Detection of Oligosaccharides Labeled with Cyanine Dyes Using

Jun 26, 2004 - Hisashi Narimatsu,‡ and Yasuro Shinohara†. Department of Research and Development, Amersham Biosciences K.K., 3-25-1 Hyakunincho,...
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Anal. Chem. 2004, 76, 4537-4542

Detection of Oligosaccharides Labeled with Cyanine Dyes Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Akihiko Kameyama,*,† Yuko Kaneda,† Hidenori Yamanaka,† Hiroshi Yoshimine,† Hisashi Narimatsu,‡ and Yasuro Shinohara†

Department of Research and Development, Amersham Biosciences K.K., 3-25-1 Hyakunincho, Shinjuku-ku Tokyo 169-0073, Japan, and Glycogene Function Team, Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Open Space Laboratory C-2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan

The sensitivity of oligosaccharides in mass spectrometry lags far behind that of peptides. This is a critical factor in realizing the high-throughput analysis of posttranslational modifications in proteomics. We here described that hydrazide derivatives of cyanine dyes (Cy3, Cy5) with a positive charge made excellent labeling reagents for the detection of oligosaccharides by matrix-assisted laser desorption/ionization mass spectrometry. Cy3-labeled standard N-glycan could be detected at 200 amol on the MALDI target plate in reflectron mode without any purification procedures after the labeling reaction, which may meet the level of sensitivity required in proteome research. Despite the general recognition that the production of signals of oligosaccharides under MALDI conditions would be highly dependent on the matrix, most of the known N-glycans from chicken ovalbumin could be detected upon Cye derivatization nearly independent of the kind of matrix tested (e.g., nor-harman, 2,5-dihydroxybenzoic acid and r-cyano-4-hydroxycinnamic acid) without spoiling the signal strength. Postsource decay afforded simple spectra mainly consisting of Y-type fragment ions, thus simplifying the sequence analysis. Insource decay afforded a similar fragmentation pattern only when acidic matrixes were used. In addition, this derivatization technique was successfully applied to the profiling of N-glycans of gel-separated glycoproteins. Characterization of the proteome, a key activity in the postgenomic era, is made extremely challenging by the microheterogeneity introduced by posttranslational modifications such as glycosylation. Glycoprotein oligosaccharides play a vital role in biological processes such as stability, protein conformation, intraand intercell signaling, and binding affinity to and specificity for other biomolecules.1,2 The analysis of constituent glycans has not reached the same degree of sophistication as that of proteins, * To whom correspondence should be addressed: (phone) +81-29-861-3198; (fax) +81-29-861-3201; (e-mail) [email protected]. † Amersham Biosciences K.K. ‡ National Institute of Advanced Industrial Science and Technology. (1) Roseman, S. J. Biol. Chem. 2001, 276, 41527-41542. (2) Dwek, R. A.; Butters, T. D. Chem. Rev. 2002, 102, 283-284. 10.1021/ac049897z CCC: $27.50 Published on Web 06/26/2004

