Assignment and Quantification of 2-Aminopyridine Derivatized

Anal. Chem. 2004, 76, 7294-7303

Assignment and Quantification of 2-Aminopyridine Derivatized Oligosaccharide Isomers Coeluted on Reversed-Phase HPLC/MS by MSn Spectral Library Yasuhiro Takegawa,† Kisaburo Deguchi,*,† Shinya Ito,‡ Shinji Yoshioka,§ Akihiro Sano, Kiyomi Yoshinari,| Kinya Kobayashi,| Hiroaki Nakagawa,† Kenji Monde,† and Shin-Ichiro Nishimura†

Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 001-0021, Japan, Hitachi High-Technologies Co., Hitachinaka 312-8504, Japan, Naka Customer Center, Hitachi Science Systems Co., Hitachinaka 312-8504, Japan, and Hitachi Research Laboratory, Hitachi Ltd., Hitachi 319-1292, Japan

2-Aminopyridine (PA)-derivatized oligosaccharides from IgG were analyzed by using reversed-phase HPLC/mass spectrometry (RP-HPLC/MS) and a MSn spectral library, in particular, focusing on two pairs of isomers incompletely separated or coeluted in chromatograms. We previously reported that MSn spectral matching considering both major fragment ions (m/z) and intensities is useful and applicable to the structural assignment of PAoligosaccharide isomers. In this study, MSn spectral matching based on the MSn spectral library was applied to the assignment of these PA-oligosaccharide isomers in IgG. Its usefulness was investigated by comparing it to the conventional two-dimensional mapping method based on retention time indexes. Specifically, we focus on the assignment and quantification of the isomers, which are coeluted in chromatograms. From this, we propose a new method using MSn spectral matching and the working curve on which are plotted the relative intensities of selected fragment ions in their MS2 spectra versus various mixtures of the isomers. This new method demonstrated that the obtained quantities coincide very well with those estimated after separating by a combination of lectin and reversed-phase columns. This means that separation by RP-HPLC/MS is greatly simplified because complete separation of the isomers is no longer required. Application of this new method was tested by using the two other pairs of fucosylated and nonfucosylated PA-oligosaccharides from IgG. The results showed that this method works for them as well.

of oligosaccharides, it is important to elucidate oligosaccharide structures and distribution in a glycoprotein. In contrast to proteins and nucleic acids, however, there is no universal method for the simple and rapid analysis of oligosaccharides, and therefore, structural and functional studies of oligosaccharides are difficult to perform.2 Conventionally, normal-phase high-performance liquid chromatography (NP-HPLC) and reversed-phase HPLC (RP-HPLC) combined with exoglycosidase digestions have been used to analyze fluorescently labeled oligosaccharides. This method is sometimes referred to as two-dimensional (2D) mapping.3 For sialylated oligosaccharides, this method was extended to 3D mapping by adding anion-exchange column separation to the NPHPLC and RP-HPLC.4,5 To date, more than 400 pyridilaminated oligosaccharides (PA-oligosaccharides) have been mapped and can be seen on the Web (http://www.glycoanalysis.info/ENG/ index.html). Although this 2D or 3D mapping method is useful for assigning and quantifying oligosaccharides, it requires tedious and time-consuming procedures. In the past decade, mass spectrometry (MS), most predominantly featuring matrix-assisted laser desorption/ionization (MALDI6,7) and electrospray ionization (ESI8), has been in widespread use as a method for the rapid analysis of oligosaccharide structures.9,10 In particular, several tandem mass spectrometry (MSn) techniques providing additional structural informationhavebeenintensivelyusedincombinationwithexoglycosidase digestions. However, despite many attempts to establish rules for elucidating oligosaccharide structures from MSn fragmentation patterns, it is still almost impossible to completely assign the

Oligosaccharides attached to a glycoprotein play an important role in numerous biological processes, e.g., protein conformation, molecular recognition, and cellar interaction.1 Structures of oligosaccharides are also associated with various developmental stages and pathological states. Therefore, to understand the roles

(2) Dwek, R, A.; Edge, C. J.; Harvey, D. J.; Wormald, M. R. Annu. Rev. Biochem. 1993, 62, 65-100. (3) Tomiya, N.; Awaya, J.; Kurono, M.; Endo, S.; Arata, Y.; Takahashi, N. Anal. Biochem. 1988, 171, 73-90. (4) Nakagawa, H.; Kawamura, Y.; Kato, K.; Shimada, I.; Arata, Y.; Takahashi, N. Anal. Biochem. 1995, 226, 130-138. (5) Takahashi, N.; Nakagawa, H.; Fujikawa, K.; Kawamura, Y.; Tomiya, N. Anal. Biochem. 1995, 226, 139-146. (6) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T. Rapid Commun. Mass Spectrom. 1988, 2, 151-153. (7) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (8) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science, 1989, 246, 64-71. (9) Harvey, D. J. Mass Spectrom. Rev. 1999, 18, 349-451. (10) Mechref, Y.; Novotny, M. V. Chem. Rev. 2002, 102, 321-370.

