JMS letters Received: 23 May 2013
Revised: 12 September 2013
Accepted: 3 October 2013
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3294
Rapid identification of disaccharides by tandem mass spectrometry Ákos Kuki, Katalin E. Szabó, Lajos Nagy, Miklós Zsuga and Sándor Kéki* Additional supporting information may be found in the online version of this article at the publisher’s web site.
Dear Sir,
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Carbohydrates are the important class of biological molecules. The wide variety of positional and anomeric structures makes it possible for saccharides to form plenty of compounds. It is well known that oligosaccharides attached to proteins or lipids (forming glycoconjugates) and provide important biological functions.[1,2] Furthermore, oligosaccharide units can also be found in flavonoid glycosides, and their mass spectrometric behaviors have been investigated in detail.[3–5] The function of the disaccharides is strongly influenced by their structures. Therefore, it is necessary to determine the stereochemistry of the monosaccharide units, the linkage position and the anomeric configuration of the isomeric disaccharides. Tandem mass spectrometry combined with soft ionization methods such electrospray ionization (ESI) and matrix-assisted laser desorption/ionization is an effective tool for the structural analysis of oligosaccharides. Determination of the linkage position and anomeric configuration has been demonstrated both in the negative and positive ion modes. For protonated oligosaccharide cleavage occurs almost exclusively at the nonreducing side of the glycosidic oxygen (referred as B-type and Y-type ions[6]).[7,8] For alkali metal adduct cross-ring fragmentation can also be observed besides the abundant B-type and Y-type ions.[9–11] The effect for the destabilization of the glycosidic bond is decreasing with the size of the cations. Hence, higher energy required to fragment the disaccharides cationized by larger ions.[12,13] In the negative ion mode, the collision-induced dissociation spectra of the deprotonated disaccharides show distinguishable fragmentation patterns with C-type and A-type ions for the different isomeric and anomeric configurations.[14,15] Moreover, derivatization (e.g. permethylation) can be applied to increase the extent of fragmentation,[16] and negative ion adducts are also used for the analysis of disaccharides.[17,18] Glucose disaccharides varying solely in their anomeric configuration were clearly distinguished on the basis of the relative abundance of the characteristic product ions arising from the cleavage of the glycosidic bond of the lithiated adduct ion at mass-to-charge ratio (m/z) 169 and 187 using wavelength-tunable infrared multiple-photon dissociation-mass spectrometry.[19] Very recently, Domingues et al. proposed that the ratio of m/z 169/187 for [M + Li]+ ions and m/z 185/203 for [M + Na]+ ions seems to be a simple criterion for the differentiation of isomeric hexose disaccharides with β-(1 → 4) linkage but distinct monosaccharide composition.[20] In this work, we have examined the use of ESI-MS/MS in positive ion mode of the lithiated, sodiated and ammoniated adducts
J. Mass Spectrom. 2013, 48, 1276–1280
for the differentiation of four glucose disaccharides: maltose (Glcα1-4Glc), cellobiose (Glcβ1-4Glc), isomaltose (Glcα1-6Glc) and gentiobiose (Glcβ1-6Glc). According to our best knowledge, no detailed report on the fragmentation of the ammoniated adducts of the glucose disaccharides has been issued. Our goal was to define simple criteria for distinguishing between the four disaccharides. Herein, we also report a highly automated data acquisition and processing method for the identification of these disaccharides by ESI-MS/MS, thereby considerably reducing the analysis time. The HPLC grade methanol (VWR International, Leuven, Belgium) was used without further purification. Water was purified by a Direct-Q water system (Millipore, Molsheim, France). NaCl, LiCl, NH4Cl and all disaccharides were received from Aldrich (Steinheim, Germany). The disaccharides were dissolved in methanol/water (4/1 V/V) at a concentration of 0.1 mg/mL. To obtain lithiated, sodiated and ammoniated adducts, LiCl, NaCl and NH4Cl were added to the disaccharide solutions to obtain 0.1 mM concentration of the salts. The MS/MS measurements were performed with a MicrOTOF-Q type Qq-TOF MS instrument (Bruker Daltoniks, Bremen, Germany) using an ESI source with positive ion mode. The sample solutions were introduced directly into the ESI source with a syringe pump (Cole-Parmer Ins. Co., Vernon Hills, IL, USA) at a flow rate of 3 μL/min. The spray voltage was set to 4 kV. The temperature of the drying gas (N2) was kept at 180 ° C. For the MS/MS experiments, nitrogen was used as the collision gas, and the laboratory frame collision energies (i.e. the kinetic energy acquired by the ions from the accelerating electric field in front of the collision cell) were varied in the range of 1–48 eV. The pressure in the collision cell was determined to be 8 × 103 mbar. The precursor ions for MS/ MS were selected with an isolation width of 1–4 m/z units. The MS/MS spectra were accumulated and recorded by a digitizer at a sampling rate of 2 GHz. The mass spectra were calibrated externally using the exact masses of clusters [(NaTFA)n + Na]+ generated from the electrosprayed solution of sodium trifluoroacetate (NaTFA). The mass spectra were evaluated with the DATAANALYSIS 3.4 software from Bruker.
* Correspondence to: Sándor Kéki, Department of Applied Chemistry, University of Debrecen, H-4032 Debrecen, Egyetem tér 1., Hungary. E-mail: keki. [email protected]
Copyright © 2013 John Wiley & Sons, Ltd.