© 2004 American Chemical Society

because the analysis of carbohydrates is much more difficult due to the problems imposed by structural complexities such as branching, linkage, and heterogeneity on a microscale. Additionally, carbohydrates in their native state are poorly detected in chromatography and mass spectrometry in general. Although matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has proven to have several advantages over other techniques for the analysis of carbohydrates due to its simplicity, speed of analysis, and comparatively increased sensitivity,3,4 the relatively low response to oligosaccharides (compared, for instance, to peptides) still limits the utility of MALDI-MS for the glycosylation analysis in large-scale proteomics. Oligosaccharides lack basic sites to which a proton could attach itself, with the result that carbohydrates become ionized by the addition of metal ions, usually Na+, with low efficiency. Derivatization has frequently been used in an attempt to redress these problems. Permethylation is one of the most important derivatizations because of the higher sensitivity on MS and abundant structural information afforded from the fragment ions.4 For analysis of sample quantities below a few micrograms, however, permethylation may be difficult to apply due to sample losses.5 Another class of derivatization is “tagging” of reducing ends of oligosaccharides. To increase sensitivity, the carbohydrate may be derivatized with a reagent that already contains a charged functional group or with one easily protonated. Many existing labeling reagents, most of which had originally been intended to introduce a fluorophore or chromophore for optical detection, have been tested for detection by MALDI-TOF.6,7 Labeling reagents that improve ionization have also been introduced based on the assumption that a higher sensitivity could be achieved if a basic ionizable group is incorporated into the derivatives. This includes the incorporation of a quaternary ammonium center by reacting sugars with Girard’s reagent T,8 trimethyl(p-aminophenyl) am(3) Harvey, D. J. Mass Spectrom. Rev. 1999, 18, 349-451. (4) Dell, A.; Morris, H. R. Science 2001, 291, 2351-2356. (5) Zaia, J. Mass Spectrom. Rev. 2004, 23, 161-227. (6) Harvey, D. J. J. Am. Soc. Mass Spectrom. 2000, 11, 900-915. (7) Okamoto, M.; Takahashi, K.; Doi, T.; Takimoto, Y. Anal. Chem. 1997, 69, 2919-2926. (8) Naven, T. J. P.; Harvey, D. J. Rapid Commun. Mass Spectrom. 1996, 10, 829-834.

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Figure 1. Structures of the cyanine hydrazides for derivatization of oligosaccharides for MALDI-TOF MS.

monium chloride,9 and benzylamine/NaBH3CN followed by N,Ndimethylation.10 The approach actually improved the sensitivity 10-20-fold, and thus, quarternized derivatives provide one extraordinarily intense signal, M+, at a high overall yield, simplifying the interpretation of the mass spectrum. Greater hydrophobicity is also considered important to increase the signal intensity, because a more hydrophobic analyte may be preferentially incorporated into the crystal structure of the hydrophobic matrix than a hydrophilic one.11 Ligating hydrophobic properties onto carbohydrate may also improve the signal intensity of oligosaccharides in MALDI-MS analysis. In our attempt to develop more feasible labeling reagents for the analysis of oligosaccharides by MALDI-MS, cyanine (Cy) reagents (Figure 1) came to be of great interest because they carry both a constitutive positive charge and hydrophobic features. Cy reagents have also been shown to be extremely useful as fluorescent labels for biological compounds.12-15 In this article, we examine the performance of the derivatization by cyanine dyes for the analysis of oligosaccharides by MALDITOF MS. To clarify the improved performance quantitatively, the detection limit of Cy-labeled oligosaccharides was compared with those derivatized by Girard’s T reagent and 2-aminopyridine (PA).7,16 Furthermore, matrix-dependent ionization and fragmentation by postsource decay (PSD) and in-source decay (ISD) were elucidated. Finally, exploring the feasibility of two-dimensional gel electrophoresis-based glycoproteomics using these dyes, we attempted to take the molecular mass profile of glycans of standard glycoproteins from a gel plug of SDS-PAGE. EXPERIMENTAL SECTION Materials and Reagents. A complex-type N-linked oligosaccharide (Galβ1-4GlcNAcβ1-2ManR1-6(Galβ1-4GlcNAcβ12ManR1-3)Manβ1-4GlcNAcβ1-4GlcNAc, NA2) and N-linked oligosaccharide library of ovalbumin were purchased from Oxford (9) Okamoto, M.; Takahashi, K.; Doi, T. Rapid Commun. Mass Spectrom. 1995, 9, 641-643. (10) Broberg, S.; Broberg, A.; Duus, J. O. Rapid Commun. Mass Spectrom. 2000, 14, 1801-1805. (11) Kratzer, R.; Eckerskorn, C.; Karas, M.; Lottspeich, F. Electrophoresis 1998, 19, 1910-1919. (12) Mujumdar, R. B.; Ernst, L. A.; Mujumdar, S. R.; Lewis, C. J.; Waggoner, A. S. Bioconjugate Chem. 1993, 4, 105-111. (13) Southwick, P. L.; Ernst, L. A.; Tauriello, E. W.; Parker, S. R.; Mujumdar, R. B.; Mujumdar, S. R.; Clever, H. A.; Waggoner, A. S. Cytometry 1990, 11, 418-430. (14) Jackson, P.; Smith, K. A.; Briggs, M. Analysis of carbohydrates. International Patent Application WO98/15829, 1998. (15) Yan, J. X.; Devenish, A. T.; Wait, R.; Stone, T.; Lewis, S.; Fowler, S. Proteomics 2002, 2, 1682-1698. (16) Hase, S.; Koyama, S.; Daiyasu, H.; Takemoto, H.; Hara, S.; Kobayashi, Y.; Kyogoku, Y.; Ikenaka, T. J. Biochem. 1986, 100, 1-10.