* Corresponding author. Telephone: +81-11-706-9030. Fax: +81-11-706-9032. E-mail: [email protected]. † Hokkaido University. ‡ Hitachi High-Technologies Co. § Hitachi Science Systems Co. | Hitachi Ltd. (1) Dwek, R. A. Chem. Rev. 1996, 96, 683-720.

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10.1021/ac0493166 CCC: $27.50

© 2004 American Chemical Society Published on Web 11/11/2004

oligosaccharide structure (i.e., the component, sequence, anomeric configuration, linkage position, and branching pattern) by MSn analysis.11-16 Recently, Royle et al.17 proposed an approach based on a structural database of 2-aminobenzamide (2AB) oligosaccharides (O-glycans), containing 2D mapping information (i.e., glucose unit (GU) and arabinose unit (AU) indexes calculated from retention times on NP-HPLC and RP-HPLC chromatograms, respectively) and MSn (n ) 1-2) spectral data obtained by NP-HPLC/ESI-MS and MALDI-MS. The major feature of their method is that it easily assigns oligosaccharide structures by simply referring to both the chromatographic and mass spectral data of the 50 standard oligosaccharides already stored in the database. This method may be a realistic approach for the assignment of oligosaccharides with a diversity of structures. In addition to the method of Royle et al., we have been trying to develop an efficient RP-HPLC/MS method by relying on both chromatographic and MSn spectral library of PA-oligosaccharide standards (N-glycans). However, a common and serious problem occurs in both approaches when oligosaccharide isomers are incompletely separated or coeluted on RP- or NP-HPLC chromatograms because it is almost impossible to distinguish the isomers on the basis of chromatographic and mass spectral data. In this case, conventional exoglycosidase digestions or 2D HPLC separation is usually required. In our previous study,18 we demonstrated that MSn (n ) 1-3) spectral matching based on correlation coefficients is useful for the structural assignment of PA-oligosaccharide isomers. In this study, MSn spectral matching is applied to the assignment of N-glycan structures in IgG, in particular, focusing on two pairs of isomers incompletely separated or coeluted on a RP-HPLC chromatogram. First, it can be seen that MSn spectral matching works even between spectral data obtained from different chromatographic elutions (e.g., water/methanol and ammonium acetate buffer/ acetonitrile gradient elutions). Second, this study discusses how the RP-HPLC/MS method can be applied to the assignment of isomers in IgG by using MSn spectral matching, compared to conventional 2D mapping. Third, this study stresses that even if two isomers coelute and completely overlap on a chromatogram, they can be correctly assigned by MSn spectral matching and roughly quantified by using a working curve of the relative intensities of selected fragment ions in their MS2 spectra versus various mixtures of these isomers and by assuming that the data are in the MSn spectral library. Although such a working curve has been simply suggested by Wheeler and Harvey19 and reported by Yamagaki and Tachibana,20 this seems to be the first set of results that discusses the assignment and quantification of (11) Hakansson, K.; Cooper, H. J.; Emmett, M. R.; Costello, C. E.; Marshall, A. G.; Nilsson, C. L. Anal. Chem. 2001, 73, 4530-4536. (12) Xie, Y.; Lebrilla, C. B. Anal. Chem. 2003, 75, 1590-1598. (13) Chai, W.; Lawson, A. M.; Piskarev, V. J. Am. Soc. Mass Spectrom. 2002, 13, 670-679. (14) Cheng, H. L.; Her, G. R. J. Am. Soc. Mass Spectrom. 2002, 13, 1322-1330. (15) Pfenninger, A.; Karas, M.; Finke, B.; Stahl, B. J. Am. Soc. Mass Spectrom. 2002, 13, 1331-1340. (16) Pfenninger, A.; Karas, M.; Finke, B.; Stahl, B. J. Am. Soc. Mass Spectrom. 2002, 13, 1341-1348. (17) Royle, L.; Mattu, T. S.; Hart, E.; Langridge, J. I.; Merry, A. H.; Murphy, N.; Harvey, D. J.; Dwek, R. A.; Rudd, P. M. Anal. Biochem. 2002, 304, 70-90. (18) Takegawa, Y.; Ito, S.; Yoshioka, S.; Deguchi, K.; Nakagawa, H.; Monde, K.; Nishimura, S. Rapid Commun. Mass Spectrom. 2004, 18, 385-391. (19) Wheeler, S. F.; Harvey, D. J. Anal. Chem. 2000, 72, 5027-5039.