JMS letters The efficiency of the fragmentation can be quantitatively described by the survival yield (SY). The SY is defined according to Eqn (1): SY ¼
Ip Ip þ ∑If
(1)
J. Mass Spectrom. 2013, 48, 1276–1280
Figure 1. Product ion spectra (electrospray ionization-MS/MS) of the ammoniated adducts of the four examined disaccharides recorded at 14 eV laboratory frame collision energy. The peaks indicated with asterisk (*) are from an impurity.
to the Y-type glycosidic cleavage are [M + NH4 NH3 C6H10O5]+ at m/z 181 and [M + NH4 C6H10O5]+ at m/z 198. Interestingly, as it can be seen in Table 1, the relative abundance of the protonated B-type ion at m/z 163 is much higher than the ammoniated B-type ion at m/z 180 and the Y-type ions at m/z 198 and 181. In addition to the glycosidic cleavages, loss of ammonia and waters from the ammoniated adduct can be observed: NH3 and H2O (35 Da) at m/z 325, NH3 and 2H2O (53 Da) at m/z 307 (with very low abundance) and NH3 and 3H2O (71 Da) at m/z 289. Two additional product ions are formed by the loss of water from the more abundant B-type ion ([M + NH4 C6H12O6]+) at m/z 145 and 127. Cross-ring cleavages cannot be detected in the ESI-MS/ MS spectra of the ammoniated adducts of the disaccharides. Although the product ion spectra of the [M + NH4]+ ions are very similar, remarkable differences can be observed between the characteristic collision energies (CE50) (Table 2). Interestingly, as seen
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where Ip is the intensity of the precursor ion and ΣIf is the sum of all the fragment ion intensities. In the MS/MS of the sodiated disaccharides the sodium ion, which is one of the main product ions, cannot be detected; therefore, the SY cannot be calculated according to Eqn (1). This ‘incomplete’ SY is denoted by SY′ and still can be interesting for expressing the extent of fragmentation of structurally similar compounds. The collision energy at which the intensity of the precursor ion is equal to the sum of that of all the fragment ions (i.e. SY′ = 0.5) was determined with appropriate accuracy by a linear two-point interpolation. This energy is noted as characteristic collision energy (CE50). For the calculation of the t (Student’s t) distributions and t-tests, the built-in functions of spreadsheet software were used. The MS/MS spectra of the four lithiated disaccharides recorded by our ESI-quadrupole-time-of-flight (QTOF) at 30 eV laboratory frame collision energy are presented in Fig. S1. The dominant process in the ESI-QTOF MS/MS of the sodiated disaccharides was the simple loss of the cation from disaccharide. The dramatic decrease of the precursor ion signal intensity without the parallel increase of the product ion signals by increasing collision energy was observed. (The increase of the sodium ion signal parallel to the decrease of the total ion intensity could not be detected because the mass of sodium ion fell below the lowest mass limit on our mass analyzer.) The MS/MS spectra of the four sodiated disaccharides recorded at 36 eV laboratory frame collision energy are presented in Fig. S2. Although some product ion peaks can be observed in the MS/MS spectra of the [M + Na]+ ions, however because of the loss of the charge, the sodiated adducts cannot be used for the differentiation of the four glucose disaccharides. For instance, the absolute intensity of the characteristic product ion arising from the cleavage of the glycosidic bond of the sodiated maltose adduct ion at m/z 185 is extremely low (below 10) and has a large relative standard deviation (around 30% on the basis of ten measurements). Nevertheless, some characteristic differences can be observed in the fragmentation spectra of the α-anomers and β-anomers of the sodiated disaccharides. The fragmentation is more extent for α-anomers (SY′ = 0.61 for maltose and SY′ = 0.62 for isomaltose at 36 eV collision energy) compared with β-anomers (SY′ = 0.81 for cellobiose and SY ′ = 0.82 for gentiobose at 36 eV collision energy). Furthermore, the relative abundance of the product ion at m/z 203 (derived by the cleavage at the glycosidic bond) is much enhanced for α-anomers (0.51 for maltose and 0.46 for isomaltose) than for β-anomers (0.05 for cellobiose and 0.04 for gentiobose). As a novel approach, we examined the use of the ESI-MS/MS of the ammoniated adducts for the differentiation between the isomeric disaccharides. The MS/MS spectra of the [M + NH4]+ ions of maltose, cellobiose, isomaltose and gentiobiose are presented in Fig. 1. The relative intensities, the elemental compositions and the calculated and the measured masses of the product ions are compiled in Table 1. As seen in Fig. 1 and Table 1, all MS/MS spectra show the same product ion peaks. The product ions originated from the B-type glycosidic cleavage are [M + NH4 NH3 C6H12O6]+ at m/z 163 and [M + NH4 C6H12O6]+ at m/z 180, whereas the ions assigned
0.10 0.71 — 0.14 0.13 0.13 0.31 1.68 0.41 4.06 31.61 100 2.49 2.90 2.25 5.81 58.44 17.65 127.041 145.051 163.061 180.087 181.073 198.096 289.090 325.112 360.150 0.27 1.05 — 0.22 0.21 0.14 0.44 1.65 0.78 5.34 31.07 100 2.73 3.03 1.82 6.29 53.16 14.55 127.039 145.051 163.061 180.088 181.072 198.098 289.090 325.112 360.151 0.18 0.57 — 0.05 0.15 0.05 0.26 1.14 0.23 3.29 28.77 100 0.45 4.82 0.43 4.74 55.71 2.64 127.039 145.050 163.060 180.086 181.070 198.097 289.088 325.111 360.149 0.19 1.16 — 0.18 0.23 0.21 0.31 1.43 0.35 4.09 32.30 100 3.16 3.12 1.98 5.07 54.53 7.53 127.039 145.050 163.060 180.086 181.071 198.096 289.088 325.111 360.148 C6H6O3H C6H8O4H C6H10O5H C6H10O5NH4 C6H12O6H C6H12O6NH4 C12H16O8H C12H20O10H C12H22O11NH4 127 145 163 180 181 198 289 325 360
127.038 145.049 163.060 180.086 181.071 198.097 289.091 325.113 360.150
Standard deviation Relative intensity Measured mass
Relative intensity
Standard deviation
Measured mass
Relative intensity
Standard deviation
Measured mass
