ANALYTICAL SCIENCES JULY 2010, VOL The Japan Society for Analytical Chemistry

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1 ANALYTICAL SCIENCES JULY 2010, VOL The Japan Society for Analytical Chemistry MALDI Mass Spectrometry Using 2,4,6-Trihydroxyacetophenone and 2,4-Dihydroxyacetophenone with Cyclodextrins: Suppression of Matrix-related Ions in Low-molecular-weight Region Takashi FUJITA, Tatsuya FUJINO, azunori HIRABAYASHI, and Takashi ORENAGA Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, 1-1 Minami-Osawa, Hachioji, Tokyo , Japan Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry of a model peptide, Substance P (SubP), was carried out using 2,4,6-trihydroxyacetophenone (THAP) and 2,4-dihydroxyacetophenone (DHAP) with cyclodextrins (cyclodextrin-supported matrix). It was found that the use of a cyclodextrin-supported matrix simplified the mass spectrum in the low-molecular-weight region. The interaction between THAP/DHAP and cyclodextrin (CD) was studied by UV-vis absorption spectroscopy and the incorporation of matrix molecules into the cyclodextrin cavity was confirmed by 1 H-NMR spectroscopy. DHAP showed tight incorporation with βcd (βcd(dhap)) rather than THAP and it was found that the matrix-related peaks could be weakened by less than one third of the peak intensity of a protonated analyte. The βcd(dhap) matrix was applied to the measurements of two low-molecular-weight compounds; adenosine and adrenaline. It became clear that the cyclic structure of the CD and the host-guest interaction between βcd and the matrix molecule were important to reduce the matrix-related peaks of THAP and DHAP. (Received February 23, 2010; Accepted May 18, 2010; Published July 10, 2010) Introduction Matrix-assisted laser desorption/ionization (MALDI) is an important analytical technique since it often permits the observation of protonated molecules, [M+H] +, with little or no fragmentation. 1,2 With recent emerging interest in proteomics and metabolomics, MALDI in combination with time-of-flight (TOF) mass spectrometry is recognized as a powerful tool for studying proteins and peptides. Although MALDI possesses much potential for the analysis of biological samples, it has a few drawbacks. First, the peak intensity of a protonated molecule, [M+H] +, is suppressed by the appearance of peaks of alkali metal ion adducts since biological samples contain intrinsically an abundance of alkali metal ions. Weakened mass peaks of protonated molecules [M+H] + and those of alkali metal ion adducts ([M+Na] + and [M+] + ) complicate the spectrum. Second, the applicability of MALDI is hampered by the fragmentation of matrix molecules as MALDI involves the laser desorption/ionization (LDI) of matrix molecules. The LDI process produces many peaks of matrix-related species and fragments, which is the reason why MALDI is not especially suited for low-molecular-weight compounds. Thirdly, because the spatial distribution of matrix-analyte crystals is inhomogeneous, searching for sweet spots where strong peaks appear is required. To overcome such drawbacks, many attempts have been made so far, including the use of sugars, 3 ammonium salts, 4,5 phosphoric acid, 6 serine, 7 and proteins 8 with conventional To whom correspondence should be addressed. fujino@tmu.ac.jp organic MALDI matrices. Such matrix-free techniques as the desorption/ionization on silicon (DIOS) 9 and the use of a nano-structured surface 10 have been proposed as well. Recently, we used zeolites 11 and α-cyclodextrin (αcd) as the host molecule for organic matrices, 12 and found that it reduced the fragmentation of a guest matrix molecule. Using typical organic matrices, such as THAP (2,4,6-trihydroxyacetophenone) and CHCA (α-cyano-4-hydroxycinnamic acid) with cyclodextrin, we successfully measured the mass peaks of only protonated matrix ions and suppressed their intensities and fragmentation. In addition, it became possible to analyze the mass peak of the analyte molecules without interference from the matrix. However, the efficiency of a cyclodextrin-supported matrix for the reduction of matrix-related peaks and fragments has not been discussed based on incorporation of matrix molecules into the cyclodextrin