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GlycoSystems (Abingdon, U.K.). A pentaoligosaccharide (lactoN-fucopentaose I) was obtained from Calbiochem-Novabiochem (San Diego, CA). PA-derivatized NA2 was purchased from Seikagaku Corp. (Tokyo, Japan). The hydrazide derivatives of cyanine dyes (Cy3-hydrazide and Cy5-hydrazide)14 were prepared by TFA treatment from Boc-protected hydrazide derivatives which were provided by Amersham Biosciences (Amersham, U.K.). Girard’s T reagent was purchased from Aldrich Chemical (Milwaukee, WI). Ovalbumin (grade VI), asialofetuin (type II) from fetal calf serum, and the MALDI matrix, nor-harman, were obtained from Sigma Chemical (St. Louis, MO). 2,5-Dihydroxybenzoic acid (2,5-DHB) and R-cyano-4-hydroxycinnamic acid (HCCA) were purchased from Bruker Daltonics (Billerica, MA). Human transferrin was obtained from Biogenesis (Poole, U.K.). PNGaseF (EC 3.5.1.52) was purchased from Prozyme (San Leandro, CA). Arthrobacter ureafaciens neuraminidase (EC 3.2.1.18) was purchased from Marukin Bio (Kyoto, Japan). Derivatization of Oligosaccharides with Labeling Reagents. The derivatization reaction was modified from the procedure for hydrazone formation described previously.17 For standard derivatization, 10 µL of 50 µM hydrazide in 30% ethanol and 1 µL of 10 µM oligosaccharide were added to 10 µL of 30 mM ammonium bicarbonate in 30% ethanol, and then the mixture was incubated at 90 °C for 1 h. After the reaction tube had cooled on ice, 0.5 µL of the resulting mixture was subjected to MALDITOF Mass analysis without any purification. MALDI-TOF Mass Spectrometry. Mass measurements were carried out using a Reflex IV TOF mass spectrometer equipped with a pulsed ion extraction system (Bruker-Daltonik GmbH, Bremen, Germany). PSD experiments were performed using an Ettan MALDI-ToF/Pro mass spectrometer (Amersham Biosciences, Buckinghamshire, U.K.) with harmonic reflectron. Ions were generated by a pulsed 337-nm nitrogen laser and were accelerated to 20 kV. All the spectra were obtained using a reflectron mode with delayed extraction of 200 ns and were the result of signal averaging of 200 laser shots. For sample preparation, 2,5-DHB, HCCA, and nor-harman were used as the matrixes. A 0.5-µL volume of the matrix solution (10 g/L in 30% acetonitrile) was deposited on the stainless steel target plate and allowed to dry. Then, 0.5 µL of appropriately diluted analyte solution was used to cover the matrix on the target plate and allowed to dry. Finally, 0.5 µL of matrix solution was added onto the deposited sample/matrix mixture on the target plate and allowed to dry. Preparation of N-Glycans from Standard Glycoprotein. Ingel releases of N-glycans from the standard glycoproteins were performed essentially as reported procedure before.18 Briefly, glycoprotein samples (5-100 pmol) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and negatively stained19 with a Negative Gel Stain MS Kit (Wako Pure Chemical Industries, Osaka, Japan). Visualized protein bands were excised as plugs 1 mm in diameter from the slab gel, washed, destained, reduced, and S-alkylated with iodoacetamide. A gel plug was dehydrated in acetonitrile, dried, and rehydrated with 4 µL (17) Shinohara, Y.; Sota, H.; Kim, F.; Shimizu, M.; Gotoh, M.; Tosu, M.; Hasegawa, Y. J. Biochem. 1995, 117, 1076-1082. (18) Ku ¨ ster, B.; Wheeler, S. F.; Hunter, A. P.; Dwek, R. A.; Harvey, D. J. Anal. Biochem. 1997, 250, 82-101. (19) Lee, C.; Levin, A.; Branton, D. Anal. Biochem. 1987, 166, 308-312.