Relative intensity
Standard deviation
Measured mass
Gentiobiose Isomaltose Cellobiose Maltose
Calculated mass Elemental composition m/z
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Table 1. The relative intensities, the elemental compositions and the calculated and the measured masses of the product ions for the electrospray ionization-quadrupole-time-of-flight MS/MS of the ammoniated adducts recorded at 14 eV laboratory frame collision energy
JMS letters
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in Table 2, the relative differences between the CE50 values of the ammoniated adducts are larger than that of the lithiated adducts. In this work, the main goal was to develop a quick method for the differentiation of the disaccharides using the ESI-MS/MS of the lithiated and ammoniated adducts. The base of our method was the ratio of product ions derived from the cleavage of the glycosidic bond of the lithiated adduct ion. The ratio of m/z 187/169 was determined and plotted at different collision energies (Fig. 2). As Fig. 2 shows, the m/z 187/169 ratio versus collision energy curves of maltose, cellobiose, isomaltose and gentiobiose are well separated in the collision energy range of 22–46 eV, and the gaps between the plots are decreasing with increasing collision energy. To have proper product ion abundances, 30 eV was chosen as collision energy for our method. The m/z 187/169 ratios were calculated by ten independent experiments for all the disaccharides. Table 3 compiles the mean values and the standard deviations estimated based on this sample. On the basis of the mean values of the m/z 187/169 ratios, simple criteria were determined for the identification of the disaccharides, as presented in Table 3. The probabilities that the measured m/z 187/169 ratios fall into the criterion intervals (Table 3) were calculated with the t (Student’s t) distribution and are listed in Table 3. To explore the weak points of the differentiation between the four disaccharides, the t-test was used to determine if the m/z 187/169 ratios of the four disaccharides were significantly different. Statistically significant differences were found between the ratios of isomaltose (4.33) and maltose (1.92) (p < 0.001), between the ratios of maltose (1.92) and gentiobiose (1.54) (p < 0.001), as well as between the ratios of gentiobiose (1.54) and cellobiose (0.72) (p < 0.001). In the next step, we studied how the ESI-MS/MS of the ammoniated adducts of the disaccharides can be used for confirming the identification of the isomers. As Fig. 1 and Table 1 show, the MS/MS spectra of the [M + NH4]+ ions are very similar. Nevertheless, the ratio of the abundance of the product ion at m/z 325 to the precursor ion at m/z 360 has proved to be useful in the differentiation between the disaccharide isomers. The ratio of m/z 325/ 360 was determined and plotted at different collision energies (Fig. 2). As seen in Fig. 2, the gap between the m/z 325/360 ratio versus collision energy curves of the disaccharides are increasing with increasing collision energy, except the isomaltose– gentiobiose pair, which run together, and show only a slight difference above 10 eV. To have proper precursor ion (m/z 360) abundances, 13 eV was chosen as collision energy for our method. The basic statistical properties were also determined by ten independent measurements. The mean values and the standard deviations of the m/z 325/360 ratios, together with the identification criteria and the probability of getting a good result with these criteria, are compiled in Table 4.
Table 2. Characteristic collision energies (CE50) of the product ion spectra (electrospray ionization-quadrupole-time-of-flight MS/MS) for the lithiated and ammoniated adducts of the disaccharides Disaccharide
CE50 (eV) + [M + Li]
Standard deviation
CE50 (eV) + [M + NH4]
Standard deviation
Maltose Cellobiose Isomaltose Gentiobiose
27.1 29.3 28.4 30.8
0.17 0.05 0.07 0.05
6.9 5.5 7.7 8.1
0.07 0.14 0.08 0.04
Copyright © 2013 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2013, 48, 1276–1280
JMS letters
a
b
6
35
5
Gentiobiose
Maltose
4
Cellobiose
3 2 1
m/z 325/360 ratio
m/z 187/169 ratio
Isomaltose
Isomaltose
30
Gentiobiose
25
Maltose
20
Cellobiose
15 10 5
0
0 22
32
42
0
Collision energy (eV)
5
10
15
Collision energy (eV)
Figure 2. The abundance ratio of m/z 187/169 (a) and m/z 325/360 (b) in the product ion spectra (electrospray ionization-quadrupole-time-of-flight MS/MS) of the lithiated and ammoniated adducts, respectively, as the function of the collision energy.
+
Table 3. The main statistical properties for the abundance ratio m/z 187/169 of [M + Li] ions calculated by ten experiments and the criteria for distinguishing between the isomers Disaccharide
Mean value of m/z 187/169 ratio
Relative standard deviation
Maltose Cellobiose Isomaltose Gentiobiose
1.92 0.72 4.33 1.54
0.05 0.03 0.06 0.05
Criterion for m/z 187/169 ratio (r) 1.7 < r < 3.0 r < 1.1 3.0 < r 1.1 < r < 1.7
Probability 0.97 1 1 0.96
The ten measurements were not repeated consecutively; the collision voltage was changed between the measurements. The column on the right gives the probability that the measured m/z 187/169 ratio fall into the criterion interval.
+
Table 4. The main statistical properties for the abundance ratio m/z 325/360 of [M + NH4] ions calculated by ten experiments and the criteria for distinguishing between the isomers Disaccharide
Mean value of m/z 325/360 ratio
Relative standard deviation
Maltose Cellobiose Isomaltose Gentiobiose
7.23 21.6 3.65 3.33
0.04 0.09 0.04 0.03
Criterion for m/z 325/360 ratio (r) 5 < r < 12 12 < r 3.45 < r < 5 r < 3.45
Probability 1 1 0.91 0.89
The ten measurements were not repeated consecutively; the collision voltage was changed between the measurements. The column on the right gives the probability that the measured m/z 325/360 ratio fall into the criterion interval.