cavity. We have only discussed the equilibrium constant for the complex formation of matrix molecules and αcd. In this study, we used β-cyclodextrin (βcd) mainly as a host molecule for THAP and 2,4-dihydroxyacetophenone (DHAP). The βcd has a larger cavity size, and it is known that its space is more hydrophobic than αcd. The equilibrium constant for complex formation was studied by UV-vis absorption spectroscopy in the liquid phase, and the incorporation of THAP and DHAP was confirmed by 1 H-NMR spectroscopy. Both compounds with βcd (cyclodextrin-supported matrix) were used and mass spectrometric measurements of low-molecular-weight compounds were carried out. The peaks of protonated THAP (DHAP) and analytes were observed without interference from the peaks of fragments and alkali metal ion adducts. Furthermore, it was found that the matrix-related peaks could be weakened by less than one third of the peak intensity of the

2 744 ANALYTICAL SCIENCES JULY 2010, VOL. 26 cyclodextrin-supported matrix and the analyte solution was pipetted onto a stainless-steel plate, and left in air for a few minutes to evaporate the solvent. Therefore, molar amount of analyte molecules on a plate (sensitivity) were calculated to be 37.1 pmol for SubP, 168 pmol for adenosine, and 273 pmol for adrenaline. The molar ratio among the analyte, THAP (DHAP), βcd, and TFA is 1:505:505:180 for measurements of SubP: 1:111:111:40 for adenosine, and 1:69:69:25 for adrenaline. Mass spectra of analyte molecules were obtained with a laboratory-made mass spectrometer, described previously. 12 Briefly, matrix-analyte crystals on the stainless-steel plate were put inside a vacuum chamber set at Pa. The fourth-harmonics generation of the fundamental laser output from an Nd:YAG laser (Spectra Physics, 266 nm, 10 Hz, 5 ns) was used as excitation light. The pulse energy of the excitation light was set to 10 μj. The produced ions were accelerated with an electric field of kv and detected with a linear TOF tube and an MCP detector. UV-vis absorption spectra were obtained by a commercial spectrometer (Hitachi, U-3400) using 1-cm optical cells. Results and Discussion Fig. 1 (a) MALDI mass spectrum of a model peptide, SubP, with THAP. (b) Mass spectrum of SubP using the βcd(thap) matrix. (c) MALDI mass spectrum of SubP/DHAP. (d) Mass spectrum of SubP using the βcd(dhap) matrix. (e) Mass spectrum of SubP using the αcd(dhap) matrix. protonated analyte by using DHAP with βcd (βcd(dhap)). The reduction of matrix-related peaks of THAP and DHAP by using CDs is discussed based on the incorporation of matrix molecules into the cyclodextrin cavity. Experimental THAP and DHAP were purchased (Wako Chemical) and recrystallized from methanol. CDs were dissolved in distilled water to make a concentration of 10 mm. THAP (DHAP) was dissolved in a mixture of acetonitrile and water (7:3 in volume) containing trifluoroacetic acid (TFA, 0.1% in volume), and a solution of 75 mm was obtained. Solutions (150 μl of CDs and 20 μl of THAP (DHAP)) were pipetted into a vial (micro tube) and sonicated with increasing temperature (~323 ). The incorporation of each molecule into the cyclodextrin cavity was confirmed by 500 MHz 1 H-NMR spectroscopy (JEOL). THAP (DHAP) with CD was dissolved into deuterated water (D 2O) and the saturated solution was used for measurements. The analyte molecules, substance P (SubP), adenosine, and adrenaline, were dissolved in a mixture of acetonitrile and water (7:3 in volume) containing TFA (0.1% in volume), and a solution was obtained. A half microliter each of the Figure 1(a) shows the MALDI mass spectrum of a model peptide, SubP, using THAP as an organic matrix. The mass peaks of protonated SubP and THAP are observed; the spectrum in the region less than 400 Da is complicated by matrix-related and analyte-related peaks: peak intensity ratio of [THAP+H] + :[2THAP+H] + is 3.3:1; [THAP+H] + :[2THAP+Na] + is 42.5:1; and [THAP+H] + :[SubP+H] + is 16.4:1. Although THAP is known to be a good matrix for oligonucleotides, 13 it is also applicable to peptides. The THAP molecule is mixed with βcd to form the cyclodextrin-supported matrix and the matrix, βcd(thap), is used for measurements of SubP. Figure 