coeluted oligosaccharide isomers based on the MSn spectral library plus the working curves for difficult-to-separate pairs of PA-oligosaccharide isomers. EXPERIMENTAL SECTION Materials. Methanol (HPLC grade), 1-butanol, ammonium bicarbonate, and phosphate buffer (Na) were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). 2-Aminopyridine, acetonitrile (HPLC grade), acetic acid, ammonium acetate, and 10 mM sodium chloride solution were purchased from Wako Pure Chemical Industries (Osaka, Japan). PA-oligosaccharides (310.2 and 310.3) (nomenclature proposed by Takahashi et al.3), glucose oligomers (4-20), β-N-acetylhexosaminidase (jack bean), and RCA120 (caster bean)-agarose were purchased from Seikagaku Kogyo Co. (Tokyo, Japan). Sodium cyanoborohydride was purchased from Aldrich Chemical Co. (Milwaukee, WI). Water was purified by Milli-Q (Millipore Co., Milford, MA). Human IgG, R-L-fucosidase, and trypsin were both purchased from SigmaAldrich Co. (St. Louis, MO). Peptide-N-glycosidase F (PNGase F, recombinant) was from Hoffman-LaRoche Chemicals (Basel, Switzerland), and both R-chymotrypsin from bovine kidney and Pronase were from Calbiochem Co. (Darmstadt, Germany). A Sephadex G-15 column was obtained from Pharmacia-LKB Biotech. Inc. (Uppsala, Sweden), Bio-Gel P-4 (200-400 mesh) was from Bio-Rad Laboratories (Hercules, CA), a ShimPack HRC-ODSsilica column (6.0 mm i.d. × 150 mm) was from Shimadzu Co. (Kyoto, Japan), TSKgel Amide-80 (4.6 mm i.d. × 250 mm) was from Tosoh Co. (Tokyo, Japan), and both Develosil C30-UG (4 mm i.d. × 10 mm) and Develosil C30-UG (2 mm i.d. × 150 mm, particle size 3 µm) were from Nomura Chemical Co. (Aichi, Japan). Apparatus. The HPLC/MS system used in this study was a Hitachi LaChrom HPLC system consisting of an L-7100 pump with low-pressure gradient capability, an L-7200 autosampler, an L-7480 fluorescence detector (Hitachi High-Technologies, Tokyo, Japan), and a Hitachi M-8000 3DQ (ion trap) with a sonic spray ionization (SSI) interface (Hitachi High-Technologies, Tokyo, Japan).21,22 Nitrogen was used as the desolvation gas, and helium was used as the damping and collision gas. Helium gas pressure inside the ion trap was kept at 10-3 Torr. The mass calibration precision (Th) was (0.3 (m/z 10-1000) and (0.5 (m/z 1001-2000). Preparation of PA-Oligosaccharides from IgG. N-Glycans were released by using 20 units of peptide-N-glycosidase F from IgG (20 mg) after a digestion of IgG with 200 µg of trypsin and chymotrypsin each at pH 8.0 for 16 h and then purified by the Bio-Gel P-4 column after a Pronase digestion. The obtained oligosaccharides were reductively aminated with 2-aminopyridine (PA) and sodium cyanoborohydride and then purified by the Sephadex G-15 column and 10 mM ammonium bicarbonate. To release the sialic acids, the PA-oligosaccharide mixtures were heated at 90 °C for 1 h with 0.01 N HCl (pH 2.0). Additionally, the PA-oligosaccharide mixtures were purified by the TSKgel Amide-80 (4.6 mm i.d. × 250 mm). A stepwise gradient elution was performed at a flow rate of 1.0 mL/min at 40 °C using 0.5 M (20) Yamagaki, T.; Tachibana, K. Resume of 49th Mass Spectrometry Conference of Japan, Tokyo, Japan, 2001; pp 52-53. (21) Hirabayashi, A.; Sakairi, M.; Koizumi. H. Anal. Chem. 1994, 66, 45574559. (22) Hirabayashi, A.; Sakairi, M.; Koizumi, H. Anal. Chem. 1995, 67, 28782882.

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Scheme 1. Structures of PA-Oligosaccharides

acetic acid-triethylamine (pH 7.3)/acetonitrile 35:65 (solvent A) and 0.5 M acetic acid-triethylamine (pH 7.3)/acetonitrile 65:35 (solvent B) (A/B ) 100:0 (0-8 min) f 0:100 (8-16 min)). After drying, the sample was dissolved in 60 µL of water and a 1-µL aliquot was injected into the RP-HPLC/MS. Preparation of Standard PA-Oligosaccharides. The standard PA-oligosaccharides were purified from PA-oligosaccharides from IgG by the ShimPack HRC-ODS (6.0 mm i.d. × 150 mm). A linear gradient elution was performed at a flow rate of 1.0 mL/ min at 55 °C using 10 mM sodium phosphate buffer (pH 3.8) (solvent A) and 10 mM sodium phosphate buffer (pH 3.8) containing 0.5% 1-butanol (solvent B) (A/B ) 80:20 (0 min) f 50:50 (60 min)). The obtained PA-oligosaccharides are listed as 210.1, 210.2, 210.3, 211.2, and 211.3 (see Scheme 1). Isomeric 211.2 and 211.3 mixtures, which coeluted on the ODS column, were separated by the RCA120-agarose (10 mm i.d. × 64 mm) at a flow rate of 0.5 mL/min at room temperature using 10 mM ammonium acetate buffer (pH 7.0). PA-oligosaccharides 200.2 and 200.3 were derived from 210.2 and 210.3 by digestion with R-L-fucosidase, respectively. PA-oligosaccharide 111.3 was derived from 211.2 by digestion with β-N-acetylhexosaminidase, and PA-oligosaccharide 101.3 was derived from 111.3 by digestion with R-L-fucosidase. 7296