J. Mass Spectrom. 2013, 48, 1276–1280
automated post-run data processing too, so the control method was extended with a data analysis part, namely a homemade software module developed in Visual Basic Script language, which is appended as supplementary data. The method was tested by a commercial nonalcoholic beer. The test analysis of the beer was performed as follows. The mass spectrometer first scans in MS mode and finds two precursor ions corresponding to the lithiated and the ammoniated disaccharide. After the short MS segment, the spectrometer automatically switches to MS/MS mode and subsequently sets the isolation mass to m/z 349 and 360. The collision voltage for each precursor ion is stored in the method. The post-run processing software module creates two MS/MS spectra with the precursor ions m/z 349 and 360 and identifies maltose (as it was expected in advance) on the basis of the criteria compiled in Tables 3 and 4. The average measurement time of a sample including data acquisition and post processing was less than 1 min. The MS/MS spectra of the lithiated and ammoniated ions of the nonalcoholic beer sample are presented in Fig. S3.
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As seen in Table 4, the identification of maltose is very reliable on the basis of the ammoniated adducts; therefore, it can confirm the distinguishing of gentiobiose and maltose. Statistically significant differences were found between the ratios of gentiobiose (3.33) and maltose (7.23); p < 0.001 was calculated. (This pair can be distinguished with the least probability on the basis of the m/z 187/169 ratio of lithiated adducts, as seen in Table 3.) Table 4 shows that the isomaltose–gentiobiose pair can be distinguished with the least probability on the basis of the m/z 325/360 ratio of ammoniated adducts, but this pair can be clearly differentiated with the criteria of the lithiated adducts. On the basis of the simple criteria presented in Tables 3 and 4, a highly automated data acquisition and processing method were developed for the differentiation of the four examined disaccharides by ESI-MS/MS. We developed a control and evaluation method using the Auto-MS/MS feature of the Bruker acquisition software for the automatic recognition, isolation and fragmentation of the lithiated and ammoniated precursor ions of a disaccharide. The evaluation of the MS/MS data requires an
JMS letters In summary, four glucose disaccharides, such as maltose, cellobiose, isomaltose and gentiobiose were distinguished by ESI-QTOF MS/MS in positive ion mode on the basis of the relative abundance of the lithiated adduct ions at m/z 187 and 169 and the relative abundance of the ammoniated adducts at m/z 325 and 360. Simple criteria were established for the differentiation of the four disaccharides, and a highly automated data acquisition and processing method were developed. Disaccharide samples, where lithiated and ammoniated adducts are also formed in the ESI source, can easily be prepared. Although significant differences were found on the basis of the product ion spectra both of the lithiated and the ammoniated adducts, the two distinguishing criteria confirm each other and eliminate the uncertainties. The criteria and the method were tested with a commercial beer sample. Our work is focused on the determination of the anomeric and isomeric configuration in samples containing only one disaccharide. If tandem mass spectrometry is combined with a separation technique (i.e. liquid chromatography), the qualitative analysis of a mixture containing these disaccharides can be performed on the basis of our simple criteria. The automated data acquisition and processing method can shorten the analysis time. A further improvement may be to extend the criteria for other glucose–glucose disaccharides. Yours, ÁKOS KUKI, KATALIN E. SZABÓ, LAJOS NAGY, MIKLÓS ZSUGA AND SÁNDOR KÉKI* Department of Applied Chemistry, University of Debrecen, H-4032 Debrecen, Egyetem tér 1., Hungary Acknowledgement This work was financially supported by the grants K-101850 given by the OTKA (National Scientific Research Fund, Hungary) and the grants TAMOP-4.2.2/B-10/1-2010-0024 and TÁMOP-4.2.2.A-11/1/ KONV-2012-0036 by the European Union.
References [1] M. E. Taylor, K. Drickamer. Introduction to Glycobiology, Oxford University Press: Oxford, 2006. [2] A. Varki, R. D. Cummings, J. D. Esko, H. H. Freeze, P. Stanley, C. R. Bertozzi, G. W. Hart, M. E. Etzler. Essentials of Glycobiology. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 2009. [3] Ø. M. Andersen, K. R. Markham. Flavonoids Chemistry, Biochemistry and Applications. CRC Press, Taylor & Francis Group: Boca Raton, 2006. [4] S. Keki, G. Deak, M. Zsuga. Fragmentation study of rutin, a naturally occurring flavone glycoside cationized with different alkali metal ions, using post-source decay matrix-assisted laser desorption/ ionization mass spectrometry. J. Mass Spectrom. 2001, 36, 1312.