1(b) shows the MALDI mass spectrum of SubP using βcd(thap). Similar to our previous research using αcd(thap), 12 the number of peaks in the region less than 400 Da is reduced, and almost only the peaks from protonated THAP and protonated SubP are observed; the peak intensity ratio of [THAP+H] + :[2THAP+H] + is 81.2:1; [2THAP+Na] + is not observed. The result reveals that βcd can facilitate the soft ionization of not only a guest molecule (THAP), but also an analyte (SubP) and simplify the mass spectrum. It is suspected that the matrix suppression effect, which is common in conventional MALDI, was observed in Fig. 1(b) rather than the effect of the cyclodextrin-supported matrix. In such a case, however, the intensities of all the peaks should decrease equally. In the obtained spectrum, the peaks of protonated THAP and SubP are clearly observed, while those of the matrix dimer and the alkali metal ion adducts almost disappear (again, the peak intensity ratio of [THAP+H] + :[2THAP+H] + is 3.3:1 for Fig. 1(a) and 81.2:1 for Fig. 1(b)). Therefore, it is considered that the observed spectrum is due to not the matrix suppression effect arising from the analyte/matrix mixing, 14 but the effect of the cyclodextrin-supported matrix. A conventional MALDI mass measurement was also carried out by using DHAP, and the result is shown in Fig. 1(c). DHAP has two hydroxyl groups in the acetophenone framework whereas THAP has three hydroxyl groups as shown in the inset. The peak of protonated peptides as well as that of protonated DHAP are observed as in the case of THAP, although the spectrum in the region less 400 Da is rather complicated due to the matrix-related and analyte-related peaks. Figure 1(d) shows the MALDI mass spectrum of SubP using the βcd(dhap) matrix. Similar to the result using the

3 ANALYTICAL SCIENCES JULY 2010, VOL where m is DHAP, and ε CD(m), ε m, and ε CD are the absorption coefficients of βcd(dhap), DHAP and cyclodextrin at 325 nm, respectively. Since CD has no absorption coefficient in this UV-visible region, ε CD[CD] is zero. When βcd forms a 1:1 complex with DHAP, the absorbance at 325 nm can be rewritten as A325 = εcd(m) ( A C) + ε m( B + C), m CD0 + A =, 2 m0 CD 0 1 B =, 2 2 CD 0 0 m m 0 C = , (2) Fig. 2 (a) Absorption spectra of βcd(dhap) in acetonitrile and water (7:3 in volume) solution with the DHAP concentration changing from to M. The concentration of βcd is kept to 10 3 M. (b) Fitting analysis of the absorbance at 325 nm by Eq. (2). βcd(thap) matrix, a simplified mass spectrum is observed. Moreover, the peak intensity of protonated DHAP is weakened although that of protonated SubP is almost comparable to the result in Fig. 1(b); the peak intensity ratio of [DHAP+H] + :[SubP+H] + is 3.38:1 for Fig. 1(c) and 0.28:1 for Fig. 1(d). By using αcd(dhap), peak intensity ratio of [DHAP+H] + :[SubP+H] + is 2.00:1 as shown in Fig. 1(e), which is almost comparable to that in the case of αcd(thap) reported in our previous paper (1.71:1). Therefore it is understood that DHAP, especially with βcd, can suppress the mass peak of the protonated DHAP molecule efficiently. DHAP has generally a linear molecular structure compared with THAP. Therefore, it is surmised that the degree of incorporation of matrix molecules into the βcd cavity plays an important role in reducing the peak intensity of the protonated matrix to almost less than one third of protonated SubP (0.28:1) as Fig. 1(d). In order to confirm the above argument, spectroscopic measurements were carried out for the cyclodextrin-supported matrices. Although MALDI mass spectrometry was carried out for matrix analyte mixture in the solid state, spectroscopic measurements were performed in the solution phase. Figure 2(a) shows the UV-vis absorption spectra of βcd(dhap) for a mixture of acetonitrile and water (7:3 in volume) with the DHAP concentration as a parameter. The concentration of βcd is kept to 10 3 M for all measurements. The obtained spectra are normalized to the peak height at 275 nm. It is observed that the peak intensity at around 325 nm enlarges with increasing concentration of the matrix molecules as the arrow indicates, because of the absorption coefficient of βcd(dhap). Therefore, the