PA-oligosaccharides 310.2 and 310.3 were purchased from Seikagaku Kogyo Co. (Tokyo, Japan) and used without any further purification. RP-HPLC Separation for MSn Spectral Library. Separations intended primarily for desalting were performed by using the Develosil C30-UG (4 mm i.d. × 10 mm), water, and methanol. The flow rate was 0.2 mL/min, and a linear gradient elution was used starting from 0% methanol at 0 min to 50% methanol at 10 min. RP-HPLC Separation for GU Indexes and Analysis of PAOligosaccharides from IgG. Separations were performed using the Devesosil C30-UG (2 mm i.d. × 150 mm, particle size 3 µm). A linear gradient elution (A/B ) 87:13 (0 min) f 60:40 (70 min)) was used at a flow rate of 0.2 mL/min at 40 °C, using 1 mM ammonium acetate buffer (pH 4.2) containing 3 µM sodium chloride solution (solvent A) and solvent A containing 10% acetonitrile (solvent B). Fluorescence was measured at 400 nm with excitation at 320 nm. The column was calibrated using an external standard of PA-glucose oligomers (4-20) (PA-isomaltooligosaccharide (4-20) mixtures); the retention time for individual glycans was converted to a glucose unit (GU) index.3 The values of the glucose unit (GU) indexes were reproducible within a 6% variation.

Figure 1. MSn (n ) 1-2) spectral comparison of PA-oligosaccharide 210.1 acquired by water/methanol gradient elution (A) and ammonium acetate buffer/acetonitrile gradient elution (B).

MS Analytical Condition. The operating conditions for MS1 were as follows: desolvation temperature 270 °C; desolvation gas pressure 400 kPa; capillary voltage +1500 V; drift voltage 80 V; scan range m/z 400-2000; scan time 500 ms; low-mass cutoff m/z 105. The conditions for MSn (n ) 2, 3) spectral acquisition were optimized by varying the collision-induced dissociation (CID) voltage for each precursor ion. They were as follows: precursor ion isolation voltage 0.10-0.16 V; precursor isolation width (5 Th; low-mass cutoff m/z 260-348 (one-third of the precursor ion’s m/z value); CID voltage 0.18-0.32 V. MSn Data Processing for MSn Spectral Matching. Only the major fragment ions in the MSn spectra of the isomers, i.e., the highest 13-14 peaks, were considered for calculating correlation coefficients after normalizing the base fragment ion intensity to 1. The intensity of the first 13C isotopic peak was added to each 12C peak intensity, as was done in our previous study.18 The correlation coefficients defined in the equation were calculated

correlation coefficient )

∑(xi - jx)(yi - jy) x∑(xi - jx) ∑(yi - jy) 2

bigger contribution than do small fragment ions. We consider the above correlation coefficient to be suitable for our purposes because all fragment ions considered are treated equally. Molecular Dynamics and Molecular Orbital Calculations. Molecular modeling dynamics calculations were performed on a PC (Pentium IV) employing the CS ChemBats3D PRO software package (Cambridge Software Co., Cambridge, MA), dynamically heating at 300 K for ∼10 000 steps (step interval, 1 fs), and optimizing molecular structures within MM2 Force Field level. During these calculations, the H+ ion was always fixed at PA because it was difficult to determine its optimum position. After that, molecular orbital (MO) calculations were performed to optimize the structural conformations of the PA-oligosaccharide molecular ions and to calculate the stabilization energies. The MO software package used was the electronic structure analyzing program for atomic clusters using density functional theory (ESPAC-DF) using Slater-type atomic orbitals and considering all electrons, which was developed by Hitachi Research Laboratory.24,25

2

X ) (x1,x2,......,xn), Y ) (y1,y2,......,yn)

using these relative intensities and Microsoft Excel (Microsoft Japan, Tokyo, Japan). Here, X and Y denote the two spectra to be compared, and xi and yi are the relative intensities of the fragment ions, respectively. If the averages, jx and jy, are eliminated, the result coincides with “contact angle” frequently used in UV-visible spectra and recently used in MS spectra23 in which dominant fragment ions make a

RESULTS AND DISCUSSION MSn Spectral Matching between Different Elutions. We first investigated whether MSn spectral matching between spectral data obtained by the different elution conditions, e.g., water/ methanol and ammonium acetate buffer/acetonitrile gradient elutions, can work. This is a sort of test of robustness of the MSn spectral library. The water/methanol gradient elution was with a (23) Wan, K. X.; Vidavsky, I.; Gross, M. L. J. Am. Soc. Mass Spectrom. 2002, 13, 85-88. (24) Kobayashi, K.; Kurita, N. Phys. Rev. Lett. 1993, 70, 3542-3544. (25) Kobayashi, K.; Tago, K.; Kurita, N. Phys. Rev. A 1996, 53, 1903-1906.