[5] S. Keki, G. Deak, A. Levai, M. Zsuga. Post-source decay matrix-assisted laser desorption/ionization mass spectrometric study of peracetylated isoflavone glycosides cationized by protonation and with various metal ions. J. Mass Spectrom. 2003, 38, 1207. [6] B. Domon, C. E. Costello. A systematic nomenclature for carbohydrate fragmentations in FAB-MS/MS spectra of glycoconjugates. Glycoconj. J. 1988, 5, 397. [7] L. C. Ngoka, J.-F. Gal, C. B. Lebrilla. Effects of cations and charge types on the metastable decay rates of oligosaccharides. Anal. Chem. 1994, 66, 692. [8] M. A. Fentabil, R. Daneshfar, E. N. Kitova, J. S. Klassen. Blackody infrared radiative dissociation of protonated oligosaccharides. J. Am. Soc. Mass Spectrom. 2011, 22, 2171. [9] G. E. Hofmeister, Z. Zhou, J. A. Leary. Linkage position determination in lithium-cationized disaccharides: tandem mass spectrometry and semiempirical calculations. J. Am. Chem. Soc. 1991, 113, 5964. [10] M. R. Asam, G. L. Glish. Tandem mass spectrometry of alkali cationized polysaccharides in a quadrupole ion trap. J. Am. Soc. Mass Spectrom. 1997, 8, 987. [11] J. Simões, P. Domingues, A. Reis, F. M. Nunes, M. A. Coimbra, M. R. M. Domingues. Identification of anomeric configuration of underivatized reducing glucopyranosyl-glucose disaccharides by tandem mass spectrometry and multivariate analysis. Anal. Chem. 2007, 79, 5896. [12] J. Zaia. Mass spectrometry of oligosaccharides. Mass Spectrom. Rev. 2004, 23, 161. [13] H. Suzuki, A. Kameyama, K. Tachibana, H. Narimatsu, K. Fukui. Computationally and experimentally derived general gules for fragmentation of various glycosyl bonds in sodium adduct oligosaccharides. Anal. Chem. 2009, 81, 1108. [14] B. Mulroney, J. C. Traeger, B. A. Stone. Determination of both linkage position and anomeric configuration in underivatized glucopyranosyl disaccharides by electrospray mass spectrometry. J. Mass Spectrom. 1995, 30, 1277. [15] C. Konda, B. Bendiak, X. Yu. Differentiation of the stereochemistry and anomeric configuration for 1–3 linked disaccharides via tandem mass 18 spectrometry and O-labeling. J. Am. Soc. Mass Spectrom. 2012, 23, 347. [16] S. Mendonca, R. B. Cole, J. Zhu, Y. Cai, A. D. French, G. P. Johnson, R. A. Laine. Incremented alkyl derivates enhance collision induced glycosidic bond cleavage in mass spectrometry of disaccharides. J. Am. Soc. Mass Spectrom. 2003, 24, 63. [17] B. Guan, R. B. Cole. MALDI linear-field reflectron TOF post-source decay analysis of underivatized oligosaccharides: determination of glycosidic linkages and anomeric configurations using anion attachment. J. Am. Soc. Mass Spectrom. 2008, 19, 1119. [18] Y. Jiang, R. B. Cole. Oligosaccharide analysis using anion attachment in negative mode electrospray mass spectrometry. J. Am. Soc. Mass Spectrom. 2005, 16, 60. [19] N. C. Polfer, J. J. Valle, D. T. Moore, J. Oomens, J. R. Eyler, B. Bendiak. Differentiation of isomers by wavelength-tunable infrared multiplephoton dissociation-mass spectrometry: application to glucosecontaining disaccharides. Anal. Chem. 2006, 78, 670. [20] C. S. R. Azenha, M. A. Coimbra, A. S. P. Moreira, P. Domingues, M. R. M. Domingues. Differentiation of isomeric β-(1–4) hexose disaccharides by positive electrospray tandem mass spectrometry. J. Mass Spectrom. 2013, 48, 548.
Supporting information Additional supporting information may be found in the online version of this article at the publisher’s web site.
1280 wileyonlinelibrary.com/journal/jms
Copyright © 2013 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2013, 48, 1276–1280
Revised: 12 September 2013
Accepted: 3 October 2013
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3294
Rapid identification of disaccharides by tandem mass spectrometry Ákos Kuki, Katalin E. Szabó, Lajos Nagy, Miklós Zsuga and Sándor Kéki* Additional supporting information may be found in the online version of this article at the publisher’s web site.
Dear Sir,
1276
Carbohydrates are the important class of biological molecules. The wide variety of positional and anomeric structures makes it possible for saccharides to form plenty of compounds. It is well known that oligosaccharides attached to proteins or lipids (forming glycoconjugates) and provide important biological functions.[1,2] Furthermore, oligosaccharide units can also be found in flavonoid glycosides, and their mass spectrometric behaviors have been investigated in detail.[3–5] The function of the disaccharides is strongly influenced by their structures. Therefore, it is necessary to determine the stereochemistry of the monosaccharide units, the linkage position and the anomeric configuration of the isomeric disaccharides. Tandem mass spectrometry combined with soft ionization methods such electrospray ionization (ESI) and matrix-assisted laser desorption/ionization is an effective tool for the structural analysis of oligosaccharides. Determination of the linkage position and anomeric configuration has been demonstrated both in the negative and positive ion modes. For protonated oligosaccharide cleavage occurs almost exclusively at the nonreducing side of the glycosidic oxygen (referred as B-type and Y-type ions[6]).[7,8] For alkali metal adduct cross-ring fragmentation can also be observed besides the abundant B-type and Y-type ions.[9–11] The effect for the destabilization of the glycosidic bond is decreasing with the size of the cations. Hence, higher energy required to fragment the disaccharides cationized by larger ions.[12,13] In the negative ion mode, the collision-induced dissociation spectra of the deprotonated disaccharides show distinguishable fragmentation patterns with C-type and A-type ions for the different isomeric and anomeric configurations.[14,15] Moreover, derivatization (e.g. permethylation) can be applied to increase the extent of fragmentation,[16] and negative ion adducts are also used for the analysis of disaccharides.[17,18] Glucose disaccharides varying solely in their anomeric configuration were clearly distinguished on the basis of the relative abundance of the characteristic product ions arising from the cleavage of the glycosidic bond of the lithiated adduct ion at mass-to-charge ratio (m/z) 169 and 187 using wavelength-tunable infrared multiple-photon dissociation-mass spectrometry.[19] Very recently, Domingues et al. proposed that the ratio of m/z 169/187 for [M + Li]+ ions and m/z 185/203 for [M + Na]+ ions seems to be a simple criterion for the differentiation of isomeric hexose disaccharides with β-(1 → 4) linkage but distinct monosaccharide composition.[20] In this work, we have examined the use of ESI-MS/MS in positive ion mode of the lithiated, sodiated and ammoniated adducts
J. Mass Spectrom. 2013, 48, 1276–1280
for the differentiation of four glucose disaccharides: maltose (Glcα1-4Glc), cellobiose (Glcβ1-4Glc), isomaltose (Glcα1-6Glc) and gentiobiose (Glcβ1-6Glc). According to our best knowledge, no detailed report on the fragmentation of the ammoniated adducts of the glucose disaccharides has been issued. Our goal was to define simple criteria for distinguishing between the four disaccharides. Herein, we also report a highly automated data acquisition and processing method for the identification of these disaccharides by ESI-MS/MS, thereby considerably reducing the analysis time. The HPLC grade methanol (VWR International, Leuven, Belgium) was used without further purification. Water was purified by a Direct-Q water system (Millipore, Molsheim, France). NaCl, LiCl, NH4Cl and all disaccharides were received from Aldrich (Steinheim, Germany). The disaccharides were dissolved in methanol/water (4/1 V/V) at a concentration of 0.1 mg/mL. To obtain lithiated, sodiated and ammoniated adducts, LiCl, NaCl and NH4Cl were added to the disaccharide solutions to obtain 0.1 mM concentration of the salts. The MS/MS measurements were performed with a MicrOTOF-Q type Qq-TOF MS instrument (Bruker Daltoniks, Bremen, Germany) using an ESI source with positive ion mode. The sample solutions were introduced directly into the ESI source with a syringe pump (Cole-Parmer Ins. Co., Vernon Hills, IL, USA) at a flow rate of 3 μL/min. The spray voltage was set to 4 kV. The temperature of the drying gas (N2) was kept at 180 ° C. For the MS/MS experiments, nitrogen was used as the collision gas, and the laboratory frame collision energies (i.e. the kinetic energy acquired by the ions from the accelerating electric field in front of the collision cell) were varied in the range of 1–48 eV. The pressure in the collision cell was determined to be 8 × 103 mbar. The precursor ions for MS/ MS were selected with an isolation width of 1–4 m/z units. The MS/MS spectra were accumulated and recorded by a digitizer at a sampling rate of 2 GHz. The mass spectra were calibrated externally using the exact masses of clusters [(NaTFA)n + Na]+ generated from the electrosprayed solution of sodium trifluoroacetate (NaTFA). The mass spectra were evaluated with the DATAANALYSIS 3.4 software from Bruker.
