Structural Characterization of Pyridylaminated Oligosaccharides

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Structural Characterization of Pyridylaminated Oligosaccharides Derived from Neutral Glycosphingolipids by HighSensitivity Capillary Electrophoresis-Mass Spectrometry Emi Ito, Kazuki Nakajima, Hiroaki Waki, Kozo Miseki, Takashi Shimada, TakaAki Sato, Kazuaki Kakehi, Minoru Suzuki, Naoyuki Taniguchi, and Akemi Suzuki Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac401460f • Publication Date (Web): 26 Jul 2013 Downloaded from http://pubs.acs.org on July 27, 2013

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Analytical Chemistry

Structural Characterization of Pyridylaminated Oligosaccharides Derived

from

Neutral

Glycosphingolipids

by

High-Sensitivity

Capillary Electrophoresis-Mass Spectrometry Emi Ito1, Kazuki Nakajima1, Hiroaki Waki2, Kozo Miseki2, Takashi Shimada3, Taka-Aki Sato3, Kazuaki Kakehi4, Minoru Suzuki3, Naoyuki Taniguchi1, and Akemi Suzuki5＊

1

Systems Glycobiology Research Group, RIKEN-Max Planck Joint Research Center, Global Research Cluster, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan

2

Analytical Division, Shimadzu Corporation, 1 Nishinokyo-Ku, Kuwabaracho, Nakagyo-ku, Kyoto 604-8511, Japan

3

Life Science Research Center, Shimadzu Corporation, 5-1-1 Tsukiji, Chuo-ku, Tokyo 105-0045, Japan

4

School of Pharmacy, Kinki University, 3-4-1 Kowakae, Higashi-Osaka 577-8502, Japan

5

Institute of Glycoscience, Tokai University, 4-1-1 Kitakaname, Hiratsuka, Kanagawa, 259-1292, Japan

Running Title: Sensitive structural characterization of PA-oligosaccharides by CE-MS

Key Words: Capillary

electrophoresis-mass

spectrometry,

Pyridylaminated

oligosaccharides,

Head-column field-amplified sample stacking, sheathless interface, high-speed MS, MS2 switching 1

ACS Paragon Plus Environment


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Abbreviations used: CE-MS,

capillary

electrophoresis-mass

spectrometry;

HC-FASS,

head-column

field-amplified sample stacking; ESI-QIT-TOF, electrospray ionization quadrupole ion trap time-of-flight; GSLs, glycosphingolipids; PA, pyridylaminated; Lac, lactose; Gb3, globotriose; Gb4, globotetraose; IV3αGalNAc-Gb4, Forssman antigen.

*Editorial correspondence should be addressed to: Akemi Suzuki, PhD. Institute of Glycoscience, Tokai University. 4-1-1 Kita-kaname, Hiratsuka Kanagawa 259-1292, JAPAN. Tel: +81-463-58-1211 Ext 4643 Fax: +81-463-50-2531 e-mail: [email protected]

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ABSTRACT:

High-sensitivity

capillary

electrophoresis-electrospray

ionization

quadrupole ion trap time-of-flight mass spectrometry (CE-ESI-QIT-TOF MS) was developed

to

structurally characterize four kinds

of

pyridylaminated

(PA)

oligosaccharides, i.e., Lac-PA, Gb3-PA, Gb4-PA, and Forssman antigen-PA, derived from neutral glycosphingolipids. The CE-MS system included the head-column field-amplified sample stacking (HC-FASS) method for effective sample injection into a capillary column in CE, a sheathless interface between CE and a mass spectrometer, and MS and tandem MS (MS2) measurements with narrow mass range repeated high-speed switching. The total sensitivity of the developed CE-MS system was about 20,000 times higher than that of the conventional CE-MS system consisting of pressure injection, a sheath-flow interface, and a wide mass range measurement. The MS and MS2 spectra of the four PA-oligosaccharides at a concentration of 25 amol/µL in mixtures (each 250 amol/10 µL in a tube) clearly showed protonated molecular ions ([M+H]+) and the fragment ions responsible for the sequential elimination of saccharides. The developed CE-MS system is a powerful method for the structural characterization of glycosphingolipids extracted from very small amounts of biological materials and could be extended to the structural characterization of oligosaccharides derived from glycoproteins.

