Sialylated

Feb 15, 2008 - 1,1,3,3-tetramethylguanidium (TMG) salt of α-cyano-4-hydroxycinnamic acid (CHCA) (G2CHCA) was reported by Tatiana et al. as a useful i...
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Anal. Chem. 2008, 80, 2171-2179

Ionic Liquid Matrixes Optimized for MALDI-MS of Sulfated/Sialylated/Neutral Oligosaccharides and Glycopeptides Yuko Fukuyama,*† Shuuichi Nakaya,‡ Yuzo Yamazaki,‡ and Koichi Tanaka†

Koichi Tanaka Mass Spectrometry Research Laboratory and Life Science Research Laboratory, Shimadzu Corporation, 1, Nishinokyo-Kuwabaracho, Nakagyo-ku, Kyoto 604-8511, Japan

1,1,3,3-tetramethylguanidium (TMG) salt of r-cyano-4hydroxycinnamic acid (CHCA) (G2CHCA) was reported by Tatiana et al. as a useful ionic liquid matrix (ILM) for sulfated oligosaccharides to suppress the loss of sulfate groups. However, the report mainly referred to positive ion spectra only and amounts of 10 pmol or more of the analyte were used. Herein, we demonstrated highly sensitive detection of sulfated/sialylated/neutral oligosaccharides and preferential ionization of glycopeptides by optimizing a newly synthesized ILM: TMG salt of pcoumaric acid (G3CA) and the existing G2CHCA in both positive and negative ion extraction modes. Sulfated oligosaccharides were detected with high sensitivity (e.g., 1 fmol) in both ion extraction modes, and the dissociation of sulfate groups was suppressed especially using G3CA. Sialylated and neutral oligosaccharides were also detected with high sensitivity (e.g., 1 fmol) with positive ion extraction while the dissociation of sialic acids was suppressed especially using G3CA. Additionally, glycopeptide ions were detected preferentially using the ILMs among the digest of a glycoprotein, ribonuclease B, in both ion extraction modes but particularly in the negative ion mode. As a result, the use of optimized ILMs provides an effective method for carbohydrate analysis due to the highly sensitive soft-ionization achieved in both ion extraction modes as well as the homogeneity of analyte-matrix mixtures. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)1,2 in addition to electrospray ionization mass spectrometry (ESI-MS)3 have developed into practical analytical tools in proteomics and glycomics as both have higher throughput and sensitivity than previous mass spectrometric techniques in this area. In MALDI-MS, one benefit is the detection of mainly singly charged ions, whereas ions are detected in a multiply charged * To whom correspondence should be addressed. Yuko Fukuyama, phone +81-75-823-1482; fax +81-75-823-3218; e-mail [email protected]. † Koichi Tanaka Mass Spectrometry Research Laboratory, Shimadzu Corporation. ‡ Life Science Research Laboratory, Shimadzu Corporation. (1) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T. Rapid Commun. Mass Spectrom. 1988, 2, 151-153. (2) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (3) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64-71. 10.1021/ac7021986 CCC: $40.75 Published on Web 02/15/2008

© 2008 American Chemical Society

state in ESI-MS. This benefit enables easy interpretation of the mass spectra acquired and thus MALDI-MS finds particular use in mixture analysis. On the other hand, a weak point of MALDI is difficulty in selecting appropriate matrixes and preparation methods for each sample. Therefore development of an analytespecific, polarity independent matrix is still required. In carbohydrate research using MALDI-MS, the analysis of sulfated and sialylated oligosaccharides is problematic because labile sulfate groups and/or sialic acids are frequently dissociated and thus ion species of intact molecules are hardly detected. So far, several improvement methods such as stabilizing by derivatization of acidic parts,4,5 addition of NaCl,5 use of atmospheric pressure (AP)-MALDI6 and high-pressure MALDI,7,8 and developments of “cool” matrixes9,10 have been reported. Meanwhile, in glycopeptide and/or glycoprotein analysis using MALDI-MS, it is necessary to consider the properties of both the peptide and oligosaccharide moieties in the search for an appropriate matrix and sample preparation technique as information regarding the peptide sequence, glycosylation site, and oligosaccharide composition is required.11-14 In this case, MSn is an essential process and therefore it is important to detect glycopeptide ions with high intensity. Additionally, preferential ionization of a specific analyte such as glycopeptides within a mixture is extremely helpful for complex analytes like digests. However, it has been a challenging task. 2,5-Dihydroxybenzoic acid (DHB) is a conventional “cool” matrix in MALDI, and it has been most widely used for carbohydrate analysis. It has also been used in glycopeptide analysis11-14 (4) Juhasz, P.; Costello, C. E. J. Am. Soc. Mass Spectrom. 1992, 3, 785-796. (5) Sekiya, S.; Wada, Y.; Tanaka, K. Anal. Chem. 2005, 77, 4962-4968. (6) Zhang, J.; LaMotte, LT.; Dodds, E. D.; Lebrilla, C. B. Anal. Chem. 2005, 77, 4429-4438. (7) O’Connor, P. B.; Costello, C. E. Rapid Commun. Mass Spectrom. 2001, 15, 1862-1868. (8) O’Connor, P. B.; Mirgorodskaya, E.; Costello, C. E. J. Am. Soc. Mass Spectrom. 2002, 13, 402-407. (9) Pitt, J. J.; Gorman, J. J. Rapid Commun. Mass Spectrom. 1996, 10, 17861788. (10) Papac, D. I.; Wong, A.; Jones, A. J. S. Anal. Chem. 1996, 68, 3215-3223. (11) Fukuyama. Y.; Wada, Y.; Yamazaki, Y.; Ojima, N.; Yamada, M.; Tanaka, K. J. Mass Spectrom. Soc. Jpn. 2004, 52, 328-338. (12) Bykova, N. V.; Rampitsch, C.; Krokhin, O.; Standing, K. G.; Ens, W. Anal. Chem. 2006, 78, 1093-1103. (13) Suzuki, Y.; Suzuki, M.; Nakahara, Y.; Ito, Y.; Ito, E.; Goto, N.; Miseki, K.; Iida, J.; Suzuki, A. Anal. Chem. 2006, 78, 2239-2243. (14) Lattova´, E.; Kapkava´, P.; Krokhin, O.; Perreault, H. Anal. Chem. 2006, 78, 2977-2984.