Figure 2. MALDI-TOF MS spectra of the underivatized and derivatized asialo-biantennary N-glycan (NA2) at various concentrations: (a) underivatized NA2; (b) PA-derivatized NA2, “I” indicates impurity; (c) Girard’s T-derivatized NA2; (d) Cy3-derivatized NA2. All spectra were acquired with 2,5-DHB as a matrix. The amount loaded on the target plate was indicated on each spectrum.

of 50 mM sodium phosphate buffer, pH 7.2 containing 1 unit of PNGase F followed by incubation overnight at 37 °C. Glycans were extracted from a gel plug with 10 µL of distilled water for 15 min three times. The combined extract was lyophilized and derivatized as described above. The labeled N-glycans derived from transferrin were further digested with A. ureafaciens sialidase under conditions previously described.20 RESULTS AND DISCUSSION Derivatization of Oligosaccharides with Cy-Hydrazide. Fluorescent trimethincyanine dyes, whose overall charge on the dyes is +1, form the basis of the dyes used (Figure 1). We employed cyanine dyes which carried a hydrazide functionality. The formation of hyrazone has several advantages over reductive amination, which is more widely used for the reducing-terminal derivatization of oligosaccharides, in microscale structural and functional analyses of oligosaccharides. The substituted hydrazide couples to oligosaccharide with high efficiency on simply heating (20) Uchida, Y.; Tsukada, Y.; Sugimori, T. J. Biochem. 1979, 86, 1573-1585.

the oligosaccharide with a severalfold molar excess of reagent,17 while reductive amination requires a large excess of NaBH3CN and amine. The former does not necessarily require a purification step. Under optimized conditions, the oligosaccharide is ligated to substituted hydrazide to predominantly give cyclic β-glycoside, which is the intrinsic glycoside linkage of N-glycans present in nature.21 The MALDI-TOF mass spectrum of asialo-biantennary Nglycan (NA2) derivatized by Cy3 hydrazide is shown in Figure 2d. NA2 was incubated with a 50-fold excess of Cy3 hydrazide, and the reaction mixture was directly combined with 2.5-DHB for the MALDI-TOF analysis. The derivatized NA2 was detected solely as M+ at m/z 2121, thus simplifying the mass spectrum. Other signals, including NA2 itself, could not be detected, indicating that the reaction proceeds with high efficiency or the signal intensity is drastically increased by the Cy derivatization. That the M+ ion species is the only ion species to be detected is in good agreement (21) Shinohara, Y.; Sota, H.; Gotoh, M.; Hasebe, M.; Tosu, M.; Nakao, J.; Hasegawa, Y.; Shiga, M. Anal. Chem. 1996, 68, 2573-2579.

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Figure 3. MALDI-TOF MS spectra of the derivatized N-glycan library with three different matrixes: (a) Girard’s T-derivatized N-glycan library (250 ng); (b) Cy3-derivatized N-glycan library (10 ng).