3



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Recent studies have demonstrated that glycosphingolipids (GSLs) play critical roles in many cellular functions such as cell–cell recognition, cell growth, and signal transduction.1–5 Carbohydrate chains function as ligands that are recognized by carbohydrate-binding proteins. Changes in their expression are related to pathological consequences of diseases.6–8 To elucidate the functions of GSLs, the development of a sensitive analytical system is a prerequisite for the characterization of carbohydrate structures of GSLs prepared from very small amounts of tissues, cells, and various types of membranes. Structural characterizations of oligosaccharides derived from GSLs have been accomplished by high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE), after enzymatic or chemical release of oligosaccharides from glycoconjugates followed by fluorescence derivatization.9–11 Pyridylaminated (PA) derivatization is widely used for oligosaccharide analysis because PA-oligosaccharides are very stable and can be separated by chromatography.12,13 The structures of PA-oligosaccharides have been characterized by multidimensional mapping by combining several chromatographic modes,14 and they have been also characterized by hyphenated

techniques

coupled

to

electrospray

ionization-mass

spectrometry

(ESI-MS).15 Many of these studies have been performed at 1–10 pmol concentrations, and no reports have described high-sensitivity analysis of extremely small amounts of oligosaccharides derived from GSLs. Capillary electrophoresis coupled with electrospray ionization mass spectrometry (CE-ESI-MS) is a useful and promising tool for the structural characterization and quantitative determination of fluorescence-labeled oligosaccharides.16–18 In that system, two types of interfaces exist between CE and ESI-MS, i.e., sheath-flow and sheathless.19–34 The sheath-flow interface provides a stable spray for ESI, but its

4


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disadvantage is low sensitivity due to dilution of the analytes by the sheath liquid prior to ionization. First, we compared the sensitivity of sheath-flow and sheathless interfaces of CE-ESI-MS with pressure injection of Gb4-PA. Second, we compared the scanning mass range (i.e., wide or narrow) to examine its effect on MS sensitivity by CE-ESI-MS with a sheathless interface and electrokinetic injection of Gb4-PA by head-column field-amplified sample stacking (HC-FASS). With wide mass range measurement, ions were scanned at m/z 200–2,000 and with narrow mass range measurement, ions were scanned from the protonated molecular ions to their isotope ions (3.5 amu). In each case, scanning was performed with repeated MS and MS2 switching. Based on the above results, we compared the developed CE-MS system consisting of a sheathless interface, narrow mass range measurement, and electrokinetic sample injection with the conventional CE-MS system having a sheath-flow interface, wide mass range measurement, and pressure sample injection. We report here that four kinds of PA-oligosaccharides (i.e., Lac-PA, Gb3-PA, Gb4-PA, and Forssman antigen-PA) were successfully characterized in a mixture at a concentration of 25 amol/µL (each 250 amol/10 µL in a tube) by the developed CE-ESI-MS method with MS and MS2 switching.

■ EXPERIMENTAL SECTION

Materials. The PA-oligosaccharides (Lac-PA, Gb3-PA, Gb4-PA, and Forssman antigen-PA) used in this study were purchased from Takara Bio Inc. (Kyoto, Japan), and formic acid, ammonium formate, and acetonitrile were purchased from Wako Pure Chemicals Industries (Osaka, Japan). All solvents used for the MS analysis were of HPLC grade. Mass Spectrometry. All experiments were performed with a LCMS-IT-TOF mass 5



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spectrometer controlled by LabSolutions LCMS Solution software (Shimadzu Corp., Kyoto, Japan). All mass spectrometric measurements were done in the positive ion mode and the detection voltage was set to 2.0 kV. To keep mass precision, the temperature inside of the instrument was controlled at 40 ± 0.3°C. For a wide mass range measurement, the mass range spanned the ions from m/z 200 to m/z 2000. The narrow mass range was set at 3.5 amu to include the protonated molecular ions ([M+H]+) and their isotope ions. The mass range of each PA-oligosaccharide was as follows: m/z 420.5–424.0 for Lac-PA, m/z 582.5–586.0 for Gb3-PA, m/z 785.0–788.5 for Gb4-PA, and m/z 988.5–992.0 for Forssman antigen-PA. For MSn switching, the event times of MS and MS2 were set to 100 ms and 150 ms, respectively. In this method, a set of eight kinds of positive ion MS and MS2 mass spectra of the four PA-oligosaccharides (Lac-PA, Gb3-PA, Gb4-PA, and Forssman antigen-PA) were recorded at approximately 1 s per cycle. Instrument of Capillary Electrophoresis-Mass Spectrometry (CE-MS). CE-MS analysis was performed by an electrospray ionization quadrupole ion trap time-of-flight (ESI-QIT-TOF) mass spectrometer equipped with a CE (P/ACE MDQ system, Beckman-Coulter, Fullerton, CA) and an uncoated fused-silica capillary (50 µm i.d. × 95 cm, GL Sciences Inc., Tokyo, Japan). Sample solutions of 10 µL were placed in microvials (PCR-02-NC, Axygen Scientific, Union City, CA). A voltage of 30 kV was applied to the uncoated fused-silica capillary at 25°C and 10 psi to obtain stable spray. CE-MS with a Sheath-Flow Interface. An ESI probe modified with a sheath-flow interface (Shimadzu Corp.) was used. An uncoated fused-silica capillary was inserted into the ESI-metal sprayer of the ESI-probe, and the location of the capillary tip was optimized. The spray voltage was 2.5 kV. The running buffer and sheath liquid, which were introduced by a syringe pump (Harvard Apparatus, Holliston, MA) at a flow rate of 4 µL/min, were 20% acetonitrile in 5 mM ammonium formate, pH 2.7. The nitrogen nebulizer gas was introduced at 1 L/min. 6