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Table 1. Carrageenan Oligosaccharides Used in This Study analyte no. 1 2 3 4 a

oligosaccharides (2Na+)

neocarratetraose-41,3-di-O-sulfate neocarrahexaose-41,3,5-tri-O-sulfate (3Na+) neocarrahexaose-24,41,3,5-tetra-sulfate (4Na+) neocarradodecaose-41,3,5,7,9,11-hexa-sulfate (6Na+)

na

FW

2 3 4 6

834.6 1242.9 1345.0 2467.9

n corresponds to the number of repeating units in the oligosaccharides (see Figure 1).

probably because it is applicable to both peptides and oligosaccharides. A weak point of DHB is the formation of inhomogeneous needle-shaped crystals, and therefore the analytes are ionized in only a few small areas on the target called “sweet spot” or “hot spot”. This has made the application of DHB in MALDI-MS very difficult because of long measurement time to find the “sweet spot” and poor reproducibility of the results. This may be one reason for the difficulty in quantitative analysis with MALDI-MS. Recently, one intriguing report demonstrated that MALDI-MS worked as reliably as chromatography in relative quantitative analysis of permethylated oligosaccharides.15 Further experimental results and practical evidence are probably needed to establish the reliability of MALDI-MS for quantitative analysis, and in that case, homogeneity of a laser-irradiated analyte-matrix mixture is essential. Ionic liquid matrices (ILMs) introduced by Armstrong et al.16-19 were reported to have not only the property to make a homogeneous spot surface of an analyte-matrix mixture but also the suitable properties for ionization of analytes. The essential point is that the ILMs consist of a conventional solid MALDI matrix, e.g., R-cyano-4-hydroxycinnamic acid (CHCA), DHB, or sinapinic acid (SA) and an organic base, e.g., tributylamine, pyridine, or 1-methylimidazole,20 which enables a relative state of “liquidity” under vacuum conditions. The constituent solid matrixes probably contribute to the ionization process. Moreover, the homogeneous property of ILMs leads to high-throughput analysis and high reproducibility of the results, and this is considered to be crucial for quantitative analysis. Although several ILMs have been reported for biopolymers and synthetic polymers, there are only a few ILMs for carbohydrate analysis.21-24 In particular, sensitivity of oligosaccharide analysis using ILMs has been restricted to the picomole level22-24 at best, while in peptide analysis, levels of sensitivity as low as femtomole or attomole have been reported.25 When considering further improvements of ILMs as an alternative to conventional solid matrixes, higher sensitivity is required in addition to the existing homogeneity of the spot surface. (15) Wada, Y.; Azadi, P.; Costello, C. E.; Dell, A.; Dwek, R. A.; Geyer, H.; Geyer, R.; Kakehi, K.; Karlsson, N. G.; Kato, K.; Kawasaki, N.; Khoo, K.-H.; Kim, S.; Kondo, A.; Lattova, E.; Mechref, Y.; Miyoshi, E.; Nakamura, K.; Narimatsu, H.; Novotny, M. V.; Packer, N. H.; Perreault, H.; Peter-Katalinic´, J.; Pohlentz, G.; Reinhold, V. N.; Rudd, P. M.; Suzuki, A.; Taniguchi, N. Glycobiology 2007, 17, 411-422. (16) Armstrong, D. W.; He, L.; Liu, Y.-S. Anal. Chem. 1999, 71, 3873-3876. (17) Armstrong, D. W.; Zhang, L.-K.; He, L.; Gross, M. L. Anal. Chem. 2001, 73, 3679-3686. (18) Anderson, J. L.; Ding, J.; Welton, T.; Armstrong, D. W. J. Am. Chem. Soc. 2002, 124, 14247-14254. (19) Carda-Broch, S.; Berthod, A.; Armstrong, D. W. Rapid Commun. Mass Spectrom. 2003, 17, 553-560. (20) Tholey, A.; Heinzle, E. Anal. Bioanal. Chem. 2006, 386, 24-37. (21) Bungert, D.; Bastian, S.; Heckmann-Pohl, D. M.; Giffhorn, F.; Heinzle, E.; Tholey, A. Biotechnol. Lett. 2004, 26, 1025-1030. (22) Mank, M.; Stahl, B.; Boehm, G. Anal. Chem. 2004, 76, 2938-2950.