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Table 1. Results of MSn Spectral Library Search for Peaks b and c correlation coefficients peaks/standards in data library peak b 210.2 210.3 111.3 peak c 210.2 210.3 111.3

Figure 2. RP-HPLC/MS chromatograms of PA-oligosaccharides from human IgG. (A) Chromatogram by FL (em, 400 nm; ex, 320 nm). (B) Base ion chromatogram in positive-ion mode. (C) Mass chromatograms of base peaks [M + H + Na]2+ of the seven main peaks (a-g).

short column, Develosil C30-UG (4 mm i.d. × 10 mm) (see Experimental Section), was used for making the MSn spectral library with the standard PA-oligosaccharides, while the ammonium acetate buffer/acetonitrile gradient elution was with a long analytical column, Devesosil UG-C30 (2 mm i.d. × 150 mm) (see Experimental Section), was used for separation of the PAoligosaccharide mixtures released from IgG. Parts A and B of Figure 1 show MSn (n ) 1-2) spectra of the PA-oligosaccharide 210.1 (see Scheme 1) recorded in the two different gradient elutions under the same MS conditions. Both MS1 spectra are very similar and have the same [M + H + Na]2+ ions (m/z 782)

MS2 spectral matching)

MS3 spectral matching

0.992 0.933 0.578

0.967 (vs 200.2) 0.588 (vs 200.3) 0.174 (vs 101.3)

0.939 0.992 0.617

0.631 (vs 200.2) 0.921 (vs 200.3) 0.564 (vs 101.3)

GU indexes (elution position) 10.8 11.2 11.6 11.8 11.1 11.2 11.6 11.8

as the base peak. Both MS2 spectra obtained by selecting the [M + H + Na]2+ ions (m/z 782) as the precursor ion are also very similar, and the correlation coefficient between these MS2 spectra (MSn spectral matching) was 0.985. In this calculation, only the highest 13-14 fragment ions (m/z values) (i.e., B- and Y-type fragment ions of Domon and Costello nomenclature26) and their intensities were considered. This good correlation means that MS2 fragmentations occurring inside the ion trap MS are not influenced by chromatographic conditions. In other words, the MS2 spectra acquired by the water/methanol gradient elution with a short column can be used for the MSn spectral library. This result greatly simplifies the making of the MSn spectral library. RP-HPLC/MS Analysis of PA-Oligosaccharides from IgG. RP-HPLC/MS analysis using MSn spectral matching was extended to structural assignments of N-glycans in a glycoprotein. Human IgG, having complex-type N-glycan isomers composed of Hex4GlcNAc4Fuc1 and Hex4GlcNAc5Fuc1,27 was used as a model glycoprotein to investigate the usefulness of our method, discussed below. The N-glycans were enzymatically released from human IgG and labeled with PA following the method previously reported.3 The obtained PA-oligosaccharide mixtures were then analyzed by RP-HPLC/MS. Parts A and B of Figure 2 show the

Figure 3. MS1, MS2, and MS3 spectra of two isomers incompletely separated. (A) Expanded view of peak b and peak c in Figure 2C. (B) and (C) MS1, MS2, and MS3 spectra derived from the indicated precursor ions of peak b and peak c areas, respectively. 7298 Analytical Chemistry, Vol. 76, No. 24, December 15, 2004

Figure 4. MS1 and MS2 spectra of isomers coeluted on RP-HPLC/MS. (A) expanded view of peak f in Figure 2C. (B) MS1 and MS2 spectra derived from precursor ion [M + H + Na]2+ (m/z 965) in peak f area. Table 2. Results of MSn Spectral Library Search for Peak f peak/standards in data library

correlation coefficients (MS2 spectral matching)