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CE-MS with a Sheathless Interface. A sheathless interface (PicoClearTM, New Objective, Inc., Woburn, MA) was used to join the uncoated fused-silica capillary to a nanospray emitter (standard coated silica tip, 30 µm tip i.d., New Objective, Inc.). The fused-silica capillary and emitter were carefully inserted into the PicoClear union to reduce the dead volume between the two ends. The running buffer of CE was 20% acetonitrile in 5 mM ammonium formate, pH 2.7. High voltage for the electrophoresis was applied in the same way as routine analysis by the P/ACE MDQ system. Sample Injection of CE-MS with the Pressure Injection Method and the Head-Column Field-Amplified Sample Stacking Injection Method (HC-FASS). For the pressure injection method, Gb4-PA was dissolved in water and injected at 0.5 psi for 60 s. The HC-FASS analytical conditions were modified according to Weng et al.35 Briefly, a short zone of acetonitrile was formed in the inlet of the capillary by the pressure method (0.5 psi, 10 s). A sample dissolved in acetonitrile/water (7:3, v/v) was then introduced to the capillary by the electrokinetic method (10 kV, 120 s). The running buffer for both methods was 20% acetonitrile in 5 mM ammonium formate, pH 2.7. Analytical Conditions for a Neutral PA-Oligosaccharide Mixture by the Developed CE-MS System. CE-MS analysis was performed with an uncoated fused-silica capillary (20 µm i.d. × 95 cm) and a nanospray emitter (10 µm tip i.d.). A short zone of acetonitrile was formed at 1 psi for 10 s and a sample was introduced at 30 kV for 120 s. Electrophoresis was performed at +30 kV at 3.5 psi to obtain stable spray. The CE running buffer was 20% acetonitrile in 5 mM ammonium formate, pH 2.7.

■ RESULTS AND DISCUSSION

Comparison of the Sheathless Interface and Sheath-Flow Interface for CE-MS. 7



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Table 1 lists the structures of the PA-oligosaccharides used in this study, i.e., Lac-PA, Gb3-PA, Gb4-PA, and Forssman antigen-PA. First, we compared the sensitivity of the sheath-flow and sheathless interfaces of CE-ESI-MS with pressure injection of Gb4-PA and wide mass range measurement. Figure 1A shows the MS mass spectrum obtained for the injection of 250 fmol/µL of Gb4-PA with the sheathless interface and wide mass range measurement. A protonated molecular ion ([M+H]+) of Gb4-PA at m/z 786.3 was clearly observed. Figure 1B shows the selected ion electropherogram for this sample: a peak selected as the protonated molecular ion of Gb4-PA was clearly observed at a migration time of about 3 min. Figure 1C and 1D show the MS mass spectrum and the selected ion electropherogram monitoring protonated molecular ions at m/z 786.3, which were obtained by the injection of a 2 pmol/µL solution of Gb4-PA with the sheath-flow interface and wide mass range measurement. The intensity of the peak at m/z 786.3 in the selected ion electropherogram obtained from the injection of 250 fmol/µL Gb4-PA in the sheathless CE-MS is comparable to that obtained by sheath-flow injection of 2 pmol/µL of Gb4-PA, as shown in Figure 1B and 1D, respectively. These results indicate that an approximately 8-fold sensitivity enhancement is achieved by using the sheathless interface. Issaq et al. reported that the sheathless interface is the optimal design for coupling CE online with ESI-MS because of the compatible flow rates of CE and ESI-MS. This leads to high ionization efficiency and correspondingly high detection sensitivity.22 Kelly et al. compared sheath-flow interfaces with sheathless interfaces,19 and their and our results indicate that sheathless interfaces produced an order of magnitude higher sensitivity than commercial sheath-flow interfaces. Therefore, we selected the sheathless interface to develop the sensitive CE-MS system described below. However, it should be noted that recent work from Dovichi’s group has demonstrated quite high sensitivity with a nano-flow sheath flow interface.36-38 That interface appears to produce very little dilution of the sample, and those results appear to be comparable to the best 8


