Production and Properties of Nanoelectrospray Emitters Used in

Production and Properties of Nanoelectrospray Emitters Used in Fourier Transform Ion Cyclotron Resonance Mass Spectrometry: Implications for Determina...
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Anal. Chem. 2004, 76, 5172-5179

Production and Properties of Nanoelectrospray Emitters Used in Fourier Transform Ion Cyclotron Resonance Mass Spectrometry: Implications for Determination of Association Constants for Noncovalent Complexes Cleidiane G. Zampronio, Anastassios E. Giannakopulos, Martin Zeller, Eleni Bitziou, Julie V. Macpherson, and Peter J. Derrick*

Department of Chemistry, University of Warwick, Coventry, CV4 7AL, U.K.

Electrospray ionization (ESI) is extensively used in the analysis of biological compounds; yet some fundamental properties of this technique are not completely understood. It is widely recognized that care should be exercised when noncovalent complexes are being studied by ESI, since weak noncovalent binding can be broken or formed during the desolvation process. In the present work, spectra from the noncovalent complex, vancomycin/diacetyl-L-lysyl-D-alanyl-D-alanine, obtained from ESI and from nanoelectrospray ionization (nanoESI), have been compared. The results indicated that the milder desolvation conditions arising as a result of the smaller sizes of droplets produced in the nanoESI source attenuated effects upon weak bonds in the desolvation process. The association constant values calculated from the relative peak intensities suggest that, when using ESI, the analyzed noncovalent complex dissociated in the condensed phase during the spraying process. The influences of experimental parameters such as tip diameter and coating for nanoESI needles were investigated. Principal component analysis, a multivariate analysis method, was applied to achieve a better evaluation of the spectra obtained using different needle diameters and coatings for the analysis of the noncovalent complex vancomycin/ diacetyl-L-lysyl-D-alanyl-D-alanine. It was found that 2-µm tip diameter resulted in more reproducible spectra than the larger tip diameters tested (6-20 µm). The development of electrospray ionization (ESI)1 has permitted the study of many biological and biochemical problems using mass spectrometry. One important example is the characterization of the stability and structural behavior of noncovalent complexes involving proteins or peptides. ESI is capable of releasing the noncovalent complex from its native solution state into the gas phase in the form of multiply charged ions and has allowed the detection of complexes and location of the binding sites for a * To whom correspondence should be addressed. E-mail: P.J.Derrick@ warwick.ac.uk. (1) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64-71.

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number of protein-protein2 and protein-ligand interactions.3 The study of these complexes is important since cellular function is often triggered by weak noncovalent interactions between enzyme and substrate, protein and ligand, and protein and protein. Although a great deal of research has been carried out on droplet formation in ESI, questions such as the influence of the desolvation conditions (high temperature and shock waves during the expansion of gas into the mass spectrometer) on the stability of complexes remain unanswered. Understanding of the fundamental physical parameters influencing the appearance of weakly bound complexes in mass spectra will allow stoichiometric binding studies of biological systems. Contaminants in the analyte solution such as salts or detergents, or the presence of other analytes, may lead to substantial suppression of the analyte signal.4,5 Nanoelectrospray ionization (nanoESI) is a variation of standard ESI, producing smaller droplets of the order of micrometer and submicrometer radius and requiring a lower degree of desolvation for the production of ions. Some advantages of nanoESI are high sensitivity (femtomolar), higher tolerance toward salt contamination, compatibility with small sample volume, fewer analyte suppression effects in mixtures,6,7 and mild conditions that allow the detection of noncovalent complexes. NanoESI spectra can, however, be highly dependent upon the properties of the needle used for droplet formation. Factors such as tip diameter and needle coating may have an important effect on signal intensities and reproducibility, although this has not yet been studied in detail. In the study presented here, the effects of tip diameter and needle coating on the ion intensities of bound and unbound peptides in spectra of a noncovalent complex are investigated. In the first part of this work, tip diameters between 2 and 20 µm with both gold- and platinum-coated needles were used in the nanoESI source, and the multivariate statistical technique of (2) Sobott, F.; Robinson, C. V. Curr. Opin. Struct. Biol. 2002, 12, 729-734. (3) Hill T. J.; Lafitte D.; Wallace J. I.; Cooper H. J.; Tsvektov P. O.; Derrick P. J. Biochem. 2000, 39, 7284-7290. (4) Tang, L.; Kebarle, P. Anal. Chem. 1993, 65, 3654-3668. (5) Juraschek, R.; Du ¨ lcks, T.; Karas, M. J. Am. Soc. Mass Spectrom. 1999, 10, 300-308. (6) Wilm, M.; Mann, M. Anal. Chem. 1996, 68, 1-8. (7) Schmidt, A.; Karas, M.; Dulcks, T. J. Am. Soc. Mass Spectrom. 2003, 14, 492-500. 10.1021/ac049569z CCC: $27.50