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A few ILMs for sulfated oligosaccharide analysis have been reported.23,24 Recently, Tatiana et al. reported 1,1,3,3-tetramethylguanidinium (TMG) salts of CHCA (G2CHCA) as a new and the most useful ILM for sulfated oligosaccharide analysis.24 This ILM demonstrated the most effective suppression of dissociation of the sulfate groups. However, its sensitivity was limited to 10 pmol or more amounts of sample, and the authors referred only to positive ion spectra because the data quality was better. In general, sulfated oligosaccharide analyses were reported in both positive and negative ion extraction mode with MALDIMS26-31 and ESI-MS.32-39 Indeed, some of them gave more significant results using negative ion extraction.29-33,35,37,39 Furthermore, it has been noted that significant structural information of some acidic oligosaccharides and glycopeptides can be obtained in the negative ion extraction mode.40-42 These facts suggest an importance of optimization and investigation not only in positive but also using negative ion extraction. On the point of sensitivity, detection of 1-5 pmol amounts of carrageenans (sulfated oligosaccharides) was reported with high sensitivity with ESI-MS analysis in the negative ion extraction mode.39 (23) Laremore, T. N.; Murugesan, S.; Park, T.-J.; Avci, F. Y.; Zagorevski, D. V.; Linhardt, R. J. Anal. Chem. 2006, 78, 1774-1779. (24) Laremore, T. N.; Zhang, F.; Linhardt, R. J. Anal. Chem. 2007, 79, 16041610. (25) Cramer, R.; Corless, S. Proteomics 2005, 5, 360-370. (26) Juhasz, P.; Biemann, K. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 4333-4337. (27) Juhasz, P.; Biemann, K. Carbohydr. Res. 1995, 270, 131-147. (28) Venkataraman, G.; Shriver, Z.; Raman, R.; Sasisekharan, R. Science 1999, 15, 537-542. (29) Ackloo, S.; Terlouw, J. K.; Ruttink, P. J. A.; Burgers, P. C. Rapid Commun. Mass Spectrom. 2001, 15, 1152-1159. (30) Fukuyama, Y.; Ciancia, M.; Nonami, H.; Cerezo, A. S.; Erra-Balsells, R.; Matulewicz, M. C. Carbohydr. Res. 2002, 337, 1553-1562. (31) Barboza, M.; Duschak, V. G.; Fukuyama, Y.; Nonami, H.; Erra-Balsells, R.; Cazzulo, J. J.; Couto, A. S. FEBS J. 2005, 272, 3803-3815. (32) Ekeberg, D.; Knutsen, S. H.; Sletmoen, M. Carbohydr. Res. 2001, 334, 4959. (33) Yu, G.; Guan, H.; Ioanoviciu, A. S.; Sikkander, S. A.; Thanawiroon, C.; Tobacman, J. K.; Toida, T.; Linhardt, R. J. Carbohydr. Res. 2002, 337, 433440. (34) Antonopoulos, A.; Favetta, P.; Helbert, W.; Lafosse, M. Carbohydr. Res. 2004, 339, 1301-1309. (35) Zaia, J.; McClellan, J. E.; Costello, C. E. Anal. Chem. 2001, 73, 6030-6039. (36) Gunay, N. S.; Tadano-Aritomi, K.; Toida, T.; Ishizuka, I.; Linhardt, R. J. Anal. Chem. 2003, 75, 3226-3231. (37) Antonopoulos, A.; Favetta, P.; Helbert, W.; Lafosse, M. Anal. Chem. 2005, 77, 4125-4136. (38) Aguilan, J. T.; Dayrit, F. M.; Zhang, J.; Nin ˜onuevo, M. R.; Lebrilla, C. B. J. Am. Soc. Mass Spectrom. 2006, 17, 96-103. (39) Yu, G.; Zhao, X.; Yang, B.; Ren, S.; Guan, H.; Zhang, Y.; Lawson, A. M.; Chai, W. Anal. Chem. 2006, 78, 8499-8505. (40) Deguchi, K.; Ito, H.; Takegawa, Y.; Nagai, S.; Nakagawa, H.; Nishimura, S.-I. Rapid Commun. Mass Spectrom. 2006, 20, 741-746. (41) Ito, H.; Takegawa, Y.; Deguchi, K.; Nagai, S.; Nakagawa, H.; Shinohara, Y.; Nishimura, S.-I. Rapid Commun. Mass Spectrom. 2006, 20, 3557-3565. (42) Ito, H.; Yamada, K.; Deguchi, K.; Nakagawa, H.; Nishimura, S.-I. Rapid Commun. Mass Spectrom. 2007, 21, 212-218.