GU indexes (elution position)

peak f 211.2 211.3 310.2 310.3

0.945 0.559 0.852 0.743

15.7 16.3 16.3 14.3 15.5

chromatogram observed by fluorescence detection (FL) and the base ion chromatogram (BIC) in the positive-ion mode of sonic spray ion trap mass spectrometry (SSI-IT MS), respectively. Figure 2C shows the mass chromatograms (MC) corresponding to the base peaks [M + H + Na]2+ of the seven main peaks (a-g) in the FL and BIC chromatograms; their MS2 and MS3 spectra were recorded during data acquisition. The MC of m/z 863 shows two isomeric peaks, b and c, composed of PA-Hex4GlcNAc4Fuc1. Although the MC of m/z 965 shows one peak, f, it is known from the conventional 2D mapping method that two isomers composed of PA-Hex4GlcNAc5Fuc1 completely overlap or coelute here. Generally, isomers incompletely separated from each other (e.g., peak resolution Rs ∼ 0.8) or coeluted (e.g., Rs < 0.6)28 on a chromatogram are difficult to assign and quantify, and therefore, tedious exoglycosidase digestions or 2D HPLC separations are additionally required.17 Below, we discuss the application of MSn spectral matching in such a case and propose a new method to solve it. Analysis of Isomers Incompletely Separated on RPHPLC/MS. Figure 3A shows an expanded view of peak b and peak c in Figure 2C. The two MS2 spectra in Figure 3B and C (26) Domon, B.; Costello, C. E. Glycoconjugate J. 1988, 5, 397-409. (27) Takahashi, N.; Ishii, I.; Ishihara, H.; Mori, M.; Tejima, S.; Jefferis, R.; Endo, S.; Arata, Y. Biochem. 1987, 26, 1137-1144. (28) Snyder, L. R.; Kirkland, J. J.; Glajch, J. L. Practical HPLC Method Development,2nd ed.; Wiley: New York, 1997.

Figure 5. Correlation coefficients of MS2 spectra between peak f and various mixtures of standards 211.2 and 211.3.

were obtained from the areas indicated on the MC chromatogram by arrows b and c, excluding the overlapping area and selecting the same precursor ion [M + H + Na]2+ (m/z 863). Similarly, the two MS3 spectra were acquired by selecting the precursor ion [M - Fuc + H + Na]2+ (m/z 790). Then, MSn spectral matching was performed between these MS2 and MS3 spectra and the MSn spectral library data. Table 1 summarizes the results of MS2 and MS3 spectral matching, including 111.3 with the same mass and their GU indexes. The GU indexes were calculated after calibrating the column with the standard PA-glucose oligomers (4-20).3 The GU indexes stored in the library are reproducible within a variation of 6%. Among these three isomers, the GU index of peak b is closest to that of 210.2; peak b also has the highest correlation with 210.2 (MS2 spectral matching, 0.992). Therefore, peak b may be identified as 210.2 from both results. However, MS2 spectral matching with 210.3 also indicates a high value (0.933). In this case, it is necessary to confirm by using MS3 spectral matching. The results are included in the third column in Table 1. From Analytical Chemistry, Vol. 76, No. 24, December 15, 2004

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Figure 6. MS2 spectra of standards 211.2 (A) and 211.3 (B) derived from precursor ion [M + H + Na]2+ (m/z 965).

Figure 7. Relative intensities of fragment ions ([M - GlcNAc + Na]+/[M - Fuc + H + Na]2+) in MS2 spectra versus various mixtures of standards 211.2 and 211.3.

these correlation coefficient values, we can safely conclude that peak b has the same core structure as 200.2 (i.e., the highest correlation coefficient 0.967), and therefore, it is 210.2. Here note that MS3 spectral matching were performed between MS3 spectra in Figure 3B and C and MS2 spectra of nonfucosyl PA-oligosaccharide isomers 200.2, 200.3, and 101.3 (see Scheme 1) stored in the MSn spectral library. In the case of peak c, the results of the GU index and the correlation coefficient are contradictory; that is, 210.2 has the closest GU index and 210.3 has the highest correlation coefficient (MS2 spectral matching, 0.992). However, both the MS2 and MS3 spectral matching results indicate that peak c is 210.3. Thus, the MSn spectral matching in these cases is more reliable than the GU index. In addition, the peak area ratio 63:37 (peak b/peak c) was estimated from the MC of m/z 863 ( 1. This is very close to the ratio of 62:38 estimated from the FL chromatogram. That is, it seems to indicate that the ionization efficiency of these isomers are almost same. 7300 Analytical Chemistry, Vol. 76, No. 24, December 15, 2004

Figure 8. Correlation coefficients between MS2 spectra of overlapped peak (i.e., peaks b and c) and various mixtures of standards 210.2 and 210.3.

Analysis of Isomers Coeluted on RP-HPLC/MS. Another pair of isomers composed of PA-Hex4GlcNAc5Fuc1 (peak f) completely overlaps on the MC (m/z 965), as shown in Figure 4A. Figure 4B shows MSn (n ) 1-2) spectra of peak f. MS2 spectral matching was performed in the same manner as described above, and the results are summarized in Table 2. Although the GU index of 310.3 is closest to that of peak f, except for 310.2, the others are still within the 6% variation. In contrast, peak f has the highest correlation with 211.2 (MS2 spectral matching, 0.945), and therefore, peak f definitely contains 211.2. However, there still remains a possibility of some contamination (or mixture) of 211.3, which has the same GU index as 211.2. A possibility of contamination of other isomers 310.2 and 310.3 can be ignored, because the GU index differences 0.8 (16.3(211.2) - 15.5(310.3)) and 2.0 (16.3(211.2) - 14.3(310.2)) are enough to separate each other. Next, we discuss and propose a method to roughly estimate the degree of contamination of 211.3.