© 2004 American Chemical Society Published on Web 08/05/2004

principal component analysis (PCA) was applied to analyze the data obtained. In the second part of this study, ion intensities in spectra of a noncovalent complex obtained using ESI and nanoESI ionization were compared. Spectra from nanoESI were obtained using metallized and nonmetallized needles. The association constant was calculated for each recorded spectrum. The noncovalent complex used in these analyses was vancomycin/diacetylL-lysyl-D-alanyl-D-alanine, which has been already analyzed by mass spectrometry in other studies and the optimum temperature and pH have been established.8,9 Vancomycin is an alternative for combating bacteria that are already resistant to other antibiotics or for patients allergic to other antibiotics (penicillin). Vancomycin acts by binding to the D-alanyl-D-alanine-terminating peptides that are metabolic intermediates in cell wall biosynthesis.10 When a large number of spectra are being analyzed, e.g., the table (or matrix) with 100 spectra each consisting of well over 1000 different peak intensities across the full mass range, it can be very difficult to extract relevant information used to provide new insight into the problem investigated. PCA is a multivariate statistical technique now commonly used in chemistry11-13 for analysis and understanding of large, usually highly collinear, data sets. In many spectrometric applications, neither qualitative nor quantitative analyses are possible using a univariate (i.e., single peak) approach, and in these cases, multivariate techniques such as PCA14-16 and partial least squares (PLS)17,18 have proved invaluable. PCA can work as a data reduction method to assist in data exploration and understanding or as a classification method for separating samples according to their chemical properties. The fundamental idea of PCA is to decompose a matrix containing a large number of correlated variables into a small set of uncorrelated variables called principal components (PCs). The first principal component (PC1) describes as much of the variability in the data as possible, and each succeeding PC, orthogonal to the others, accounts for as much of the remaining variability as possible.14-16 Thus, a large data set is decomposed into a product of two matrices, one of which contains the information about the samples (scores) and the other contains information about the variables (loadings). In matrix notation

X ) TPT + E

(1)

where X (I × J) is the data matrix, T (I × R) is the scores matrix, P (J × R) is the loadings matrix, and E (I × J) is a matrix of residuals. The number of PCs, R, is chosen to be as low as (8) Jørgensen, T. J. D.; Roepstorff, P.; Heck, A. J. R. Anal. Chem. 1998, 70, 4427-4432. (9) Zhang, Y.-B. Ph.D. Thesis, University of Warwick, 2002. (10) Popieniek, P. H.; Pratt, R. F. J. Am. Chem. Soc. 1991, 113, 2264-2270. (11) Alberici, R. M.; Zampronio, C. G.; Poppi, R. J.; Eberlin, M. N. Analyst 2002, 127, 230-234. (12) Kirkor, E. S.; Scheeline, A. Anal. Chem. 2000, 72, 1381-1388. (13) Wagner, M. S.; Tyler, B. J.; Castner, D. G. Anal. Chem. 2002, 74, 18241835. (14) Joliffe, I. T. Principal Component Analysis; Springer-Verlag: New York, 1986. (15) Wold, S.; Esbensen, K.; Geladi, P. Chemom. Intell. Lab. Syst. 1987, 2, 3752. (16) Massart, D. L.; Vandeginste, B. G. M.; Buydens, L. M. C.; De Jong, S.; Lewi, P. J.; Smeyers-Verbeke, J. Handbook of chemometrics and qualimetrics: part A; Elsevier Science B. V.: Amsterdam, The Netherlands, 1997; p 20A. (17) Geladi, P.; Kowalski, B. Anal. Chim. Acta 1986, 185, 1-17. (18) Martens, P.; Næs, T. Multivariate Calibration; Wiley: Chichester, 1989.