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sheathless interface results. Comparison between the Narrow and Wide Mass Range Measurements of CE-MS with a Sheathless Interface Using the HC-FASS Method. We compared the peak intensities of the protonated molecular ion ([M+H]+) at m/z 786.3 of Gb4-PA obtained by the narrow and wide mass range measurements. Figure 2A and 2B show the MS mass spectrum and the selected ion electropherogram obtained from the injection of 25 amol/µL solution of Gb4-PA with the repeated narrow mass range measurement for m/z 785.0–788.5. Figure 2C and 2D show the spectrum and electropherogram obtained from the injection of 500 amol/µL solution of Gb4-PA with the repeated wide mass range measurement for m/z 200–2000. In these selected ion electropherograms, the m/z 786.3 peak intensities were almost the same. Therefore, we concluded that the sensitivity of the repeated narrow mass range measurement was about 20 times higher than that with the repeated wide mass range measurement. The theoretical plate counts for CE-MS shown in Figure 1 and 2 were 2,520, which is very low and this is due to the pressure application for electrospray ionization. Comparison between the Developed CE-MS System and the Conventional CE-MS System. We compared the sensitivity of the developed CE-MS system consisting of HC-FASS sample injection, sheathless interface, and narrow mass range measurement with the conventional CE-MS system consisting of pressure sample injection, sheath-flow interface, and wide mass range measurement. The analytical conditions were the same as noted above. Figure 3A and 3B show the MS mass spectrum and selected ion electropherogram obtained with the developed CE-MS system for the injection of 100 amol/µL of Gb4-PA. Figure 2C and 2D show the MS mass spectrum and selected ion electropherogram obtained by the conventional CE-MS system for the injection of 2 pmol/µL of Gb4-PA. The protonated molecular ion, [M+H]+, was detected at m/z 786.3 in the mass spectra and the selected ion electropherogram. As shown in Figure 3B and 3C, the signal-to-noise ratios (S/N) and 9



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intensities of the peaks at m/z 786.3 were almost the same in these selected ion electropherograms, indicating that the developed CE-MS system was about 20,000 times more sensitive than the conventional CE-MS system. The 20,000-fold sensitivity increase was achieved by the summation of the effects of the sheathless interface (8-fold), narrow mass range measurement (20-fold), and HC-FASS sample injection (125-fold). In CE, sample solutions are generally introduced with the pressure method into a capillary, but only nanoliter levels of sample solutions can be introduced for actual analysis.39 Various approaches of online sample concentration have been developed,40-49 and head-column field-amplified sample stacking (HC-FASS) has been shown to provide the highest sensitivity enhancement among electrokinetic concentration techniques.46-49 Analysis of a Neutral PA-Oligosaccharide Mixture by the Developed CE-MS System with MSn Switching. We applied the developed CE-MS system to the analysis of a PA-oligosaccharide mixture composed of Lac-PA, Gb3-PA, Gb4-PA, and Forssman antigen-PA. A group of eight MS and MS2 positive ion mass spectra was obtained in about 1 s. The narrow mass range of the MS measurement was set as described above. The analytical conditions were modified to improve the separation of the mixture components; briefly, the inside diameters of the uncoated fused-silica capillary and the nanospray emitter were decreased and electrophoresis was performed at 3.5 psi. Consequently, the migration time of each PA-oligosaccharide was increased. Figure 4A to 4D show the MS2 spectra obtained for the injection of a mixture of 25 amol/µL of each of Lac-PA, Gb3-PA, Gb4-PA, and Forssman antigen-PA (each 250 amol/10 µL in a tube), selecting as the precursor ions those at m/z 421.2, 583.2, 786.3, and 989.4, respectively. Each MS2 mass spectrum provided significant fragment ions based on the successive elimination of saccharide or saccharides, i.e., m/z 259.1 as [MH–Gal]+ for Lac-PA (Figure 4A); m/z 421.2 as [MH–Gal]+ and 259.1 as [MH–Gal-Gal]+ for Gb3-PA (Figure 4B); m/z 583.2 as [MH – GalNAc]+, 421.2 as [MH – GalNAc-Gal]+, and 259.1 10