Table 2. Sialylated and Neutral Oligosaccharides Used in This Studya

Figure 1. Structure of carrageenan oligosaccharides used in this study. n corresponds to the number of repeating units in the oligosaccharides (see Table 1).

a GlcNAc, 9; galactose, O; glucose, B; mannose, B; fucose, 1 NeuAc, (; PA, aminopyridine.

Figure 2. Structure of matrixes used in this study. The location of the third negative charge on the CA moiety in G3CA has not been clarified (see Results and Discussion).

In this study, we demonstrated the highly sensitive detection of sulfated/sialylated/neutral oligosaccharide molecules and preferential ionization of glycopeptides among digests of a glycoprotein by optimizing a newly synthesized TMG salt of pcoumaric acid (G3CA) and using the existing G2CHCA.24 Optimization of the ILMs was carried out using an appropriate matrix solvent and focusing of an analyte-matrix mixture on a mirrorpolished stainless target. As a result, all carrageenan oligosaccharides were detected with high sensitivity (e.g., 1 fmol) using the ILMs, especially using G3CA, in both positive and negative ion extraction modes. Dissociation of sulfate groups was suppressed particularly when using G3CA. All sialylated and neutral oligosaccharides were detected with high sensitivity (e.g., 1 fmol) using the ILMs in the positive ion extraction mode and some of them were also observed with negative ion extraction. Dissociation of sialic acids was also particularly suppressed when using G3CA. Additionally, glycopeptide ions were detected preferentially in digest mixtures using the ILMs in both ion extraction modes but especially with negative ion extraction. Remarkable reproducibility of ion detection was also observed using the ILMs due to their ability to form homogeneous spot surfaces. The improvements observed when using ILMs such as G3CA provide additional potential in carbohydrate research using MALDI-MS. EXPERIMENTAL SECTION Materials. p-Coumaric acid, purified 2,5-dihydroxybenzoic acid (DHB), and 1,1,3,3-tetramethylguanidine (TMG) were purchased from Sigma-Aldrich Co. (St. Louis, MO). TMG is a flammable hazardous liquid with amine odor, and appropriate safety precautions should be observed. Purified R-cyano-4-hydroxy-cinnamic acid (CHCA) was obtained from LaserBio Labs (Sophia-Antipolis Cedex, France). Neocarrageenan oligosaccharides (Table 1,

Figure 1) including several sulfate groups as sodium salts were obtained from Dextra Laboratories (U.K.) and Sigma-Aldrich Co. PA-labeled and nonlabeled sialylated or neutral oligosaccharides (Table 2) were obtained from Takara Bio Inc., Japan, and Cosmo Bio Co., Japan, respectively. Ribonuclease B (RNase B) was obtained from Sigma-Aldrich Co. All solvents were analytical grade, and Milli-Q water was used. Ionic Liquid Matrixes (ILMs) and a Solid Matrix. Matrixes used in this study (DHB, G2CHCA, and G3CA) are shown in Figure 2. Especially, G3CA was a newly developed ILM. ILMs were prepared according to the method of Tatiana et al.24 CHCA was mixed with TMG at a 1:2 molar ratio in methanol, and p-coumaric acid (CA) was mixed with TMG at a 1:3 (or 1:1) molar ratio in methanol. After evaporation of the methanol in a SpeedVac for 2-3 h, they were left in vacuum overnight and dissolved in methanol again at a concentration of 9 mg/0.1 mL. The solution was further diluted 5 or 10% with methanol to be used as ILMs, G2CHCA and G3CA. The freshly synthesized ILMs were used as soon as possible (at least within a few days). On the other hand, purified DHB was dissolved in 50% aqueous acetonitrile at 10 mg/ mL and used as a conventional solid matrix for comparisons with the ILMs. Analytes and Preparation. Oligosaccharides (Tables 1 and 2) were dissolved in water and diluted using a serial dilution method. The analyte solution was mixed with ILMs at a 1:1 ratio (v/v). An amount of 0.5 µL of the mixture was spotted on a mirrorpolished stainless-steel target and analyzed with a MALDI-QITTOF mass spectrometer in both the positive and negative ion extraction modes. Furthermore, 0.5 µL of DHB solution was mixed with 0.5 µL of analyte solution on the target. From 1 pmol to 1 fmol amounts of the analytes were analyzed to evaluate the sensitivity. RNase B is reported to contain a single oligosaccharide chain, which is linked to 34Asn and consists of two GlcNAc residues and Analytical Chemistry, Vol. 80, No. 6, March 15, 2008

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Figure 3. Stereoscopic microscope photographs of the analytematrix (DHB, G2CHCA, and G3CA) mixtures (A) and the time course of the analyte-ILM (G3CA) mixture (B) on a mirror-polished stainlesssteel target.