possible, yet sufficient for TPT to describe all of the chemically significant information in X. The residuals, E, should describe only chemically irrelevant information (i.e., noise). When all the species contained in the samples are mutually independent, i.e., each species has a characteristic spectrum, the number of PCs will equal the total number of species present. The scores are commonly plotted against each other (e.g., PC1 vs PC2, PC1 vs PC3), allowing a visual evaluation of the similarities and differences among the samples in the reduced-dimension subspace. Generally speaking, samples with similar chemical properties are found to lie close to one another within the scores plot. The scores for each PC are a weighted sum of original variables. These weights are called loadings, and one can write the equation as follows: J

tir )

∑p x

(2)

jr ij

j)1

where tir is the score for the sample i for the rth principal component, prj is the loading for variable j for the rth principal component, and xij is the intensity from sample i at variable j. Note that variables which have a high loading have an important influence on the scores value and can thus be considered important variables. Similarly, a small loading denotes a relatively unimportant variable. More detailed explanations of PCA (and closely related techniques such as singular value decomposition and eigenanalysis) and its application to chemical data can be found in the literature.14-16 EXPERIMENTAL SECTION Chemicals. Vancomycin antibiotic (V) and diacetyl-L-lysyl-Dalanyl-D-alanine peptide (KAA) were purchased from Sigma Chemical Co. (St Louis, MO). Stock solutions from these ligands were made using deionized water obtained from a Milli-Q system (Millipore Corp., Bedford, MA). The noncovalent complex [V + KAA] was prepared in 5 mmol L-1 ammonium acetate buffer (pH 5.1) with three different concentration ratios of V/KAA (1:2, 1:1, 2:1). Prior to analysis, each sample was centrifuged in a Whatman Micro-Centrifuge at 9000 rpm/g for 10 min. Manufacture of NanoESI Emitters. NanoESI emitters (needles) with different tip diameters were created in-house from borosilicate glass capillaries (Harvard Apparatus, 1.2-mm o.d., 0.68mm i.d.) using a microcapillary puller (Sutter model P-2000). A microscope equipped with a calibration grid with accuracy (0.5 µm was used to determine the tip size. Tip diameters of 2, 6, 10, 15, and 20 µm were produced. Both metallic-coated needles and nonmetallized needles were made. Gold and platinum coatings were prepared using organometallic paint from Johnson Matthey Noble Metals (GBV02954/08 and PBV00158, respectively). Each needle was manually painted using a rotating device and heated in an oven at 600 °C for 0.5 h.19 This process was repeated twice to give a uniform coating. A flow of nitrogen was used to purge the capillaries while the needles were being painted, thus preventing paint from blowing the capillary opening. Nonmetallized needles consisted of a wire inserted through the capillary. Three different types of wire (platinum, tungsten, silver) were (19) Macpherson, J. V.; Unwin, P. R. Anal. Chem. 1998, 70, 2914-2921.

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Figure 1. Scheme of the nonmetallized needle setup for nanoESI.