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as [MH – GalNAc-Gal-Gal]+ for Gb4-PA (Figure 4C); and m/z 786.3 as [MH – GalNAc]+,

583.2

as

[MH

–

GalNAc-GalNAc]+,

and

421.2

as

[MH

–

GalNAc-GalNAc-Gal]+ for Forssman-PA (Figure 4D). The selected ion electropherograms shown in Figure 5 monitored the molecular protonated ion, [M+H]+, in MS. The fragment ions of each PA-oligosaccharide detected in MS2 mass spectra are shown in Figure 4A–D. The developed CE-MS system separated and structurally characterized the four PA-oligosaccharides at a concentration of 25 amol/µL within 8 min. We are working on establishing a sample preparation method which includes two major steps. One is endoglycoceramidase treatment to liberate oligosaccharides from GSLs, and the other is to remove excess reagents used for pyridylamination. For the former process, we applied the method reported by Ito and Yamagata50 and Ishibashi et al.,51 and it was confirmed to work nicely. We are now testing for the latter process which is performed by preparative-CE with a wide boar capillary (0.32mm x 2m). Oligosaccharide-PA derivatives are separately eluted from a large excess of the reagents in preparation. Glycomic profiling or mapping is used for the analysis of oligosaccharides.52-55 It is very useful for the comparative analysis of major components. However, “omic” methods are not suitable for the analysis of oligosaccharides as minor components. Our method might be useful for such targeted oligosaccharide analysis.

■ CONCLUSIONS

We have described a highly sensitive system for the structural characterization of a neutral PA-oligosaccharide mixture derived from neutral GSLs. The system used CE-ESI-QIT TOF MS with sample injection by HC-FASS, a sheathless interface, and a narrow mass range measurement. The sensitivity of the developed CE-MS system was 11



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20,000 times greater than that with the conventional CE-MS system. With our system, the four PA-oligosaccharides, Lac-PA, Gb3-PA, Gb4-PA, and Forssman antigen-PA, were clearly separated and characterized by their MS and MS2 spectra within 8 min. The detection limit of the mixture was 25 amol/µL. The developed CE-MS system is useful for the structural characterization of targeted PA-oligosaccharides and could be applied to analyze glycosphingolipids prepared from small amounts of tissues, isolated cells, and membrane fragments including microdomains.

■ ACKNOWLEDGMENT

This work was supported by a MEXT grant for Supporting Research Centers in Private Universities and by a Grant-in-Aid for Scientific Research (A).

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■ REFERENCES (1) Karlsson, K. A. Annu. Rev. Biochem. 1989, 58, 309–350. (2) Zhao, J. F. K.; Fukumoto, S.; Okada, M.; Furugen, R.; Miyazaki, H.; Takamiya, K.; Aizawa, S.; Shiku, H.; Matsuyama, T.; Furukawa, K. J. Biol. Chem. 1999, 274, 13744–13747. (3) Yu, R. K.; Tsai, Y. T.; Ariga, T. Neurochem Res. 2012, 37, 1230-1244. (4) Bucior, I.; Burger, M. M. Curr. Opin. Struct. Biol. 2004, 14, 631–637. (5) Podbielska, M.; Krotkiewski, H.; Hogan, E. L. Neurochem. Res. 2012, 37, 1154–1169. (6) Nakagawa, T.; Takeishi, S.; Kameyama, A.; Yagi, H.; Yoshioka, T.; Moriwaki, K.; Masuda, T.; Matsumoto, H.; Kato, K.; Narimatsu, H.; Taniguchi, N.; Miyoshi, E. J. Proteome Res. 2010, 9, 4888–4896. (7) Gao, C.; Maeno, T.; Ota, F.; Ueno, M.; Korekane, H.; Takamatsu, S.; Shirato, K.; Matsumoto, A.; Kobayashi, S.; Yoshida, K.; Kitazume, S.; Ohtsubo, K.; Betsuyaku, T.; Taniguchi, N. J. Biol. Chem. 2012, 287,16699–16708. (8) Mechref, Y.; Hu, Y.; Garcia, A.; Hussein, A. Electrophoresis 2012, 33, 1755–1767. (9) Anumula, K. R. Anal. Biochem. 2006, 350, 1–23. (10) Ma, S.; Lau, W.; Keck, R. G.; Briggs, J. B.; Jones, A. J.; Moorhouse, K.; Nashaben, W. Methods Mol. Biol. 2005, 308, 397–409. (11) Suzuki, M.; Yamakawa, T.; Suzuki, A. J. Biochem. 1991, 109, 503–506. (12) Hase, S.; Ikenaka, T.; Matsushima, Y. Biochem. Biophys. Res. Commun. 1978, 85, 257–263. (13) Hase, S. Methods Mol. Biol. 1993, 14, 69–80. 13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 25

