peptides react to doubly labeled species, suggesting that this reaction is blocked by the competing oxidation of the cysteine residues to sulfinic acid.29 The total concentration of protein, [P], is lower in the case of the mutant peptide due to poorer solubility in DMSO. Substitution of [S]I and [S]II in eq 3 yields the decomposed spectrum (Figure 2C). It can be seen that the line shape of the spin-labeled peptide I changes considerably when applying the decomposition. The broadening of the decomposed spectrum is more pronounced, especially in the outer peaks, and the intensity of the middle peaks is reduced, as expected for a dipolarbroadened spectrum.2 Interspin Distance Calculations. Distances were calculated with eqs 4 and 5 by using the ESR spectrum of peptide I before (Figure 2A) and after (Figure 2C) decomposition. For the interpretation of the interspin distance, calculated with the second moment analysis, it is crucial that the dipolar broadening is exclusively due to intra- and not intermolecular interaction. We feel it is safe to proceed with this assumption for the following reasons. First, the singly labeled peptides II and III did not have observable line broadening due to intermolecular dipolar interactions. Moreover, the sample with peptide I was diluted by adding concentrated SDS stock solution to the sample, changing the (26) Edelhoch, H. Biochemistry 1967, 6, 1948-1954. (27) Morrisett, J. D. In Spin labeling: theory and applications; Berliner, L. J., Ed.; Academic Press: New York, 1976; Vol. I, pp 292-293. (28) Camoretti-Mercado, B.; Frydman, R. B. Eur. J. Biochem. 1986, 156, 317325. (29) Men, L.; Wang, Y. Rapid Commun. Mass Spectrom. 2005, 19, 23-30. (30) Wegener, A. A.; Klare, J. P.; Engelhard, M.; Steinhoff, H.-J. EMBO J. 2001, 20, 5312-5319.
Table 4. Parameters for the Interspin Distance Calculation Using the Second Moment Spectral Analysis MS/10-8 T2 MD/10-8 T2 ∆M2/10-8 T2 original decomposed a
390 ( 390 ( 5a 5a
599 ( 5* 622 ( 5*
209 ( 7 232 ( 7
r/nm 0.95 ( 0.06 0.94 ( 0.06
Taken from ref 30.
peptide ratio between 1:200, 1:900, and 1:1600. The distance calculated with the second moment analysis was not affected by the peptide to SDS ratio, suggesting that the dipolar interaction is intra- and not intermolecular. The relevant parameters are presented in Table 4. The second moment of the original spectrum of the spin-labeled peptide I is slightly smaller than that of the decomposed spectrum. Consequently, the interspin distance decreases slightly, from 0.95 nm for the original spectrum to 0.94 nm for the decomposed spectrum. Although this change in interspin distance appears marginal for this case, the correction would be larger already for a slightly larger interspin distance. For instance, in the case of an interspin distance of 1.20 nm, the same correction in second moment would give rise to an increase in an apparent interspin distance to 1.32 nm.
In summary, we present a quantitative chemical method to decompose an ESR spectrum into a nonbroadened component and in a dipolar-broadened component based on MALDI-TOF mass spectrometry. The resulting decomposed dipolar-broadened spectrum can be used to calculate the interspin distance using different methods, including the fitting of the spectral line shape 13 and a second moment analysis.17 Although the method is applied for a small peptide here, we believe that the method is applicable for large proteins as well, for instance, after cutting the protein into smaller parts with trypsin digestion. ACKNOWLEDGMENT This work was supported by contract QLG-CT-2000-01801 of the European Commission (MIVase-New Therapeutic Approaches to Osteoporosis: targeting the osteoclast V-ATPase). We thank Heinz-Ju¨rgen Steinhoff (Fachbereich Physik, Universita¨t Osnabru¨ck) for the software for the integration of the firstderivative absorption ESR spectrum and calculation of the first and second moments. We thank the members of the MIVase consortium for useful discussion. We acknowledge Dr. Igor V. Borovykh for useful discussion and help with the ESR measurements. Received for review December 6, 2005. Accepted May 31, 2006. AC0521448
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