used, all with diameter 0.125 mm. First, each wire was spot welded to a stainless steel cylinder and cut to length. Next, the wire was inserted into the needle. The end of the wire was set close to the tip by fixing the cylinder at the back of the needle as shown in the Figure 1. Mass Spectrometry. In this work, two different Fourier transform ion cyclotron resonance (FT-ICR) spectrometers were used: a 9.4-T FT-ICR mass spectrometer (Bruker Daltonics APEX II) and a 3.0-T FT-ICR mass spectrometer (Bruker Daltonics APEX II). On the 9.4-T FT-ICR, nanoESI spectra were recorded in order to investigate the reproducibility of the signal obtained using different needle coatings and tip diameters. The sample analyzed was an equimolar V/KAA solution at 135 µmol L-1. Each needle was positioned at a distance of 2 mm from the counter electrode, and a voltage between 700 and 1500 V was applied. The capillary exit voltage (27.8 V), the offset (1.72 V), the residence time in the hexapole (2 s), and the ion injection time in the cell (3500 µs) were maintained constant throughout. Gold- and platinumcoated needles with tip diameters of 2, 6, 10, 15, and 20 µm were tested. Under these conditions, there was a significant degree of fragmentation of the complex in the gas phase. Each measured spectrum was a sum of 32 scans and contained 448 679 data points, describing an m/z range between 300 and 2000. Five spectra were recorded for each needle tested. With two types of needle coatings and five different tip diameters, a total of 50 spectra were recorded. Thus, the data from the first experiment were stored in a 50 × 448 679 matrix. This data set was analyzed on a PC using routines written in the MATLAB, version 6.5 (The MathWorks, Natick, MA) programming environment. A second experiment was performed on the 9.4-T FT-ICR in order to optimize the conditions to analyze the complex from vancomycin and peptide by nanoESI. Two different samples were analyzed, an equimolar V/KAA solution at 50 µmol L-1 and a pure vancomycin solution at 50 µmol L-1. Platinum-coated needles with 2-µm tip diameter were used. The needle was positioned at a distance of 2 mm from the counter electrode, and a voltage between 700 and 800 V was applied. The capillary exit voltage (91.60 V), the offset (1.45 V), the residence time in the hexapole (3 s), and the ion injection time in the cell (2800 µs) were optimized to obtain an intense peak for the complex and were maintained constant throughout. Three spectra were recorded for each experiment and each measured spectrum being a sum of 32 scans. During both experiments on the 9.4-T FT-ICR, no back pressure gas was used on the needles and the ion source operated without desolvation gas at a temperature of 25 °C. On the 3.0-T FT-ICR, three different experiments were performed. In the first experiment, spectra from ESI and nanoESI were recorded and compared. Different types of needle were used for nanoESI. The sample analyzed was an equimolar V/KAA solution with a concentration of 50 µmol L-1. The ESI mass spectra were recorded using nitrogen drying gas at a temperature of 150 5174

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°C for efficient desolvation of the spray droplets produced. A flow rate of 100 µL/h was required for stable spray production at the voltage and solution conditions used. The ESI source was then replaced by the nanoESI source, and five types of needles were used: platinum-coated, gold-coated, and three nonmetallized needles with different types of wire (platinum, tungsten, silver). All needles investigated were made with 2-µm tip diameters. The needles were positioned at a distance of 2 mm from the counter electrode, and a voltage between 900 and 1100 V was applied. The capillary exit voltage (37.7 V), the offset (1.80 V), the residence time in the hexapole (5 s), and the ion injection time in the cell (1700 µs) were optimized to obtain an intense peak for the complex and maintained constant for all the ESI and nanoESI experiments. Three spectra were recorded for each needle with each measured spectrum being a sum of 32 scans. During all the experiments on the 3.0-T FT-ICR, no back pressure gas was used on the needles and the ion source operated without desolvation gas at a temperature of 25 °C. A second experiment on the 3.0-T FT-ICR was performed, which both ESI and nanoESI spectra were recorded in the same conditions of the first experiment on the 3-T FT-ICR. However, in this case, the samples analyzed had different ratios V/KAA (1: 2, 1:1, 2:1) at concentrations 25 and 50 µmol L-1. A 2-µm platinumcoated needle was used in the nanoESI source for each different solution. This experiment was performed to investigate whether different concentrations influenced the relative intensities of the peaks. Finally, a spectrum was obtained from sustained off-resonance irradiation for collision-activation dissociation (SORI-CAD) to investigate how the complex dissociated in the gas phase. An equimolar V/KAA solution at 50 µmol L-1 was analyzed using a 2-µm nonmetallized needle with platinum wire in the nanoESI source. RESULTS AND DISCUSSION Effect of Needle Coating and Tip Diameter in NanoESI. The suitability of nanoESI for noncovalent complex analysis was investigated. In particular, the influence of physical properties of emitters such as type of coating and tip diameter was considered. In this first experiment performed on the 9.4-T FT-ICR, an equimolar solution V/KAA was analyzed using two different types of needle coatings and five different tip diameters. The spectra for the 2-, 10-, and 20-µm-diameter gold-coated needles are shown in Figure 2, and those for 2-, 10-, and 20-µmdiameter platinum-coated needles are displayed in Figure 3. In each case, the spectrum shown represents a mean of the five spectra actually recorded. As can be observed, the peak ratios changed when different tip diameters were used. For both the 10- and 20-µm diameter needles, a difference was seen between the spectra for gold- and platinum-coated needles (in terms of the ratio between the complex and the free vancomycin ion intensities). However, for the 2-µm-diameter needle, the spectra for gold and platinum coatings did not exhibit any significant difference from each other. Note that the presence of potassium in the vancomycin peak was from contamination of the buffer solution. For a deeper analysis of the recorded spectra and the effect of using different needles, PCA was used. The data were organized into a matrix with dimensions 50 × 448 679, where 50 is the number of spectra (two types of coating, five tip diameters, and

Figure 2. Mass spectra of [V] ) [KAA] ) 135 µmol L-1 for goldcoated needles with tip diameters of (a) 2, (b) 10, and (c) 20 µm.