(14) Takahashi, N.; Nakagawa, H.; Fujikawa, K.; kawamura, Y.; Tomiya, N. Anal. Biochem. 1995, 226, 139–146. (15) Takegawa, Y.; Deguchi, K.; Ito, S.; Nakagawa, H.; Nishimura, S. Anal. Chem. 2005, 77, 2097–2106. (16) Gennaro, L. A.; Delaney, J.; Vouros, P.; Harvey, D. J.; Domon, B. Rapid Commun. Mass Spectrom. 2002, 16, 192–200. (17) Kamoda, S.; Kakehi, K. Electrophoresis 2006, 27, 2495–2504. (18) Zamfir, A.; Peter-Katalinić, J. Electrophoresis 2004, 25, 1949–1963. (19) Kelly, J. F.; Ramaley, L.; Thibault, P.; Anal. Chem. 1997, 69, 51–60. (20) Zamfir, A. D.; Dinca, N.; Sisu, E.; Peter-Katalinic, J. J. Sep. Sci. 2006, 29, 414–422. (21) Shi, L. H.; Jin, Y. X.; Moon, D. C.; Kim, S. K.; Park, S. R. Electrophoresis 2009, 30, 1661–1669. (22) Issaq, H. J.; Janini, G. M.; Chan, K. C.; Veenstra, T. D. J. Chromatogr. A 2004, 1053, 37–42. (23) Wahl, J. H.; Gale, D. C.; Smith, R. D. J. Chromatogr. A 1994, 659, 217–222. (24) Fang, L.; Zhang, R.; Williams, E. R.; Zare, R. N. Anal. Chem. 1994, 66, 3696–3701. (25) Petersson, M. A.; Hulthe, G.; Fogelqvist, E. J. Chromatogr. A 1999, 854, 141–154. (26) Settlage, R. E.; Russo, P. S.; Shabanowitz, J.; Hunt, D. F. J. Microcolumn Sep. 1998, 10, 281–285. (27) Moini, M.; Demars, S. M.; Huang, H. Anal. Chem. 2002, 74, 3772–3776. 14


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(28) Janini, G. M.; Conrads, T. P.; Wilkens, K. L.; Issaq, H. J.; Veenstra, T. D. Anal. Chem. 2003, 76, 1615–1619. (29) Janini, G. M.; Zhou, M.; Yu, L. R.; Blonder, J.; Gignac, M.; Conrads, T. P.; Issaq, H. J.; Veenstra, T. D. Anal. Chem. 2003, 75, 5984–5993. (30) Cao, P.; Moini, M. J. Am. Soc. Mass Spectrom. 1997, 8, 561–564. (31) Faserl, K.; Sarg, B.; Kremser, L.; Lindner, H. Anal. Chem. 2011, 83, 7297–7305. (32) Ramautar, R.; Busnel, J. M.; Deelder, A. M.; Mayboroda, O. A. Anal. Chem. 2012, 84, 885–892. (33) Campa, C.; Coslovi, A.; Flamigni, A.; Rossi, M. Electrophoresis 2006, 27, 2027–2050. (34) Zamfir, A. D.; Seidler, D. G.; Schonherr, E.; Kresse, H.; Peter-Katalinic, J. Electrophoresis 2004, 25, 2010–2016. (35) Weng, Q.; Xu, G.; Yuan, K.; Tang, P. J. Chromatogr. B 2006, 835, 55–61. (36) Wojcik, R.; Li, Y.; MacCoss, M.; Dovichi, N. J. Talanta. 2012, 88, 324-329. (37) Wojcik, R.; Dada, O. O.; Sadilek, M.; Dovichi, N. J. Rapid Commun. Mass Spectrom. 2010, 24, 2554-2560. (38) Sun, L.; Li, Y.; Champion M. M.; Zhu, G.; Wojcik, R.; Dovichi, N. J. Analyst. 2013, 138, 3181-3188. (39) Zamfir, A. D. J. Chromatogr. A 2007, 1159, 2–13. (40) Yang, Y.; Boysen, R. I.; Hearn, M. T. Anal. Chem. 2006, 78, 4752–4758. (41) Shihabi, Z. K. J. Chromatogr. A 2005, 1066, 205–210.