absorbance at 325 nm can be written as A 325 = ε CD(m)[CD(m)] + ε (m)[m] + ε CD[CD], (1) where m 0 and CD 0 are the initial concentrations of DHAP and βcd, respectively, and is the equilibrium constant for the complex formation of βcd(dhap). By using Eq. (2), the absorbance at 325 nm is fitted and the result is shown in Fig. 2(b). The absorbance at 325 nm is fairly reproducible by Eq. (2), indicating that βcd forms a 1:1 complex with DHAP. By the fitting analysis, = 2745 M 1 and ε CD(m) = 9228 M 1 cm 1 are obtained. The same analysis is also performed for βcd(thap), and = 1739 M 1 and ε CD(m) = 6725 M 1 cm 1 are obtained as a result of the 1:1 complex formation of βcd with THAP. The finding that the equilibrium constant for βcd(dhap) is larger than that for βcd(thap) suggests that DHAP, rather than THAP, makes a stable complex with βcd. The incorporation of matrix molecules in the cyclodextrin cavity was then confirmed by 1 H-NMR spectroscopy, the results of which are summarized in Table 1 (some spectra are depicted in Fig. S1 (Supporting Information) for example). For a comparison, 1 H-NMR spectroscopy was also performed for p-hydroxyacetophenone (phap), which only has one hydroxyl group at the para position. It is mentioned that phap does not have the hydroxyl group that forms an intramolecular hydrogen bond with an acetophenone substituent, and phap shows less efficiency as an organic matrix for MALDI mass spectrometry (data not shown). In this argument phap is used only for a comparison. The βcd is a cyclic oligosaccharide composed of seven linked glucoses, whereas αcd and γcd are composed of six and eight linked glucoses. The orientation of methine protons (H-3 and H-5 in Table 1) in its cavity produces a relatively hydrophobic space. Protons of CDs (indicated by H-3 and H-5) show large chemical shifts compared with the other protons, upon the incorporation of guest molecules. 15 From Table 1 for βcd(thap), βcd(dhap) and βcd(phap), it is recognized that the chemical shifts of H-3 and H-5 are larger than those of the other protons upon the incorporation of guest molecules. Furthermore, H-3 and H-5 protons show large chemical shifts according to the order phap > DHAP > THAP, indicating tight incorporation into βcd in this order. However, for αcd(thap), αcd(dhap) and αcd(phap), the chemical shifts of H-3 and H-5 are not large compared with those of the other protons. For αcd(thap), the chemical shifts of the H-6 proton are large whereas those of other protons are small. For αcd(dhap) and αcd(phap), all protons show large chemical shifts. This result means that αcd does not efficiently incorporate the matrix molecules although αcd forms a 1:1

4 746 ANALYTICAL SCIENCES JULY 2010, VOL. 26 Table 1 1 H-NMR chemical shifts by the formation of a cyclodextrin-supported matrix H-1 H-2 H-3 H-4 H-5 H-6 δ(free) δ(βcd(thap)) δ(βcd(dhap)) δ(βcd(phap)) δ(free) δ(αcd(thap)) δ(αcd(dhap)) δ(αcd(phap)) δ(free) δ(γcd(thap)) δ(γcd(dhap)) δ(γcd(phap)) complex with those guests according to the equilibrium constant revealed by UV-vis absorption measurements. 12 The van der Waals volume of THAP is estimated 16 to be Å 3 and that of DHAP is Å 3. When we assume a spherical molecular structure, the diameters can be calculated to be nm for THAP and nm for DHAP. The diameter of cyclodextrin is nm for α, nm for β, and nm for γ, and it is suggested that αcd or βcd is suited for the incorporation of THAP and DHAP. Therefore, it is considered that the weakened peak intensity of the protonated matrix ([DHAP+H] + ) as observed in Fig. 1(d) (almost one third of protonated analytes) is fairly due to the tight incorporation of DHAP into the βcd cavity compared with THAP. When γcd, which has a larger cavity size than βcd, is used to form a cyclodextrin-supported matrix, a reduction of the peak intensity of the protonated matrix is not so expected. Figure 3 shows the MALDI mass spectra of SubP using γcd(thap) and γcd(dhap) matrices. Although the spectra in the region less than 400 Da are simplified, as in the case of βcd, the peak intensity of the protonated DHAP is increased, as shown in Fig. 3(b) (peak intensity ratio of [DHAP+H] + :[SubP+H] + = 2.3:1 and [DHAP+H] + :[SubP+H] + = 