Figure 3. Mass spectra of [V] ) [KAA] ) 135 µmol L-1 for platinumcoated needles with tip diameters of (a) 2, (b) 10, and (c) 20 µm.

five replicas) and 448 679 is the number of m/z intensities in each spectrum. The spectra were mean-centered, with no other preprocessing being applied, and a PCA was performed. Two principal components were selected, together describing 44% of the variation in the data. Further principal components were found to describe low-level variation in the spectra and were discarded. Each spectrum is represented in the new variable space described by the principal components as shown in Figure 4. Spectra from needles with 2-µm diameter are closely clustered, for both gold and platinum coatings, indicating more similarity between the spectra than for larger diameter needles. In other words, 2-µm-tip diameter needles exhibited the best reproducibility. Ion intensities seemed to vary greatly during the spraying (20 min) for needles with diameters between 6 and 20 µm, resulting in a wide scattering of these spectra in the scores plot. This observed difference between 2-µm-diameter tips and wider tips is possibly due to the fact that smaller tip diameters produced smaller droplets that underwent fewer fission events.20 Thus, the desolvation process from 2-µm tip diameter can result in more reproducible spectra.

For 2-µm tip diameter, gold- and platinum-coated needles gave very similar spectra. Despite the lack of reproducibility for larger tip diameters, a systematic difference between the spectra measured using gold and platinum coatings was sometimes seen, although there was no overall pattern across all tip diameters. In general, spectra from smaller tip diameters were found to the left of the scores plot (Figure 4) and spectra from larger tip diameters to the right. To further understand why the scores plot in Figure 4 shows this distribution for the spectra, it is necessary to consider the loadings for PCs 1 and 2 given in Figure 5. The most important factor in the data is described by PC1 (32%). The loadings for PC1 show two large, negative peaks corresponding to free vancomycin ([V + H]+ and [V + K]+). Therefore, spectra that contain free vancomycin in relatively large intensity are found to the left of the scores plot in Figure 4. In general, it was also observed that, in spectra obtained from smaller tip diameter needles, the complex peak at m/z 911 ([V + KAA + 2H]2+) was relatively more intense and they are found to left of the scores plot in Figure 4. This supports the theory that small droplets preserve more faithfully the original solution composition, as discussed by Karas et al..20 The most significant loadings for PC2 correspond to the singly charged complex ([V + KAA + H]+ and [V + KAA + K]+). Spectra below the horizontal line in the scores plot exhibit higher peaks for these ions. From both the PC1 and PC2 loadings, it can be concluded that the 2-µm-tip diameter cluster found in the top-left corner of the scores plot represent spectra with intense peaks for free vancomycin and the doubly charged complex ([V + KAA + 2H]2+). These results demonstrate the important effect that tip diameter had on both the signals itself and the signal reproducibility for nanoESI. This means that different spectra result from different tip diameters and that large diameters might result in lower reproducibility as well. One implication of this is found in the common procedure in the case of a blocked needle, which is to gently touch the tip against the counter electrode to break it up, thus widening the tip diameter; this could clearly result in a considerable change in the subsequently observed spectra. Optimization of Mass Spectra by NanoESI. Spectra obtained in the second experiment on the 9.4-T FT-ICR by nanoESI are shown in Figure 6. Based on the results above, all the experiment used needles with 2-µm tip diameter. The spectrum in Figure 6a obtained from equimolar V/KAA solution differed from those obtained by nanoESI in the first experiment, Figures 2 and 3. First, contamination by potassium was not present. Second, an intense peak from the doubly charged complex [V + KAA + 2H]2+ m/z 911 was obtained, while in the previous experiment, this peak was less intense than both ligands (peptide, antibiotic). This is because, in the second experiment, the parameters, e.g., capillary exit voltage, offset, residence time in the hexapole, and ion injection time in the cell, were adjusted to maximize the peak for the complex [V + KAA + 2H]2+ 21-23 In (20) Karas, M.; Bahr, U.; Dulcks, T. Fresenius’ J. Anal. Chem. 2000, 366, 669676. (21) McDonnell, L. A.; Giannakopulos, A. E.; Derrick, P. J.; Tsybin, Y. O.; Hakansson, P. Eur. J. Mass Spectrom. 2002, 8, 181-189. (22) Sannes-Lowery, K. A.; Hofstadler, S. A. J. Am. Soc. Mass Spectrom. 2000, 11, 1-9. (23) Håkansson, K.; Axelsson, J.; Palmblad, M.; Håkansson, P. J. Am. Soc. Mass Spectrom. 2000, 11, 210-217.