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Page 16 of 25

(42) Dawod, M.; Breadmore, M. C.; Guijt, R. M.; Haddad, P. R. J. Chromatogr. A 2010, 1217, 386–393. (43) Chien, R. L.; Burgi, D. S. Anal. Chem. 1992, 64, 489A–496A. (44) Quirino, J.P.; Terabe, S. Anal. Chem. 2000, 72, 1023–1030. (45) Breadmore, M. C.; Thabano, J. R.; Dawod, M.; Kazarian, A. A.; Quirino, J. P.; Guijt, R. M. Electrophoresis 2009, 30, 230–248. (46) Chien, R. L.; Burgi, D. S. J. Chromatogr. 1991, 559, 141–152. (47) Chien, R. L.; Burgi, D. S. J. Chromatogr. 1991, 559, 153–161. (48) Zhang, C. X.; Thormann, W. Anal. Chem. 1996, 68, 2523–2532. (49) Zhang, C. X.; Thormann, W. Anal. Chem. 1998, 70, 540–548. (50) Ito, M.; Yamagata, T. J. Biol. Chem. 1989, 264, 9510-9519. (51) Ishibashi, Y.; Kobayashi, U.; Hijikata, A.; Sakaguchi, K.; Goda, HM.; Tamura, T.; Okino, N.; Ito, M. J. Lipid Res. 2012, 53, 2242-2251 (52) Jmeian, Y.; Hammad, L. A.; Mechref, Y. Anal. Chem. 2012, 84, 8790–8796. (53) Canis, K.; Mckinnon, T. A.; Nowak, A.; Haslam, S. M.; Panico, M.; Morris, H. R.; Laffan, M. A.; Dell, A. Biochem. J. 2012, 447, 217–228. (54) Hu, Y.; Mechref, Y. Electrophoresis 2012, 33, 1768–1777. (55) Tharmalingam, T.; Adamczyk, B.; Doherty, M. A.; Royle, L.; Rudd, P. M. Glycoconj. J. 2013, 30, 137-146.

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Table 1. Structures of CDH-PA, CTH-PA, Globoside-PA, and Forssman antigen-PA

Lactosyl-PA, Lac-PA

:

Galβ1-4Glc-PA

Globotriosyl-PA, Gb3-P

:

Galα1-4Galβ1-4Glc-PA

Globotetraosyl-PA, Gb4-PA

:

GalNAcβ1-3Galα1-4Galβ1-4Glc-PA

Forssman antigen-PA

: GalNAcα1-3GalNAcβ1-3Galα1-4Galβ1-4Glc-PA

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FIGURE LEGENDS Figure 1. Comparison of CE-MS interfaces: sheathless and sheath-flow. Sheathless CE-mass spectrum (A) and selected ion electropherogram (B) obtained for the injection of 250 fmol/µL of Gb4-PA, and sheath-flow CE-mass spectrum (C) and selected ion electropherogram (D) obtained for the injection of 2 pmol/µL of Gb4-PA. Acetonitrile (20%) in 5 mM ammonium formate (pH 2.7) was used as the running buffer with an uncoated fused-silica capillary (50 µm i.d. × 95 cm) and nanospray emitter (30 µm tip i.d.). CE was done at 10 psi at a voltage of 30 kV. Gb4-PA was injected with pressure injection at 0.5 psi for 60 s. All data were obtained in the positive ion mode for m/z 200–2000.

Figure 2. Comparison of sheathless CE-MS with narrow mass range measurement to wide mass range measurement using the HC-FASS method. Narrow mass range measurement (m/z 786–789.5) CE-mass spectrum (A) and selected ion electropherogram (B) obtained for the injection of 25 amol/µL of Gb4-PA, and wide mass range measurement (m/z 200–2,000) CE-mass spectrum (C) and selected ion electropherogram (D) obtained for the injection of 500 amol/µL of Gb4-PA. Gb4-PA was injected with electrokinetic injection at 10 kV for 120 s after preinjection of acetonitrile at 0.5 psi for 10 s.