five to nine mannose residues.43-45 An amount of 10 mg of dithiothreitol was added in 950 µL of 8 M urea dissolved in TrisHCl (pH 9.0), and then 50 µL of RNase B (200 pmol/ µL in 8 M urea) was added for reduction at 37 °C for 30 min. Following this, the RNase B was carboxyamidomethylated by adding 25 mg of iodoacetamide at 37 °C for 60 min in the dark. The obtained solution was dialyzed in 50 mM ammonium hydrogencarbonate three times at room temperature, and then the RNase B was digested by lysylendopeptidase at 37 °C for 16 h. The resultant digests were mixed with ILMs at a 1:1 ratio (v/v) without any prepurification such as desalting. The mixture was analyzed by mass spectrometry in both the positive and negative ion extraction modes. Mass Spectrometry. MALDI-MS was performed using an AXIMA-QIT (Shimadzu Biotech, Manchester, U.K.) MALDI-QITTOF mass spectrometer equipped with a nitrogen UV laser (337 nm). Argon gas (Ar) was used for CID fragmentation, and helium gas (He) was used for ion cooling. MS and MS/MS were carried out in both the positive and negative ion extraction modes. All analyses were performed in a high vacuum condition of 5 × 10-5 Pa or less. Typically, mass spectra were obtained by accumulation of 100 laser shots for each analysis. External mass calibration was carried out using some general standard peptides. RESULTS AND DISCUSSION Ionic Liquid Matrix. Among several structural analogues of (43) Bernard, B. A.; Newton, S. A.; Olden, K. J. Biol. Chem. 1983, 258, 1219812202. (44) Reid, G. E.; Stephenson, J. L., Jr.; McLuckey, S. A. Anal. Chem. 2002, 74, 577-583. (45) Gotte, G.; Libonati, M.; Laurents, D. V. J. Biol. Chem. 2003, 278, 4624146251.

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Figure 4. Ion intensity distributions of the analyte-matrix surface (810 µm × 810 µm) using DHB and ILMs (G2CHCA and G3CA) for analyte 1 (in Table 1).

CHCA, p-coumaric acid provided the most effective ILM for sulfated oligosaccharide analysis in combination with TMG. Thus the TMG salt of p-coumaric acid was investigated. When either CHCA or CA was mixed with TMG at a molar ratio of 1:n (n ) 1∼5) as shown in Experimental Section, G2CHCA was obtained for n g 2 and G3CA was obtained for n g 3. Tatiana et al. reported that G2CHCA afforded higher sensitivity than any ILM including GCHCA.24 Additionally, the application of the GnCA (n ) 1∼3) for a sulfated oligosaccharide (analyte 1 in Table 1) demonstrated that it showed better results in terms of sensitivity and suppression of dissociation of sulfate groups than others (data not shown). Therefore, G2CHCA and G3CA were mainly used as representatives in this study and discussed below. The matrixes used here (DHB, G2CHCA, and G3CA) are shown in Figure 2. Structures of ILMs, GnCHCA (n ) 1, 2) and GnCA (n ) 1∼3) were confirmed by proton NMR spectroscopy (data not shown). NMR spectra of GCHCA, G2CHCA, GCA, and G2CA demonstrated that the first negative charge was located on carboxyl groups, and the second negative charge was located on hydroxyl groups of CHCA and CA, respectively. Although it was confirmed that the third negative charge was located somewhere on the CA moiety in G3CA, unfortunately its location has not been

Figure 5. Positive and negative ion mass spectra of analyte 2 (100 fmol/well) with DHB (A and D), G2CHCA (B and E), and G3CA (C and F). [Fn]+ ) [M + Na - nSO3Na + nH]+. [Fn]- ) [M - Na - nSO3Na + nH]-. Table 3. Detection Limits of Sulfated Oligosaccharide Molecules (Analytes 1-4 in Table 1) Used in This Studya positive: [M + Na]+

negative: [M - Na]-

analyte no.

DHB

G2CHCA

G3CA

DHB

G2CHCA

G3CA

1 (2S) 2 (3S) 3 (4S) 4 (6S)

1p 1p ND ND

100 f 10 f 100 f 10 f

1f 5f 10 f 1f

100 f 100 f 1p ND

10 f 1f 10 f 1f

1f 1f 1f 1f

a The highest sensitivity (mol/well) is shown for each analysis when 1 pmol-1 fmol/well analytes were analyzed. ND denotes that analyte molecular ions are not detected.

Figure 6. Negative ion mass spectra of analyte 4 (100 fmol/well) with G2CHCA (A) and G3CA (B). [Fn]- ) [M - Na – nSO3Na – nH]-.

clarified yet. The analysis is still under investigation. Nevertheless, it is clear that the G3CA worked effectively in this study as described later.