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Figure 4. Scores plot from PCA analysis of the 50 mass spectra. Numbers from 2 to 20 represent the tip diameter used; letters “g” and “p” indicate gold or platinum coating.

Figure 5. Loadings for (a) PC1 and (b) PC2.

Figure 6. Mass spectra from 2-µm platinum-coated needle: (a) [V] ) [KAA] ) 50 µmol L-1; (b) [V] ) 50 µmol L-1.

the first experiment, the main objective was to optimize parameters (tuning) to obtain stable signals. However, in the conditions of the first experiment, a proportion of the complex present in solution fragmented in the gas phase as evidenced by the intense peaks for singly charged unbound vancomycin and peptide. In Figure 6b is shown the spectrum from pure vancomycin obtained with the same conditions as used for the spectrum in Figure 6a. Note that under these conditions the peak for doubly charged vancomycin [V + 2H]2+ is the most intense and [V + H]+ does 5176

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not exist. Beyond the tuning used to acquire this spectrum, the charge state in this case has considerable influence in the spectrum profile. As described by Marshall et al.,24 the signal increases linearly with ion charge, so that ICR is increasingly more sensitive for multiply charge ions. Comparison of ESI and NanoESI. Mass spectra from ESI and nanoESI were recorded using the same equimolar solution in which the complex [V + KAA] was formed. NanoESI measurements were made using five different types of needle. In all the analyses by nanoESI, needles with 2-µm tip diameter were used. The spectra obtained on the 3.0-T FT-ICR from the different sources and different emitters investigated are shown in Figure 7. The experimental conditions were maintained exactly the same for the different sources used. While the ESI spectrum exhibited strong peptide and doubly charged free vancomycin peaks, these were much less intense in all the nanoESI spectra. The same results were found for ESI and nanoESI spectra recorded from solutions with different ratios of the antibiotic and peptide (V/ KAA 1:2, 1:1, and 2:1). As can be observed in Figures 8c and 9c, in solutions where free vancomycin was present in excess, this manifested itself in the [V + 2H]2+ ion. When the ligands were in equimolar concentration (Figures 8b and 9b), the [V + 2H]2+ peak was still very intense in the ESI spectra, but it was almost not present in the nanoESI spectra. The intense peak for [V + 2H]2+ in the ESI spectra indicated the presence of free vancomycin in the droplet that was from the dissociation of the noncovalent complex during the spraying and desolvation process. Even when free vancomycin was deficient (i.e., V:KAA ) 1:2), a small peak for the ion [V + 2H]2+ was observed for ESI (Figure 8a). In the last experiment, an equimolar solution was analyzed by SORI-CAD.25 This experiment was performed to confirm how the complex dissociated in the gas phase. The spectrum obtained is shown in Figure 10, and it can be observed that in the gas phase the complex breaks to form single-charged vancomycin and (24) Marshall, A. G.; Hendrickson, C. L.; Jackson, G. S. Mass Spectrom. Rev. 1998, 17, 1-35. (25) Gauthier, J. W.; Trautman, T. R.; Jacobson, D. B. Anal. Chim. Acta 1991, 246, 211-225.

Figure 7. Mass spectra obtained by (a) ESI, (b) nanoESI platinum wire needle 2 µm, (c) nanoESI tungsten wire needle 2 µm, (d) nanoESI silver wire needle 2 µm, (e) nanoESI platinum-coated needle 2 µm, and (f) nanoESI gold-coated needle 2 µm.

peptide peaks ([V + H]+ and [KAA + H]+). Thus, it is concluded that the strong peak found for [V + 2H]2+ in the ESI spectra was not a result of fragmentation of the complex in the gas phase. This free vancomycin was formed in the spraying and desolvation process, when the complex was still in a solution-like environment.