with previous observations for those compounds having a quaternary ammonium center and may be explained by quaternary amines containing a permanent positive charge. The purification of Cy-labeled oligosaccharides prior to MALDITOF analysis was by no means necessary. If necessary, the excess reagent could be easily removed using ZipTip C18 (Millipore), where the labeled glycans remained in the solution while the reagent adsorbed to the resin using H2O as a solvent (data not shown). Such purification may expand the application of Cy-labeled oligosaccharide as a probe for examining molecular interactions,22 as a substrate for highly sensitive analyses of enzyme activity,23 and so forth. Sensitivity of Cy-Labeled Oligosaccharide on MALDI-TOF MS. To objectively evaluate the effect of Cy labeling, the improved sensitivity was compared with underivatized, PA, and Girard’s T-labeled oligosaccharide, using NA2 as a model oligosaccharide (Figure 2). The approximate detection limits for the derivatized oligosaccharides on MALDI-TOF MS were evaluated by acquiring the spectra of the derivatized NA2 applied with 2,5-DHB on the target plate in decreasing amounts by serial dilution. The Cy3 derivative could be detected down to 200 amol loaded on the target (Figure 2d). To obtain spectra with a similar signal-to-noise ratio, 100, 10, and 2-10 fmol of underivatized NA2, the PA derivative, and Girard’s T derivative of NA2 were required, respectively. Thus, a ∼500-fold increase in sensitivity was observed for the Cy3 derivative over that of underivatized NA2. Correspondingly, a ∼25fold and 10-25-fold increase in sensitivity was found for the Cy3 derivative over the PA derivative and Girard’s T derivative. The better sensitivity of the Cy3 derivative over Girard’s T might be due to physical properties other than the constitutive positive charge, such as hydrophobicity and optical properties. In addition, acquiring the MALDI mass spectra of the Cy3 derivative made it

relatively easier to hit the so-called “sweet spot” on the 2,5-DHB crystal, which may also substantially improve the signal-to-noise ratio. Derivatization by Cy5 hydrazide proceeded in the same manner as that by Cy3 hydrazide, while the Cy5-derivatized oligosaccharide appeared to be 2-3-fold less sensitive (data not shown). One limitation of Cye derivatization is that such a remarkable signalenhancing effect upon Cy labeling was not observed for acidic oligosaccharides such as sialylated oligosaccharides (data not shown). This might be due to canceling of the positive charge of the dye with negative charge(s) of the acidic moiety. This inconsistency will be solved by methyl esterification of the sialic acid residue to render sialylated oligosaccharides chemically equivalent to neutral oligosaccharides as reported.24 To expand the feasibility of Cye labeling to acidic oligosaccharides, we are currently studying the rapid and easy method for esterification applicable to a trace amount of sialylated oligosaccharides. Matrix Dependency. It is worth noting that we did obtain signals, albeit of poor quality, from the Cy derivative of NA2 also in the absence of matrix in the sample. This observation prompted us to investigate whether the production of signal under MALDI conditions would be dependent on the nature of the matrixes. 2,5-DHB is the most popular MALDI matrix for carbohydrate analysis. When 2,5-DHB is used as a matrix, the so-called sweet spot has to be sought carefully because 2,5-DHB forms long, thick, needle-shaped crystals and the central region of the target spot consists of a nonhomogeneous amorphous mixture of sugar, contaminants, and salts, which disturbs the high-throughput measurement and mass accuracy. To evaluate the compatibility with matrixes other than 2,5-DHB, MALDI-TOF analysis of a Cy3labeled N-linked oligosaccharide library (Oxford Glycosystems), which contains a large variety of N-glycans, was performed with 2,5-DHB, HCCA, and nor-harman25 as the matrixes.

(22) Jacob, G. S.; Kirmaier, C.; Abbas, S. Z.; Howard, S. C.; Steininger, C. N.; Welply, J. K.; Scudder, P. Biochemistry 1995, 34, 1210-1217. (23) Whittal, R. M.; Palcic, M. M.; Hindsgaul, O.; Li, L. Anal. Chem. 1995, 67, 3509-3514.

(24) Powell, A. K.; Harvey, D. J. Rapid Commun. Mass Spectrom. 1996, 10, 10271032. (25) Nonami, H.; Fukui, S.; Erra-Balsells, R. J. Mass Spectrom. 1997, 32, 287296.

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Figure 4. PSD and ISD spectra of the Cy5-derivatized lacto-neo-fucopentaose I (LNFP-I). These spectra were acquired with HCCA as a matrix. P, • and •• in ISD spectrum indicates parent ion, [M + 16]+ and [M - 209]+, respectively.