Synthesized G2CHCA was a highly viscous yellow liquid, and G3CA was a light brown viscous liquid like honey at room temperature. The pH values of G2CHCA and G3CA in water were 9.8 and 11.9, respectively. After spotting the analyte-ILM mixture solution on a target and then evaporating the solvent, a small liquid droplet of analyte-ILM mixture remained on the target (Figure 3A). Visually, the mixture droplet of G2CHCA was a small yellow liquid and that of G3CA was a small light brown liquid on the target. These small droplets were irradiated by UV laser light and analyzed in the mass spectrometer. Optimized Preparation. Some reported photographs of analyte-ILM mixtures showed the droplets spread homogeneously throughout a well of a target.17,20,22 In this research, we tried to make not only a uniform analyte-ILM mixture but also decrease the overall sample droplet diameter for a simple and Analytical Chemistry, Vol. 80, No. 6, March 15, 2008

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Figure 7. Negative ion MS/MS spectrum of analyte 4 (1 fmol/well; [M - Na]-, m/z 2443.4) with G3CA.

sensitive MALDI analysis. For that purpose, the following things were devised; (1) ILM solutions were diluted to 9 g/mL or less concentration. This corresponds to 1/10 or less of what has been reported previously. (2) The analyte-ILM mixture was dissolved in 1:1 methanol/water. Including water in the solution mixture was one of the key points. (3) A mirror-polished stainless-steel target was used as a MALDI target. As a result of 1-3, focusing of the analyte-ILM mixture droplet was achieved on the target (Figure 3B). Right after spotting the mixture on a target, the droplet spread homogeneously across the well and then gradually focused to a smaller diameter during solvent evaporation (Figure 3B). Finally, a small droplet of an analyte-ILM mixture was formed in the well and analyzed with a mass spectrometer. Actually, the sensitivity using the diluted ILM solution (9 g/mL) was more than 10 times higher than that reported using the 90 g/mL (data not shown). Additionally a smaller droplet was formed when using the more dilute ILM solution (data not shown). Furthermore, the inclusion of water in the solvent probably affects surface tension of the analyte-ILM mixture when the focusing is progressing on a mirror-polished target. When a standard (relatively rough) stainless-steel target was used, the mixture did not focus to a small area (data not shown). The small droplets formed by the combination of these factors may lead to an increase in analytical sensitivity using ILMs as shown below. A comparative study of the laser energy (laser fluence) for the ionization of oligosaccharides and glycopeptides when using ILMs and DHB, showed that it was approximately 25% lower for G2CHCA and almost the same for G3CA when compared to DHB. This shows that the ionization efficiency of the ILMs is higher than or equal to conventional matrixes. Additionally, mass resolution (fwhm) of the ion peaks for the ILMs was about 6000-8000 which was same for DHB. Homogeneity. ILMs have been reported to form homogeneous analyte-matrix mixtures. Considering that solid matrixes usually tend to form inhomogeneous analyte-matrix crystals, the property of ILMs seems to be an attractive benefit for simple and 2176 Analytical Chemistry, Vol. 80, No. 6, March 15, 2008

effective operation with MALDI, as the homogeneity of the ILMs is maintained under vacuum conditions due to their physical characteristic. Here, we compared ion intensity distributions on analyte-matrix surfaces of ILMs (G2CHCA and G3CA) and DHB for [M - Na]- of analyte 1 in Table 1 using an incorporated automatic analytical function. The results shown in Figure 4 illustrate the difference between the matrixes. The ion [M - Na]- was uniformly detected across the sample spot when using the ILMs whereas it was detected in only a few small areas called “sweet-spots” using DHB (Figure 4). G2CHCA, in particular, gave near-perfect uniform distribution (Figure 4). It was noted that all analyses using the ILMs were carried out easily and rapidly when compared to DHB. A fact demonstrates that a homogeneous matrix distribution across the sample spot is beneficial when using MALDI-MS. Sulfated Oligosaccharide Analysis. Four commercial carrageenan oligosaccharide sodium salts (Table 1 and Figure 1) were evaluated using a conventional solid matrix (DHB) and optimized ILMs, G2CHCA and G3CA. Investigation was carried out to assess the extent of dissociation of sulfate groups and the detection sensitivity of intact molecular ions in both positive and negative ion extraction modes. Results indicate that the loss of sulfate groups for all four sulfated oligosaccharides was suppressed when using ILMs regardless of the extraction polarity. Figure 5 shows the mass spectra obtained for analyte 2 in Table 1 as a representative example. The intact molecules were confirmed as [M + Na]+ and [M - Na]-. The fragmentation occurred mainly by dissociation of the sulfate groups according a loss of 102 Da from [M + Na]+ and/or [M - Na]- to yield [Fn]+ ) [M + Na - nSO3Na + nH]+ and/or [Fn]- ) [M - Na - nSO3Na + nH]-, respectively. The degree of the fragmentation was less in the positive ion mode than with negative ion extraction (Figure 5). Suppression of dissociation of the sulfate groups using the ILMs was observed more clearly in negative ion mode than with positive ion extraction (Figure 5D-F). In particular, G3CA was more effective in sup-

Figure 8. Positive and negative ion mass spectra of analyte 5 (100 fmol/well) with DHB (A and D), G2CHCA (B and E), and G3CA (C and F). Table 4. Detection Limits of Sialylated and Neutral Oligosaccharide Molecules (Analytes 5-10 in Table 2) Used in This Studya positive: [M + Na]+

negative: [M - Na]-

analyte no.