To study effects in the sprays obtained from the different sources, the association constant for the complex analyzed was calculated from each spectra shown in Figure 7. As described above, all spectra were obtained in the same condition on the same day. Different methods have been applied to determine association

Figure 8. Mass spectra obtained by ESI of V/KAA: (a) 1:2; (b) 1:1; (c) 2:1.

constants using electrospray ionization mass spectrometry.26,27 A typical method is to monitor a solution-phase titration and to Analytical Chemistry, Vol. 76, No. 17, September 1, 2004

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Table 1. Association Constants for the Complex [V + KAA + 2H]2+ Calculated by Eq 5, in an Equimolar V/KAA Concentration at 50 µmol L-1 KV+KAA (L mol-1)

Figure 9. Mass spectra obtained by nanoESI (2-µm platinum-coated needle) of V/KAA: (a) 1:2; (b) 1:1; (c) 2:1.

Figure 10. SORI mass spectrum using [V] ) [KAA] ) 50 µmol L-1.

determine the association constant by graphic linearization such as Scatchard plots.28 In the present work, the association constants were calculated from the measurement of the relative peak intensities of the free host (vancomycin) and the complex. The association constant of the vancomycin complex in solution is given by eq 3:

K[V+KAA] ) [V + KAA]/[V][KAA]

(3)

for an equimolar mixture,

[V] ) [KAA]

(4)

K[V+KAA] ) [V + KAA]/[V]2

(5)

[V + KAA] and [V] were obtained from

[V + KAA] )

[V] )

I[V+KAA+2H]2+[V]0 I[V+H]+ + I[V+2H]2+ + I[V+KAA+2H]2+ I[V+2H]2+[V]0

I[V+H]+ + I[V+2H]2+ + I[V+KAA+2H]2+

(6)

(7)

(26) Daniel, J. M.; Friess, S. D.; Rajagopalan, S.; Wendt, S.; Zenobi, R. Int. J. Mass Spectrom. 2002, 216, 1-27. (27) Gabelica, V.; Galic, N.; Rosu, F.; Houssier, C.; Pauw, E. J. Mass Spectrom. 2003, 38, 491-501. (28) Greig, M. J.; Gaus, H.; Cummins, L. L.; Sasmor, H.; Griffey, R. H. J. Am. Chem. Soc. 1995, 117, 10765-10766.

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experiment

before correction

after correction

ESI nanoESI Pt wire nanoESI W wire nanoESI Ag wire nanoESI Pt coating nanoESI Au coating

(7.1 ( 0.2) × 104 (1.3 ( 0.1) × 106 (2.4 ( 0.1) × 106 (1.9 ( 0.1) × 106 (3.4 ( 0.1) × 106 (2.0 ( 0.1) × 106

(7.8 ( 0.2) × 104 (1.4 ( 0.1) × 106 (2.6 ( 0.1) × 106 (2.0 ( 0.1) × 106 (3.6 ( 0.1) × 106 (2.2 ( 0.1) × 106