3.4:1). For SubP/γCD(DHAP), even the peaks of the alkali metal ion adducts of SubP are observed. From Table 1, γcd shows incorporation with DHAP; chemical shifts of H-3 and H-5 protons are and ppm, respectively, and these values are larger than those of other protons. However, these values are smaller than the chemical shifts of H-3 and H-5 protons observed for βcd(dhap). Therefore, it can be Fig. 3 Mass spectra of SubP using cyclodextrin-supported matrix: (a) γcd(thap) and (b) γcd(dhap). The peak from the protonated matrix is strongly observed. mentioned that peak intensity of the protonated matrix in the mass spectrum is related to the values of the chemical shifts of the H-3 and H-5 protons, meaning the degree of incorporation of the matrix molecules into the cyclodextrin cavity. It should be mentioned that we used 266-nm pulses for excitation nevertheless βcd(thap) or βcd(dhap) show a characteristic

5 ANALYTICAL SCIENCES JULY 2010, VOL Fig. 4 Mass spectra of low-molecular-weight compounds (a) adenosine and (b) adrenaline using the βcd(dhap) matrix. The peak from the protonated DHAP almost disappears. absorption band at 325 nm. By photoexcitation with 266-nm pulses, we excite the ensemble of matrix molecules with and without CD. The peaks of βcd(thap) and βcd(dhap) are not observed in the mass spectra, indicating that the interaction between THAP and DHAP with the cyclodextrin cavity being collapsed by photoexcitation. We carried out conventional MALDI mass measurements of βcd(thap) and βcd(dhap) with synapinic acid (SA) as a matrix; however, the peaks of βcd(thap) and βcd(dhap) were not observed. In our previous paper, we also used α-cyano-4-hydroxycinnamic acid (CHCA) with αcd. 12 In that case, the peak from protonated CHCA was not observed completely although the equilibrium constant for the formation of αcd(chca) was the smallest value of 930 M 1. Because of the low solubility of CHCA to D 2O, it was not possible to distinguish the NMR spectrum of αcd incorporated by CHCA. By considering the molecular structure of CHCA, which is similar to that of phap, it is possible to assume that CHCA does not efficiently incorporate into αcd cavity. If so, such a mechanism as the matrix and analyte reaction is operative rather than the incorporation of CHCA into αcd to reduce the peak from the protonated matrix. The proton transfer mechanism is different between acetophenones and CHCA, so that further study such as picosecond/femtosecond time-resolved fluorescence spectroscopy is needed to completely understand the proton transfer reaction among the matrix, analyte and solvent. According to the above arguments, it is suggested that βcd(dhap) may be a good matrix for measurements of low-molecular-weight compounds because the matrix-related peaks are almost eliminated. The βcd(dhap) matrix is applied to low-molecular-weight compounds. Figure 4(a) shows the spectrum of adenosine in the mass region less than 400 Da. As in Fig. 1(d), the matrix-related peaks almost disappear and the peak of the protonated matrix and that of the Na-adducted DHAP dimer are barely recognized; the peak intensity ratio of [DHAP+H] + :[adenosine+h] + is 0.09:1, and that of [2DHAP+Na] + :[adenosine +H] + is 0.20:1. On the other hand, the peak of the protonated adenosine [M+H] + and that of the fragment, protonated adenine, are clearly observed without any interference from the matrix-related peaks. Then, the βcd(dhap) matrix is also applied to adrenaline; the result is Fig. 5 Mass spectra of SubP using DHAP and (a) maltohexaose or (b) maltoheptaose as a co-matrix. shown in Fig. 4(b). As in the case of adenosine, the peak of the protonated matrix and that of the Na-adducted DHAP dimer are scarcely recognized, whereas the peak of the protonated adrenaline is clearly observed; the peak intensity ratio of [DHAP+H] + :[adrenaline +H] + is 0.13:1, and that of [2DHAP+Na] + :[adrenaline +H] + is 0.10:1. It is generally known that organic matrices have merits and demerits for the ionization of a variety of analyte molecules. For example, THAP is known as a good matrix for oligonucleotides, as we mentioned above. By using the cyclodextrin-supported matrix, however, the peak from the protonated