DHB

G2CHCA

G3CA

DHB

G2CHCA

G3CA

5 6 7 8 9 10

1p ND 100 f 100 f 10 f 10 f

10 f 100 f 10 f 10 f 1f 1f

10 f 100 f 10 f 10 f 10 f 1f

50 f 1p 100 f ND 10 f 10 f

1f 100 f 25 f ND 100 f 100 f

1f 100 f 10 f ND 1f 10 f

a The highest sensitivity (mol/well) is shown for each analysis when 1 pmol-1 fmol/well analytes were analyzed. ND denotes that analyte molecular ions are not detected.

pressing the fragmentation than G2CHCA (Figure 5E,F) for all four sulfated oligosaccharides in Table 1. The more sulfate groups the analyte contained such as analytes 3 and 4, the greater is the difference of the suppression effect between G2CHCA and G3CA (Table 1, Figure 6). An important point is that the ILMs, especially G3CA, allowed detection of intact sulfated oligosaccharide ions not only in the positive ion mode but also the negative ion mode in which the dissociation of sulfate groups was suppressed. These results may constitute the basis for the development of the analysis of this category of oligosaccharides in negative ion extraction mode using ILMs.

Table 3 shows detection limits of the four carrageenan oligosaccharides in Table 1 using the ILMs and DHB when a 1 pmol to 1 fmol amount of analyte was analyzed. This demonstrates that all the analytes were detected with higher sensitivity when using the ILMs, especially G3CA, in both the positive and negative ion extraction modes than when using DHB. On the whole, the sensitivity was higher with negative than with positive ion extraction. Even with as little as 1 fmol of analyte, ions were detected with enough intensity to perform MS/MS successfully (Figure 7, Domon and Costello nomenclature was referred to46). The results so far can be summarized as follows. In sulfated oligosaccharide analysis, optimized ILMs, G2CHCA and G3CA, were shown to suppress the dissociation of the sulfate groups and exhibited detection with high sensitivity with both ion extraction polarities. They allowed femtomole level analysis for sulfated oligosaccharides. Furthermore, these effects were increased when using the G3CA rather than the G2CHCA. It is thought that the high basicity of these ILMs (pH values were 11.9 for G3CA and 9.8 for G2CHCA in the water phase as described previously) may relate to the effective analysis of acidic oligosaccharides in the negative ion extraction mode, even though the relationship between the pH (or pK) value of a matrix and the ionization process remains unclear. On the other hand, the ILMs tend to (46) Domon, B.; Costello, C. E. Glycoconjugate J. 1998, 5, 397-409.

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Figure 9. Positive and negative ion mass spectra of RNase B digests (approximately 1 pmol/well) with DHB (A and E), G2CHCA (B and F), G3CA (C and G), and GCA (D and H). The peaks indicated with arrows are derived from glycopeptide ions.

include more sodium ions compared to solid matrixes. This property is useful for stabilizing the sulfate groups and yielding [M + Na]+ species in the positive ion mode. In addition, the long lifetimes (arising from the low internal energy) of [M + Na]+ and [M - Na]- using the ILMs may lead to the suppression effect regardless of the polarity of the ion modes. Basically, keeping the internal energy of the ions low, stabilizing the unstable parts, and ionizing them with high intensity are needed to suppress the fragmentation in MS analysis. The fragmentation observed here using the ILMs is probably due to the incomplete stabilization of sulfate groups. Sialylated/Neutral Oligosaccharide Analysis. The optimized ILMs (G2CHCA and G3CA) were also applied to sialylated and neutral oligosaccharide analyses. Table 2 shows the oligosaccharides used here. In sialylated oligosaccharide analysis using 2178

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MALDI-MS, dissociation of sialic acids is observed and thus intact molecular ions are difficult to detect. Consequently, the softionization and highly sensitive detection properties of the ILMs in sulfated oligosaccharide analysis were expected to improve the issue with sialylated oligosaccharides. Figure 8 shows a comparison of the suppression effect on dissociation of sialic acids using the ILMs and DHB. For analyte 5 containing two sialic acids in Table 2, the dissociation of the sialic acid moieties was suppressed using the ILMs when compared with DHB in both the positive and negative ion extraction modes (Figure 8). Although the fragmentation was not completely eliminated, ions of the intact molecule such as [M + 3Na - 2H]+ and [M + Na - 2H]- were detected using the ILMs but not with DHB. The same result was obtained for analyte 6 which is a PA labeled-sialylated oligosaccharide (data not shown).