Figure 5 plots and curve fits the correlation coefficients between the MS2 spectra of peak f and mixtures of standards 211.2 and 211.3. The horizontal axis corresponds to the mixture ratios of these isomers and the error bars indicate the standard deviations (2 SD) of the three experiments on each mixture ratio. Although it can be seen from this best-fitt plotting curve that the mixture ratio of peak f is between 10:0 and 6:4 (211.2:211.3), the mixture ratio cannot be estimated exactly because peak f indicates almost the same correlation coefficients in the mixture ratios from 10:0 to 6:4 (211.2:211.3). This limitation is caused by the present method of calculating correlation coefficients considering the highest 13-14 fragment ion intensities. That is, it dilutes the difference in the MS2 spectra of these isomers. For such cases, it would be better to focus on specific fragment ions indicating clear differences in intensities in these MS2 spectra, as shown previously.19,20 Figure 6 shows MS2 spectra of standards 211.2 and 211.3 derived from each precursor ion [M + H + Na]2+ (m/z 965). Here, we focus on two specific Y-type fragment ions [M - GlcNAc + Na]+ (m/z 1725) and [M - Fuc + H + Na]2+ (m/z 892). The reason for this choice is that the fragment ion [M - GlcNAc + Na]+ caused by the loss of GlcNAc in the antenna may strongly reflect a structural difference between these isomers, while the fragment ion [M - Fuc + H + Na]2+ caused by the loss of reducing-end fucose may not do so much. Therefore, we plotted and curve fitted the relative intensities of these fragment ions ([M - GlcNAc + Na]+/[M - Fuc + H + Na]2+) versus various mixtures of these isomers (see Figure 7). This curve shows good linearity (R2 ) 0.9894) and implies the possibility of roughly quantifying a mixture ratio of these isomers (although the plotting curves are not shown, the relative intensities of the fragment ions (([M - GlcNAc + Na]+ + ([M - GlcNAc + H + Na]2+/[M - Fuc + H + Na]2+) also showed a good linear relationship as well as Figure 7, but others did not). By using the above linear relationship (or working curve) and from the relative

Figure 10. Relative intensities of fragment ions ([M - GlcNAc + Na]+/[M - Fuc + H + Na]2+) in MS2 spectra versus various mixtures of standards 210.2 and 210.3.

intensity 0.735 of the fragment ions ([M - GlcNAc + Na]+/[M - Fuc + H + Na]2+) on the MS2 spectrum of peak f in Figure 4B, a mixture ratio of 88:12 (211.2:211.3) was obtained. To confirm this result, we separated the two isomers by using a lectin (RCA120-agarose29) affinity chromatography after fractionation of peak f (data not shown) and then obtained a mixture ratio of 86: 14 (211.2:211.3) from the corresponding FL chromatogram. Both results coincided remarkably well. To further confirm that this method of quantification is applicable to other cases, we similarly investigated the pair of isomers 210.2 (peak b) and 210.3 (peak c) under the elution condition in which these isomers coelute. Figure 8 plots and curve fits the correlation coefficients between the MS2 spectra of the overlapped peaks (i.e., peaks b and c) and the mixtures of (29) Beanzinger, J. U.; Fiete, D. J. Biol. Chem. 1979, 254, 9795-9799.


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standards 210.2 and 210.3. Although it can be seen that the mixture ratio of peaks b and c is between 8:2 and 4:6 (210.2:210.3), we were unable to estimate it exactly from this curve. Figure 9 shows the MS2 spectra of standards 210.2 and 210.3 derived from each precursor ion [M + H + Na]2+ (m/z 863). Here, we focused on two specific Y-type fragment ions [M - GlcNAc + Na]+ (m/z 1522) and [M - Fuc + H + Na]2+ (m/z 790) for the same reason mentioned above. Figure 10 shows the fitting curve of relative intensities of these fragment ions ([M - GlcNAc + Na]+/[M Fuc + H + Na]2+) versus various mixtures of these isomers, and there is good linearity (R2 ) 0.9861). By using this curve and the relative intensity of 0.122 of the fragment ions ([M - GlcNAc + Na]+/[M - Fuc + H + Na]2+) on the MS2 spectrum from the coeluted peaks b and c, we obtained a mixture ratio of 65:35 (210.2: 210.3). This value nearly coincides with the mixture ratio of 62: 38 (210.2:210.3) estimated from the FL chromatogram (Figure 2A) and the mixture ratio of 63:37 estimated from the MC of m/z 863 ( 1 (Figure 2C). In addition, we applied this method to the nonfucosylated isomers of 200.2 and 200.3 (see Scheme 1), which was reported previously.20 Figure 11 shows the MS2 spectra of standards 200.2 and 200.3 derived from each precursor ion [M + H + Na]2+ (m/z 790). Here, we focused on the two specific fragment ions [M GlcNAc + Na]+ (m/z 1376) and [M - (GlcNAcPA) + Na]+ (m/z 1280). The Y-type fragment ion [M - GlcNAc + Na]+ caused by the loss of GlcNAc in the antenna and the B-type fragment ion [M - (GlcNAcPA) + Na]+ caused by the loss of reducing-end GlcNAcPA were chosen for the same reason as mentioned above. We plotted and curve fitted the relative intensities of these fragment ions ([M - GlcNAc + Na]+/[M - (GlcNAcPA) + Na]+ versus various mixtures of these isomers (see Figure 12). The obtained curve also shows good linearity (R2 ) 0.9948) as well as in the cases of the fucosylated isomers (211.2 and 211.3) and (210.2 and 210.3). Yamagaki and Tachibana20 focused on the two specific fragment ions [M - GlcNAc + Na]+ (m/z 1376) and [M 7302 Analytical Chemistry, Vol. 76, No. 24, December 15, 2004