As 2,5-DHB is highly compatible with carbohydrate analysis, the quality of the MALDI-MS spectra of Girard’s reagent T derivatives (250 ng of N-glycan mixture was used for the derivatization) was drastically spoiled when HCCA or nor-harmane was used as a matrix (Figure 3a). On the other hand, they were found to work as matrixes as good as 2,5-DHB for Cy3 derivatives (10 ng of N-glycan mixture was used for the derivatization) of oligosaccharides (Figure 3b). And the signal profiles of these spectra were almost the same, which suggested that the matrix dependency on sensitivity was independent of oligosaccharide structures. Although Cy dyes themselves could be somewhat labile in acidic matrixes (e.g., HCCA, 2,5-DHB) and repeated laser desorption could cause unfavorable decomposition (e.g., oxidation of alkenes), we found that they are quite stable when nor-harman, a weakly basic compound, was used as the matrix. To the authors’ knowledge, no other derivatization has achieved such broad compatibility with many matrixes without spoiling the signal strength. PSD and ISD. To characterize the behavior on fragmentation of Cy-labeled oligosaccharide, lacto-neo-fucopentaose I, which consists of three monosaccharides with different molecular weights, was chosen as a model compound. As shown in Figure 4, only the fragment ions bearing the cyanine moiety could be observed in the PSD spectrum of the Cy5-labeled oligosaccharide. Considering that the cyanine moiety possesses the constitutive positive charge, these ion species should be present in high abundance in the PSD spectra. The relatively simple spectrum mainly consisting of Y-type fragment ions simplifies the sequence analysis of oligosaccharide. Three lower signals (m/z 538.6, 481.7, and 466.7) were assigned as 0,2X0, N-N cleavage and amide bond cleavage of Cy-hydrazone, respectively. MALDI mass spectra of Cy5-labeled oligosaccharide obtained using HCCA or 2,5-DHB as a matrix showed prominent in-source

Figure 5. Proposed degradation of cyanine moiety during MALDI process with HCCA.

fragment ions, which mainly resulted from glycosidic cleavage. Two other signals, which appeared independently of the oligosaccharide structure at 16 mass units above and 209 mass units below the molecular ion M+, might come from the degradation of the cyanine moiety, which is easily oxidized under acidic conditions. Figure 5 shows the proposed degradation for the cyanine moiety. Using nor-harman as the matrix, in-source fragment signals were not observed in the spectra of the same compounds. Although Naven and co-workers reported that in-source fragmentation of nonlabeled oligosaccharide during the MALDI Analytical Chemistry, Vol. 76, No. 15, August 1, 2004

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Table 1. Observed Signals of Cy3-Labeled Oligosaccharides Released from the Standard Glycoproteins Separated by SDS-PAGE peak no. glycoprotein 1 2 3 4 5 6 7 8 9 10

Figure 6. MALDI-TOF MS spectra of Cy3-derivatized N-glycans released from a gel plug of standard glycoproteins separated by SDS-PAGE: (a) asialofetuin; (b) ovalbumin; (c) transferrin. This spectrum was obtained after neuraminidase treatment. The small peaks beside 4 and 10 were dehydrated ions [M - 18]+.

process could be observed by using time lag focusing (delayed extraction) with a delay in the order of microseconds,26 this fragmentation of Cy-labeled oligosaccharides was observed within 200 ns of the delayed time. Feasibility Study for the N-Glycan Analysis of Glycoproteins Separated by Gel Electrophoresis. Gel electrophoresis is currently the most commonly used protein separation technique in proteomics. As a result, strategies for glycosylation analysis in proteomics should be compatible with this format. To elucidate the feasibility of Cy derivatization for the glycosylation analysis of gel-separated proteins, N-glycans obtained in an in-gel release manner with PNGase F from a gel plug of well-characterized glycoproteins (asialofetuin, ovalbumin, transferrin) separated by SDS-PAGE were analyzed. Figure 6 shows the MALDI spectra of the Cy3-labeled extracts from the gel plug, which were excised from the band negatively stained with a Negative Gel Stain MS kit (Wako Chemical). N-Glycans on transferrin could be detected only after further digestion with A. ureafaciens sialidase posterior to derivatization, which suggested that a positive charge of dye was compensated with a negative charge of sialic acid as described above. The observed signals are summarized and assigned in Table 1, consistent with the N-glycan profiles of the glycoprotein reported.27-29 Major oligosaccharides could be clearly detected after Cy3-labeling when as little as 5 pmol of model protein was used. The achieved N-glycan profiling in the range of 5 pmol of glycoproteins applied to a gel is nearly compatible to the conventional Coomassie Blue staining. Though sufficient signal intensity should be theoretically obtained from femtomole levels of glycoproteins considering the detection sensitivity of Cy3labeled oligosaccharide, low picomoles of gel-separated glycopro(26) Naven, T. J. P.; Harvey, D. J.; Brown, J.; Gritchley, G. Rapid Commun. Mass Spectrom. 1997, 11, 1681-1686. (27) Green, E. D.; Adelt, G.; Baenziger, J. U.; Wilson, S.; Van Halbeek, H. J. Biol. Chem. 1988, 263, 18253-18268. (28) Yamashita, K.; Tachibana, Y.; Kobata, A. J. Biol. Chem. 1978, 253, 38623869. (29) Yamashita, K.; Ideo, H.; Ohkura, T.; Fukushima, K.; Yuasa, I.; Ohno, K.; Takeshita, K. J. Biol. Chem. 1993, 268, 5783-5789.