Comparison of the positive ion spectra (Figure 8A-C) and the negative ion spectra (Figure 8D-F) shows that the intact molecules were detected with higher intensity with negative than with positive ion extraction. As for the suppression of the dissociation of the sialic acid, G3CA in the negative ion extraction mode was proved to be the most effective. Detection limits of sialylated and neutral oligosaccharides using three matrixes were compared when a 1 pmol to 1 fmol amount of analyte was analyzed (Table 4). The result shows that the ILMs had higher sensitivity than DHB, except for the analysis of fucosylated oligosaccharides in the negative ion extraction mode (analytes 9 and 10 in Table 4). Nevertheless, femtomole level analysis of all sialylated and neutral oligosaccharides in Table 2 was achieved using optimized ILMs. Even for 1 fmol amounts of analyte, MS/MS was successfully performed (data not shown). The fact that these ILMs were useful for sialylated and neutral oligosaccharides in addition to sulfated ones suggests their applicability to any type of oligosaccharide analyses. In particular, G3CA was demonstrated as an effective matrix in almost all areas of carbohydrate research. Glycopeptide Analysis. In addition to the oligosaccharide analysis, optimized ILMs were applied to glycopeptide analysis. RNase B digests analyzed here constitute a mixture containing several peptides and glycopeptides (see Experimental Section). Previously, in the analysis of these digests, it has been difficult to find glycopeptide ion peaks in the mass spectrum because of the inherent complexity arising from the many ion peaks and preferential ionization of peptide molecules. Here we investigated applicability of the ILMs (G2CHCA and G3CA) and DHB to the ionization of glycopeptides among the RNase B digests in both positive and negative ion extraction modes. The results are described in Figure 9. The glycopeptide ions were detected as a series of five ion peaks of [M + H]+ or [M H]- at an interval of 162 Da (peaks in Figure 9 with arrows). These peaks have been identified as the amino acid sequence SRNLK modified with high mannose type N-glycans.11 This data also correlates with the fact that RNase B is known to possess a single oligosaccharide chain consisting of two GlcNAc residues and five to nine mannose residues.43-45 These results demonstrate that glycopeptides were detected with both positive and negative ion extractions using ILMs (Figure 9B,C,F,G) whereas they were detected only with positive ion extraction using DHB (Figure 9A,E). In the positive ion extraction mode using an identical amount of analyte, the glycopeptides were detected with higher intensity using ILMs (Figure 9B,C) than DHB (Figure 9A). The best relative ionization of the glycopeptides to the peptides was however observed in negative extraction mode using ILMs (Figure 9F,G). On this basis, it may be possible to confirm the presence or absence of glycopeptides in a mixture by comparing the negative ion mass spectra obtained using the ILMs and DHB. Interestingly, the most successful ionization of glycopeptides in

a mixture using either extraction polarities was demonstrated using GCA, synthesized in a similar manner to G3CA (see Experimental Section) (Figure 9D,H). It is thought that GCA may have higher affinity with the glycopeptides than other matrixes and that the pH value of GCA (7.6 in water) may also play a role in the result. Further rational interpretation will be necessary. On the other hand, we confirmed that glycopeptides among the sialylated glycoprotein digests were ionized especially using G3CA (data not shown). In that case, most of them were detected without sialic acids and a few acidic glycopeptides were detected using the ILM. The optimization for the acidic glycoprotein analysis using the ILM is progressing now as a next subject. CONCLUSION Newly synthesized G3CA and optimization of the ILMs (G3CA and G2CHCA) method improved sensitivity and suppressed the degree of dissociation of sulfate groups and sialic acids in carbohydrate analysis with MALDI. Results presented here show the detection of femtomole amounts of analyte can be achieved when using the optimized ILMs. Furthermore, the ions generated from femtomole amounts of analyte were detected with enough signal intensity for MS/MS and MS3 (data not shown) analysis. It is foreseen, therefore, that the ILMs will be applied to MSn analysis which is the essential process for the structural determination of carbohydrates and post-translational modification analysis. Through these experiments a new ILM, G3CA (or GCA for glycopeptides) was demonstrated to be especially effective among the ILMs tested. The interesting thing is the fact that the solid constituent, p-coumaric acid, of G3CA did not work as effectively as a matrix compound. This means that the properties of ILMs do not always reflect that of the solid constituent matrix. The exact reasons for the efficient works of G3CA as a matrix are not clear; however a more systematic study of these ILMs may provide a solution. The optimized preparation method for ILMs is relatively straightforward and could easily be adopted in any analytical laboratory. The 3-D graphs of analyte-ILM mixture clearly proved their homogeneity, which is thought to be quite useful for automated and quantitative analysis using MALDI-MS. Suppression of dissociation for labile sulfate groups and/or sialic acids using ILMs was significant but not complete and therefore further study is necessary to address this. For the analysis of glycopeptides, further research is also needed using ILMs. Our long-term goal is the development of a rapid and highly sensitive analysis method which is useful for any analyte and can easily be applied. ILM research may open a new insight into this difficult but attractive goal.

Received for review December 20, 2007.

October

25,

2007.

Accepted

AC7021986

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