analyte could be clearly observed even though its intensity is quite weak, because the peaks from matrix-related ions including alkali metal ion adducts and fragments almost disappear. Although the molar amount of this study is fairly high and the sensitivity is considered to be low (37.1 pmol for SubP, 168 pmol for adenosine, and 273 pmol for adrenaline), it is understood that this finding shows the potential of the βcd(dhap) matrix for applications to a variety of low-molecular-weight compounds. Finally, mass spectrometric measurements of SubP with maltohexaose or maltoheptaose were carried out to understand the reason for the advantages of the cyclodextrin-supported matrix. Maltohexaose and maltoheptaose are sugars having linear structures, and are composed of 8 or 9 glucose molecules, in contrast to CD, which has a cyclic structure. The CDs are composed of six linked glucose molecules for αcd, seven for βcd, and eight for γcd. By using DHAP and those sugars as a co-matrix, the mass spectra of SubP were measured. Contrary to the results using the cyclodextrin-supported matrix, the peaks of protonated DHAP and its fragment (OH loss) are observed with intensity ([DHAP OH+H] + :[DHAP+H] + = 0.39:1 for Fig. 5(a), and = 0.05:1 for Fig. 5(b)) and the mass spectrum in the region less than ~200 Da becomes complicated. This result is observed for both measurements using maltohexaose and maltoheptaose. Therefore, it has been confirmed that the cyclic structure and the host-guest interaction between CD and a matrix molecule are one of the important factors to reduce matrix-related peaks and to prevent the ionization of matrix molecules. The peak of the akali metal ion adduct of the matrix molecule also disappeared upon using maltohexaose and maltoheptaose. Therefore, it is understood that glucose molecules in those sugars capture the sodium and potassium atoms and prevent alkali metal-ion adduct formation.

6 748 ANALYTICAL SCIENCES JULY 2010, VOL. 26 Conclusions In summary, we carried out the matrix-assisted laser desorption/ionization (MALDI) mass spectrometry of low-molecular-weight compounds by using cyclodextrinsupported matrices. The mass spectrum in the low-molecular-weight region (by ~400 Da) was simplified, and it was found that the matrix-related peaks could be weakened by less than one third of the peak intensity of the protonated analyte by using DHAP with βcd (βcd(dhap)). The βcd(dhap) matrix was applied to measurements of two low-molecular-weight compounds: adenosine and adrenaline. It became clear that the cyclic structure of CD and the host-guest interaction between βcd and the matrix molecule were important to reduce the matrix-related peaks of THAP and DHAP. Acknowledgements Tatsuya Fujino acknowledges a Grant-in-Aid for Scientific Research on Priority Area (477) from MEXT. Supporting Information Figure S1, 1 H-NMR spectra of (a) βcd, (b) βcd(thap) and (c) βcd(dhap). This material is available free of charge on the web at References Anal. Chem., 1991, 63, 1193A. 2. F. Hillenkamp and M. aras, Int. J. Mass. Spectrom., 2000, 200, C. oester, J. A. Castoro, and C. L. Wilkins, J. Am. Chem. Soc., 1992, 114, J. Asara and J. Allison, J. Am. Soc. Mass Spectrom., 1999, 10, X. Yang, H. Wu, T. obayashi, R. J. Solaro, and R. B. van Breemen, Anal. Chem., 2004, 76, S. jellstroem and O. N. Jensen, Anal. Chem., 2004, 76, T. Nishikaze and M. Takayama, Rapid Commun. Mass Spectrom., 2007, 21, T. obayashi, H. awai, T. Suzuki, T. awanishi, and T. Hayakawa, Rapid Commun. Mass Spectrom., 2004, 18, R. Arakawa, Y. Shimomae, H. Morikawa,. Ohara, and S. Okuno, J. Mass Spectrom., 2004, 39, H. awasaki, T. Yonezawa, T. Watanabe, and R. Arakawa, J. Phys. Chem. C, 2007, 111, Y. omori, H. Shima, T. Fujino, J. N. ondo,. Hashimoto, and T. orenaga, J. Phys. Chem. C, 2010, 114, S. Yamaguchi, T. Fujita, T. Fujino, and T. orenaga, Anal. Sci., 2008, 24, A. Meyer, N. Spinelli, J.-L. Imbach, and J.-J. Vasseur, Rapid Commun. Mass Spectrom., 2000, 14, M. nochenmuss, F. Dubois, M. J. Dale, and R. Zenobi, Rapid Commun. Mass Spectrom., 1996, 10, Y. Yamamoto and Y. Inoue, J. Carbohydr. Chem., 1989, 8, J. T. Edward, J. Chem. Educ., 1970, 47, F. Hillenkamp, M. aras, R. C. Beavis, and B. T. Chait,