* Correspondence to: Sándor Kéki, Department of Applied Chemistry, University of Debrecen, H-4032 Debrecen, Egyetem tér 1., Hungary. E-mail: keki. [email protected]
Copyright © 2013 John Wiley & Sons, Ltd.
JMS letters The efficiency of the fragmentation can be quantitatively described by the survival yield (SY). The SY is defined according to Eqn (1): SY ¼
Ip Ip þ ∑If
(1)
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Figure 1. Product ion spectra (electrospray ionization-MS/MS) of the ammoniated adducts of the four examined disaccharides recorded at 14 eV laboratory frame collision energy. The peaks indicated with asterisk (*) are from an impurity.
to the Y-type glycosidic cleavage are [M + NH4 NH3 C6H10O5]+ at m/z 181 and [M + NH4 C6H10O5]+ at m/z 198. Interestingly, as it can be seen in Table 1, the relative abundance of the protonated B-type ion at m/z 163 is much higher than the ammoniated B-type ion at m/z 180 and the Y-type ions at m/z 198 and 181. In addition to the glycosidic cleavages, loss of ammonia and waters from the ammoniated adduct can be observed: NH3 and H2O (35 Da) at m/z 325, NH3 and 2H2O (53 Da) at m/z 307 (with very low abundance) and NH3 and 3H2O (71 Da) at m/z 289. Two additional product ions are formed by the loss of water from the more abundant B-type ion ([M + NH4 C6H12O6]+) at m/z 145 and 127. Cross-ring cleavages cannot be detected in the ESI-MS/ MS spectra of the ammoniated adducts of the disaccharides. Although the product ion spectra of the [M + NH4]+ ions are very similar, remarkable differences can be observed between the characteristic collision energies (CE50) (Table 2). Interestingly, as seen
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where Ip is the intensity of the precursor ion and ΣIf is the sum of all the fragment ion intensities. In the MS/MS of the sodiated disaccharides the sodium ion, which is one of the main product ions, cannot be detected; therefore, the SY cannot be calculated according to Eqn (1). This ‘incomplete’ SY is denoted by SY′ and still can be interesting for expressing the extent of fragmentation of structurally similar compounds. The collision energy at which the intensity of the precursor ion is equal to the sum of that of all the fragment ions (i.e. SY′ = 0.5) was determined with appropriate accuracy by a linear two-point interpolation. This energy is noted as characteristic collision energy (CE50). For the calculation of the t (Student’s t) distributions and t-tests, the built-in functions of spreadsheet software were used. The MS/MS spectra of the four lithiated disaccharides recorded by our ESI-quadrupole-time-of-flight (QTOF) at 30 eV laboratory frame collision energy are presented in Fig. S1. The dominant process in the ESI-QTOF MS/MS of the sodiated disaccharides was the simple loss of the cation from disaccharide. The dramatic decrease of the precursor ion signal intensity without the parallel increase of the product ion signals by increasing collision energy was observed. (The increase of the sodium ion signal parallel to the decrease of the total ion intensity could not be detected because the mass of sodium ion fell below the lowest mass limit on our mass analyzer.) The MS/MS spectra of the four sodiated disaccharides recorded at 36 eV laboratory frame collision energy are presented in Fig. S2. Although some product ion peaks can be observed in the MS/MS spectra of the [M + Na]+ ions, however because of the loss of the charge, the sodiated adducts cannot be used for the differentiation of the four glucose disaccharides. For instance, the absolute intensity of the characteristic product ion arising from the cleavage of the glycosidic bond of the sodiated maltose adduct ion at m/z 185 is extremely low (below 10) and has a large relative standard deviation (around 30% on the basis of ten measurements). Nevertheless, some characteristic differences can be observed in the fragmentation spectra of the α-anomers and β-anomers of the sodiated disaccharides. The fragmentation is more extent for α-anomers (SY′ = 0.61 for maltose and SY′ = 0.62 for isomaltose at 36 eV collision energy) compared with β-anomers (SY′ = 0.81 for cellobiose and SY ′ = 0.82 for gentiobose at 36 eV collision energy). Furthermore, the relative abundance of the product ion at m/z 203 (derived by the cleavage at the glycosidic bond) is much enhanced for α-anomers (0.51 for maltose and 0.46 for isomaltose) than for β-anomers (0.05 for cellobiose and 0.04 for gentiobose). As a novel approach, we examined the use of the ESI-MS/MS of the ammoniated adducts for the differentiation between the isomeric disaccharides. The MS/MS spectra of the [M + NH4]+ ions of maltose, cellobiose, isomaltose and gentiobiose are presented in Fig. 1. The relative intensities, the elemental compositions and the calculated and the measured masses of the product ions are compiled in Table 1. As seen in Fig. 1 and Table 1, all MS/MS spectra show the same product ion peaks. The product ions originated from the B-type glycosidic cleavage are [M + NH4 NH3 C6H12O6]+ at m/z 163 and [M + NH4 C6H12O6]+ at m/z 180, whereas the ions assigned