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asialofetuin asialofetuin asialofetuin asialofetuin ovalbumin ovalbumin ovalbumin ovalbumin obalbumin transferrin

m/z obsd

theor

composition

1959.91 2122.03 2325.35 2487.12 1715.75 1877.85 2121.99 2204.00 2324.94 2121.72

1959.89 2121.94 2325.02 2487.07 1715.78 1877.84 2121.94 2203.99 2325.02 2121.94

HexHexNAc2Man3GlcNAc2 Hex2HexNAc2Man3GlcNAc2 Hex2HexNAc3Man3GlcNAc2 Hex3HexNAc3Man3GlcNAc2 Hex2Man3GlcNAc2 Hex3Man3GlcNAc2 Hex2HexNAc2Man3GlcNAc2 HexNAc4Man3GlcNAc2 Hex2HexNAc3Man3GlcNAc2 Hex2HexNAc2Man3GlcNAc2

tein was required for the N-glycan profiling so far evaluated. This may be attributable to incomplete in-gel enzymatic deglycosylation, sample loss during sample preparation step(s), reduced reaction yield under the presence of contaminants such as salt, and so forth. Improvement in small-scale sample handling (i.e., in-gel digestion, recovery of glycans from gel, derivatization, desalting, and affinity enrichment) would allow glycan detection compatible with silver or fluorescent staining. We are currently studying these improvements for application of this method to two-dimensional gel electrophoresis-based glycoproteomics. CONCLUSION Derivatization by Cy hydrazides, whose overall charge on the dyes is +1, was found to enhance the MALDI-TOF sensitivity of oligosaccharides by ∼500-fold, enabling subfemtomole oligosaccharide detection. Considering that the sensitivity of the Cy derivative is better than that of Girard’s reagent T derivative by ∼25-fold, not only the constitutive positive charge but also other factors (i.e., hydrophobicity) should substantially contribute to the significant enhancement of sensitivity. The derivatization is extremely simple, and no cleanup step was required prior to the analysis. Unlike intact oligosaccharides or derivatized oligosaccharides with other reagents, the sensitivity of Cy derivatives was nearly independent of the kind of matrix. Cy-labeled oligosaccharide was detected as a single M+ ion, and PSD and ISD afforded simple spectra mainly consisting of Y-type fragment ions, thus simplify the profiling and sequencing analyses. The outstanding nature in MALDI-TOF analysis together with the cyanine dyes’ high sensitivity for optical detection will make these dyes of special importance in protein glycosylation analysis. ACKNOWLEDGMENT The authors thank Drs. Mark Briggs, Jon W Cummins, Karen Williams, and Edward Hawkins for the provision of Boc-protected Cy3 and 5 hydrazide derivatives and for useful discussions. A part of this work was performed as part of the R&D Project of the Industrial Science and Technology Frontier Program (R&D for Establishment and Utilization of a Technical Infrastructure for Japanese Industry) supported by the New Energy and Industrial Technology Development Organization (NEDO). Received for review January 15, 2004. Accepted May 18, 2004. AC049897Z