where I is the relative peak intensity and [V]0 is the initial concentration of vancomycin in the solution, which in this experiment was 50 µmol L-1. However, as described by Marshall et al.,24 the signal increases linearly with ion charge, so the sensitivity for the double charge state is twice more intense than the single charge state. To correct this influence of the charge state in the spectra profile, the intensities from I[V+2H]2+ and I[V+KAA+2H]2+ were divided by 2 in the eqs 6 and 7, to calculate the concentration of [V + KAA] and [V] in the solution. Table 1 shows the values of the association constants for the [V + KAA + 2H]2+ complex calculated from the different spectra in Figure 7. In the first column are the results obtained using eqs 6 and 7. In the second column, are the results obtained adding I[V+H]+ to I[V+KAA+2H]2+ in the numerator to calculate [V + KAA] in eq 6. As can be observed, the association constant for the complex [V + KAA + 2H]2+ obtained by ESI is lower than those obtained by nanoESI, for the different calculations. The value for the association constant given in the literature is 1.5 × 106 L mol-1 and was obtained indirectly by determining the absorption values (E283) of the complex formed from a titration curve of vancomycin by peptide. The data were treated by Scatchard method as described by Nieto and Perkins.29,30 The association constants obtained in this experiment from nanoESI spectra were closer to the value in the literature than those from the ESI spectra. The association constants in the second column probably are the most reliable. Because as shown in the spectrum obtained by SORI in Figure 10, when the complex is fragmented in the gas phase, the [V + H]+ ion is formed. By adding I[V+H]+ to I[V+KAA+2H]2+, correction was made for the fragmentation in the gas phase. As observed in Figure 7, there was no significant difference between the nanoESI spectra, whether metallized or nonmetallized needles were used. It is possible that if the measurement period for each sample were increased from 15 min to, for example, more than 1 h, a change in recorded spectra would have been observed as has been seen in previous works.31,32 When the sample is in contact with the metal for a long time, electrochemical reactions may take place yielding new species and may change the association constant. It is concluded that, in ESI, the high number of fission events, the high temperature (150 °C), and the high gas pressure (30 (29) Nieto, M.; Perkins, H. R. Biochem. J. 1971, 124, 845-852. (30) Nieto, M.; Perkins, H. R. Biochem. J. 1971, 123, 773-787. (31) Berkel, G. J. V.; Asano, K. G.; Schnier, P. D. J. Am. Soc. Mass Spectrom. 2001, 12, 853-862. (32) Wetterhall, M.; Klett, O.; Markides, K. E.; Nyholm, L.; Bergquist, J. Analyst 2003, 128, 728-733.

psi) required to reach a stable spray broke the weak bond before the complex was desolvated and charged.33 Thus, when the ions were realized from the droplets’ intense peaks for [V + 2H]2+, [KAA + H]+ and [KAA + K]+ were observed in the spectra. The comparisons between desolvation of droplets of substantially different diameters such as those produced by ESI (100 µm) and those produced by nanoESI (a few micrometers or smaller) indicated that the desolvation conditions experienced by the smaller droplets can have a significant impact on the quality of the information obtained. Smaller droplets do not require desolvation gas of high temperature and suffer fewer fission events, thus perhaps maintaining lower internal energy in the complex and preserving it to be released in the gas phase. For the large droplets found in conventional ESI, at least one more generation of offspring droplets is necessary for the droplets to be sufficiently small for ion release to occur. This means that formation of mass spectral ions occurs later in comparison to the smaller droplets found in nanoESI and there is, consequently, more time for the noncovalent complex to break. The present results with nanoESI satisfy the “mass flux sensitive” criterion34 (i.e., response is proportional to the absolute quantity of material present); conditions attributed to extremely low flow rates (10-100 nL/min) make nanoESI a suitable technique for the study of noncovalent complexes in biological systems. CONCLUSION NanoESI experiments performed with different tip diameters indicated that a needle diameter of 2 µm resulted in reproducible (33) Loo, J. A. Mass Spectrom. Rev. 1997, 16, 1-23. (34) Covey T. In Biochemical and Biotechnological Applications of Electrospray Ionization Mass Spectrometry; Snyder, A. P., Ed.; American Chemical Society: Washington, DC, 1995; pp 21-59.

spectra in the analysis of noncovalent complexes. It was found that the different type of needles tested (metallized and nonmetallized) did not show any significant effect on the spectra recorded. However, due to the different fission processes within conventional ESI and nanoESI, different peak intensities for the bound and unbound vancomycin and peptide ions were in the mass spectra obtained. The association constant values found for ESI and nanoESI were very different, and we conclude that, in ESI, the complex was broken during the spraying and desolvation process. This presumably is because the time spent in the source before release into the gas phase is relatively long for ESI and the noncovalent complex undergoes the effects of temperature and high pressure that dissociate the noncovalent bond. In nanoESI, the desolvation time is much shorter, and high temperature and pressure are not necessary. The spray, in this case, preserves more faithfully the original solution composition, and thus, more complex ion is realized in the gas phase. The interesting effects of desolvation of droplets observed on the ratios of bound and unbound vancomycin and peptide indicate that efforts should be directed toward understanding the effects of fundamental physical parameters on the stoichiometry of the noncovalent complexes detected using ESI and nanoESI. ACKNOWLEDGMENT The authors acknowledge the University of Warwick, GlaxoSmithKline, and EU Marie Curie Training Site for financial support. We also thank Mark Barrow for helpful discussions. Received for review March 19, 2004. Accepted June 24, 2004. AC049569Z

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