Figure 3. Comparison of the developed CE-MS system and the conventional CE-MS system. CE-mass spectrum (A) and CE-selected ion electropherogram (B) of Gb4-PA by the developed CE-MS system consisting of HC-FASS injection, sheathless interface, and narrow mass range measurement, obtained for the injection of 100 amol/µL of Gb4-PA. CE-mass spectrum (C) and CE-selected ion electropherogram (D) of Gb4-PA by the conventional CE-MS system consisting of pressure injection, sheath-flow interface, and 18


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wide mass range measurement, obtained for the injection of 2 pmol/µL of Gb4-PA.

Figure 4. Analysis of a mixture of four kinds of neutral PA-oligosaccharides by the developed CE-MS system with repeated MS and MS/MS (MS2) switching. CE-MS2 spectra of Lac-PA (A), Gb3-PA (B), Gb4-PA (C), and Forssman antigen-PA (D), selecting the ions at m/z 421.2, 583.2, 786.3, and 989.4 as the respective precursor ions. A fused silica capillary (20 µm i.d. x 95 cm length) and nanospray emitter (10 µm tip i.d.) were used. Each of 25 amol/µL of the four kinds of PA-oligosaccharides (each 250 amol/10µL in a tube) was injected by the electrokinetic method at 30 kV for 120 s after preinjection of acetonitrile at 1 psi for 10 s. Separation was at 3.5 psi at 30 kV.

Figure 5. MS and MS2 selected ion electropherograms of a mixture of four kinds of neutral PA-oligosaccharides by the developed CE-MS system with repeated MS and MS2 switching. MS and MS2 selected ion electropherograms of Lac-PA (A), Gb3-PA (B), Gb4-PA (C), and Forssman antigen-PA (D) selecting the protonated molecular ions at m/z 421.2 for Lac-PA, m/z 583.2 for Gb3-PA, m/z 786.3 for Gb4-PA, and m/z 989.4 for Forssman antigen-PA as the respective precursor ions.

19


Figure 1 Page 20 of 25


B

A Relative intensity(%)

780

Detector response

m/z 786.3

100

800

m/z

m/z 786.3

0 200

400

600

800

1000

1200

1400

1600

1800

2000

2.0

m/z

Migration time (min) 4.0

D

C

780

Detector response

m/z 786.3

100

Relative intensity(%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Ito et al.

800

m/z

m/z 786.3

0 200

400

600

800

1000

1200

m/z

1400 ACS

1600 1800 Environment 2000 Paragon Plus

2.0

Migration time (min)

4.0

Page 21 of 25

A

B

m/z 786.3

Detector response


100

m/z 786.3

0

700 800 m/z

2.0


4.0

D

C

780

Detector response

m/z 786.3

100


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Figure 2 Ito et al.


800

m/z

m/z 786.3

0 200

400

600

800

1000

1200

m/z

ACS 1400

Paragon Plus 1600 1800 Environment 2000

2.0


4.0



A

B

m/z 786.3

Detector response


100

m/z 786.3

0

700 800 m/z

2.0

4.0


D

C

780

Detector response

m/z 786.3

100


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Ito et al.

800

m/z

m/z 786.3

0 200

400

600

800

1000

1200

m/z

1400 ACS

1600 1800 Environment 2000 Paragon Plus

2.0


4.0

Page 23 of 25

259.1

100 Relative intensity(%)

A

Hex

0 200

400

500

600

700

800

900

1000

700

800

900

1000

800

900

1000

m/z

259.1

100

421.2


B

300

Hex

Hex

0 200

300

400

500

600

m/z

259.1

100

421.2 583.2


C

HexNAc

Hex

Hex

0 200

300

400

500

600

700

m/z

D

583.2

421.2

100 Relative intensity(%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Figure 4 Ito et al.


786.3 HexNAc

HexNAc

Hex

0 200

300

400

500

600

700

ACS Paragon Plus Environment m/z

800

900

1000



A m/z 421.2 m/z 259.1

B

m/z 583.2 m/z 421.2 m/z 259.1

Detector response

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Ito et al.

C

m/z 786.3 m/z 583.2 m/z 421.2 m/z 259.1

D

m/z 989.4 m/z 786.3 m/z 583.2 m/z 421.2 5

6

7

8

9

10

ACS Paragon Plus Environment Migration time (min)

Page 25 of 25

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+

CE

HC-FASS

-

MS

sheathless interface

narrow mass range, high speed switching Developed CE-MS with 100 amol/µL of Gb4-PA Conventional CE-MS with 2 pmol/µL of Gb4-PA


Structural Characterization of Pyridylaminated Oligosaccharides

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