0.10 0.71 — 0.14 0.13 0.13 0.31 1.68 0.41 4.06 31.61 100 2.49 2.90 2.25 5.81 58.44 17.65 127.041 145.051 163.061 180.087 181.073 198.096 289.090 325.112 360.150 0.27 1.05 — 0.22 0.21 0.14 0.44 1.65 0.78 5.34 31.07 100 2.73 3.03 1.82 6.29 53.16 14.55 127.039 145.051 163.061 180.088 181.072 198.098 289.090 325.112 360.151 0.18 0.57 — 0.05 0.15 0.05 0.26 1.14 0.23 3.29 28.77 100 0.45 4.82 0.43 4.74 55.71 2.64 127.039 145.050 163.060 180.086 181.070 198.097 289.088 325.111 360.149 0.19 1.16 — 0.18 0.23 0.21 0.31 1.43 0.35 4.09 32.30 100 3.16 3.12 1.98 5.07 54.53 7.53 127.039 145.050 163.060 180.086 181.071 198.096 289.088 325.111 360.148 C6H6O3H C6H8O4H C6H10O5H C6H10O5NH4 C6H12O6H C6H12O6NH4 C12H16O8H C12H20O10H C12H22O11NH4 127 145 163 180 181 198 289 325 360
127.038 145.049 163.060 180.086 181.071 198.097 289.091 325.113 360.150
Standard deviation Relative intensity Measured mass
Relative intensity
Standard deviation
Measured mass
Relative intensity
Standard deviation
Measured mass
Relative intensity
Standard deviation
Measured mass
Gentiobiose Isomaltose Cellobiose Maltose
Calculated mass Elemental composition m/z
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Table 1. The relative intensities, the elemental compositions and the calculated and the measured masses of the product ions for the electrospray ionization-quadrupole-time-of-flight MS/MS of the ammoniated adducts recorded at 14 eV laboratory frame collision energy
JMS letters
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in Table 2, the relative differences between the CE50 values of the ammoniated adducts are larger than that of the lithiated adducts. In this work, the main goal was to develop a quick method for the differentiation of the disaccharides using the ESI-MS/MS of the lithiated and ammoniated adducts. The base of our method was the ratio of product ions derived from the cleavage of the glycosidic bond of the lithiated adduct ion. The ratio of m/z 187/169 was determined and plotted at different collision energies (Fig. 2). As Fig. 2 shows, the m/z 187/169 ratio versus collision energy curves of maltose, cellobiose, isomaltose and gentiobiose are well separated in the collision energy range of 22–46 eV, and the gaps between the plots are decreasing with increasing collision energy. To have proper product ion abundances, 30 eV was chosen as collision energy for our method. The m/z 187/169 ratios were calculated by ten independent experiments for all the disaccharides. Table 3 compiles the mean values and the standard deviations estimated based on this sample. On the basis of the mean values of the m/z 187/169 ratios, simple criteria were determined for the identification of the disaccharides, as presented in Table 3. The probabilities that the measured m/z 187/169 ratios fall into the criterion intervals (Table 3) were calculated with the t (Student’s t) distribution and are listed in Table 3. To explore the weak points of the differentiation between the four disaccharides, the t-test was used to determine if the m/z 187/169 ratios of the four disaccharides were significantly different. Statistically significant differences were found between the ratios of isomaltose (4.33) and maltose (1.92) (p < 0.001), between the ratios of maltose (1.92) and gentiobiose (1.54) (p < 0.001), as well as between the ratios of gentiobiose (1.54) and cellobiose (0.72) (p < 0.001). In the next step, we studied how the ESI-MS/MS of the ammoniated adducts of the disaccharides can be used for confirming the identification of the isomers. As Fig. 1 and Table 1 show, the MS/MS spectra of the [M + NH4]+ ions are very similar. Nevertheless, the ratio of the abundance of the product ion at m/z 325 to the precursor ion at m/z 360 has proved to be useful in the differentiation between the disaccharide isomers. The ratio of m/z 325/ 360 was determined and plotted at different collision energies (Fig. 2). As seen in Fig. 2, the gap between the m/z 325/360 ratio versus collision energy curves of the disaccharides are increasing with increasing collision energy, except the isomaltose– gentiobiose pair, which run together, and show only a slight difference above 10 eV. To have proper precursor ion (m/z 360) abundances, 13 eV was chosen as collision energy for our method. The basic statistical properties were also determined by ten independent measurements. The mean values and the standard deviations of the m/z 325/360 ratios, together with the identification criteria and the probability of getting a good result with these criteria, are compiled in Table 4.
Table 2. Characteristic collision energies (CE50) of the product ion spectra (electrospray ionization-quadrupole-time-of-flight MS/MS) for the lithiated and ammoniated adducts of the disaccharides Disaccharide
CE50 (eV) + [M + Li]
Standard deviation
CE50 (eV) + [M + NH4]
Standard deviation
Maltose Cellobiose Isomaltose Gentiobiose
27.1 29.3 28.4 30.8
0.17 0.05 0.07 0.05
6.9 5.5 7.7 8.1
0.07 0.14 0.08 0.04
Copyright © 2013 John Wiley & Sons, Ltd.