Figure 12. Relative intensities of fragment ion ([M - GlcNAc + Na]+/[M - (GlcNAcPA) + Na]+) in MS2 spectra versus various mixtures of standards 200.2 and 200.3.

- GlcNAc - Hex + Na]+ (m/z 1214) obtained by quadrupoleTOF MS and showed a working curve with good linearity. However, this combination of fragment ions in our MS2 spectra did not show good linearity. Thus, this method seems to depend on MS and MS conditions used. Figure 13 shows structural conformations of sodium-coordinated 210.2 and 210.3 molecular ions [M + H + Na]2+, which were obtained by molecular dynamics calculation followed by molecular orbital calculation. The hydrogen ion (H+) was fixed at PA during the calculations because it was very difficult to optimize its position. The sodium ion (Na+) was always localized on the side of the antenna structure in both molecular ions, and their stabilization energies were almost the same (-3.4 eV). An important point is that PA and fucose residues do not contribute much to sodium coordination in both molecular ions. These results indicate that the B-type cleavage of fucose (Fuc) and Y-type cleavage of GlcNAc(Fuc)PA may occur in a way similar to both

quantity is very useful from a practical point of view. The method does not require time-consuming 2D HPLC separations using ODS and amide columns and, as the need arises, combining exoglycosidase digestions. It also does not require separations sufficient for peak integration. However, it should be noted that an application of this method is limited to two coeluting isomers.

Figure 13. Structural conformations of sodium-coordinated 210.2 and 210.3 molecular ions obtained by molecular dynamics and molecular orbital calculations.

the 210.2 and 210.3 molecular ions without disturbing the sodiumcoordinated residues. The results may also support our selection of the Y-type fragment ion [M - Fuc + H + Na]2+ as a reference for this quantification method in the above discussion. This explanation may be applied to both cases of the 211.2 and 211.3 molecular ions and the 200.2 and 200.3 molecular ions (in this case, the B-type fragment ion [M - (GlcNAcPA) + Na]+). From these three results, we may conclude that even if two isomers completely overlap, they can easily be identified by MSn spectral matching. They can also be roughly quantified by the relative intensity of selected fragment ions in the MS2 spectrum if the corresponding working curve of relative intensities versus various mixtures of their standards is available in the MSn spectral library. This method, which is based on RP-HPLC/MS and the MSn spectral library, with the capabilities of both assignment and

CONCLUSIONS In this study, we have investigated how the PA-oligosaccharide MSn spectral library and MSn spectral matching previously proposed18 can be applied to real oligosaccharide analysis, by using N-glycans of IgG as a model sample and, in particular, by focusing on two pairs of isomers incompletely separated or coeluted on a RP-HPLC chromatogram. To summarize, there are three important conclusions. First, we demonstrated that MSn spectral library data acquired by standard PA-oligosaccharides under simple separation conditions (i.e., a short column and water/methanol solution) can be used for the assignment of PA-oligosaccharides analyzed in different HPLC separation conditions. Second, we confirmed that peak assignment by MSn spectral matching is more reliable than that obtained by the conventional GU index. Third, we pointed out that the two isomers that are not sufficiently separated on a chromatogram can be assigned and quantified if the MSn spectral library contains the corresponding working curve that includes the relative intensities of the selected fragment ions. These results are significant and useful in developing an efficient RP-HPLC/MS method based on the MSn spectral library for oligosaccharides, which is currently underway in our laboratory. ACKNOWLEDGMENT This work was supported in part by the National Project on Functional Glycoconjugate Research Aimed at Developing New Industry from the Ministry of Education, Science, Sport and Culture of Japan. Parts of this study were presented at the 52nd ASMS Conference on Mass Spectrometry, Nashville, TN, 2004. Received for review May 9, 2004. Accepted September 18, 2004. AC0493166


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Assignment and Quantification of 2-Aminopyridine Derivatized

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