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JMS letters
a
b
6
35
5
Gentiobiose
Maltose
4
Cellobiose
3 2 1
m/z 325/360 ratio
m/z 187/169 ratio
Isomaltose
Isomaltose
30
Gentiobiose
25
Maltose
20
Cellobiose
15 10 5
0
0 22
32
42
0
Collision energy (eV)
5
10
15
Collision energy (eV)
Figure 2. The abundance ratio of m/z 187/169 (a) and m/z 325/360 (b) in the product ion spectra (electrospray ionization-quadrupole-time-of-flight MS/MS) of the lithiated and ammoniated adducts, respectively, as the function of the collision energy.
+
Table 3. The main statistical properties for the abundance ratio m/z 187/169 of [M + Li] ions calculated by ten experiments and the criteria for distinguishing between the isomers Disaccharide
Mean value of m/z 187/169 ratio
Relative standard deviation
Maltose Cellobiose Isomaltose Gentiobiose
1.92 0.72 4.33 1.54
0.05 0.03 0.06 0.05
Criterion for m/z 187/169 ratio (r) 1.7 < r < 3.0 r < 1.1 3.0 < r 1.1 < r < 1.7
Probability 0.97 1 1 0.96
The ten measurements were not repeated consecutively; the collision voltage was changed between the measurements. The column on the right gives the probability that the measured m/z 187/169 ratio fall into the criterion interval.
+
Table 4. The main statistical properties for the abundance ratio m/z 325/360 of [M + NH4] ions calculated by ten experiments and the criteria for distinguishing between the isomers Disaccharide
Mean value of m/z 325/360 ratio
Relative standard deviation
Maltose Cellobiose Isomaltose Gentiobiose
7.23 21.6 3.65 3.33
0.04 0.09 0.04 0.03
Criterion for m/z 325/360 ratio (r) 5 < r < 12 12 < r 3.45 < r < 5 r < 3.45
Probability 1 1 0.91 0.89
The ten measurements were not repeated consecutively; the collision voltage was changed between the measurements. The column on the right gives the probability that the measured m/z 325/360 ratio fall into the criterion interval.
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automated post-run data processing too, so the control method was extended with a data analysis part, namely a homemade software module developed in Visual Basic Script language, which is appended as supplementary data. The method was tested by a commercial nonalcoholic beer. The test analysis of the beer was performed as follows. The mass spectrometer first scans in MS mode and finds two precursor ions corresponding to the lithiated and the ammoniated disaccharide. After the short MS segment, the spectrometer automatically switches to MS/MS mode and subsequently sets the isolation mass to m/z 349 and 360. The collision voltage for each precursor ion is stored in the method. The post-run processing software module creates two MS/MS spectra with the precursor ions m/z 349 and 360 and identifies maltose (as it was expected in advance) on the basis of the criteria compiled in Tables 3 and 4. The average measurement time of a sample including data acquisition and post processing was less than 1 min. The MS/MS spectra of the lithiated and ammoniated ions of the nonalcoholic beer sample are presented in Fig. S3.
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As seen in Table 4, the identification of maltose is very reliable on the basis of the ammoniated adducts; therefore, it can confirm the distinguishing of gentiobiose and maltose. Statistically significant differences were found between the ratios of gentiobiose (3.33) and maltose (7.23); p < 0.001 was calculated. (This pair can be distinguished with the least probability on the basis of the m/z 187/169 ratio of lithiated adducts, as seen in Table 3.) Table 4 shows that the isomaltose–gentiobiose pair can be distinguished with the least probability on the basis of the m/z 325/360 ratio of ammoniated adducts, but this pair can be clearly differentiated with the criteria of the lithiated adducts. On the basis of the simple criteria presented in Tables 3 and 4, a highly automated data acquisition and processing method were developed for the differentiation of the four examined disaccharides by ESI-MS/MS. We developed a control and evaluation method using the Auto-MS/MS feature of the Bruker acquisition software for the automatic recognition, isolation and fragmentation of the lithiated and ammoniated precursor ions of a disaccharide. The evaluation of the MS/MS data requires an
JMS letters In summary, four glucose disaccharides, such as maltose, cellobiose, isomaltose and gentiobiose were distinguished by ESI-QTOF MS/MS in positive ion mode on the basis of the relative abundance of the lithiated adduct ions at m/z 187 and 169 and the relative abundance of the ammoniated adducts at m/z 325 and 360. Simple criteria were established for the differentiation of the four disaccharides, and a highly automated data acquisition and processing method were developed. Disaccharide samples, where lithiated and ammoniated adducts are also formed in the ESI source, can easily be prepared. Although significant differences were found on the basis of the product ion spectra both of the lithiated and the ammoniated adducts, the two distinguishing criteria confirm each other and eliminate the uncertainties. The criteria and the method were tested with a commercial beer sample. Our work is focused on the determination of the anomeric and isomeric configuration in samples containing only one disaccharide. If tandem mass spectrometry is combined with a separation technique (i.e. liquid chromatography), the qualitative analysis of a mixture containing these disaccharides can be performed on the basis of our simple criteria. The automated data acquisition and processing method can shorten the analysis time. A further improvement may be to extend the criteria for other glucose–glucose disaccharides. Yours, ÁKOS KUKI, KATALIN E. SZABÓ, LAJOS NAGY, MIKLÓS ZSUGA AND SÁNDOR KÉKI* Department of Applied Chemistry, University of Debrecen, H-4032 Debrecen, Egyetem tér 1., Hungary Acknowledgement This work was financially supported by the grants K-101850 given by the OTKA (National Scientific Research Fund, Hungary) and the grants TAMOP-4.2.2/B-10/1-2010-0024 and TÁMOP-4.2.2.A-11/1/ KONV-2012-0036 by the European Union.
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Supporting information Additional supporting information may be found in the online version of this article at the publisher’s web site.
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