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Facile MALDI-MS Analysis of Neutral Glycans in NaOH-Doped Matrixes: Microwave-Assisted Deglycosylation and One-Step Purification with Diamond Nanoparticles Yan-Kai Tzeng,† Cheng-Chun Chang,†,‡ Chien-Ning Huang,§ Chih-Che Wu,§ Chau-Chung Han,†,| and Huan-Cheng Chang*,†,‡,| Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 106, Taiwan, Department of Chemistry, National Taiwan Normal University, Taipei 106, Taiwan, Department of Applied Chemistry, National Chi Nan University, Puli, Nantou 545, Taiwan, and Genomics Research Center, Academia Sinica, Taipei 115, Taiwan A streamlined protocol has been developed to accelerate, simplify, and enhance matrix-assisted laser desorption/ ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) of neutral underivatized glycans released from glycoproteins. It involved microwave-assisted enzymatic digestion and release of glycans, followed by rapid removal of proteins and peptides with carboxylated/oxidized diamond nanoparticles, and finally treating the analytes with NaOH before mixing them with acidic matrix (such as 2,5dihydroxybenzoic acid) to suppress the formation of both peptide and potassiated oligosaccharide ions in MS analysis. The advantages of this protocol were demonstrated with MALDI-TOF-MS of N-linked glycans released from ovalbumin and ribonuclease B. Much effort has been made in the past to analyze neutral glycans released from glycoproteins and glycolipids, or neutral underivatized oligosaccharides in general, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF-MS).1-3 However, up to now, the sensitivity achieved for MS analysis of neutral glycans still lags far behind that for peptides and oligonucleotides by nearly 2 orders of magnitude. Additionally, the recorded mass spectra are always complicated by the presence of both Na+ and K+ adduct ions. Moreover, the procedures involved in the glycan release are time-consuming and the ensuing sample separations are laborious. Therefore, there is a need for further improvement of the glycan release, purification, and analysis methods since the availability of MS-based technologies with both high-sensitivity and high-throughout potentials is crucial in delineating the processes of glycosylation, which is one of the * To whom correspondence should be addressed. E-mail: hcchang@ po.iams.sinica.edu.tw. † Institute of Atomic and Molecular Sciences, Academia Sinica. ‡ National Taiwan Normal University. § National Chi Nan University. | Genomics Research Center, Academia Sinica. (1) Harvey, D. J. Mass Spectrom. Rev. 1999, 18, 349–450. (2) Zaia, J. Mass Spectrom. Rev. 2003, 23, 161–227. (3) Harvey, D. J. Mass Spectrom. Rev. 2006, 25, 595–662. 10.1021/ac801137g CCC: $40.75 2008 American Chemical Society Published on Web 08/01/2008
most important but yet complex steps in post-translational modifications of proteins.4,5 To facilitate the glycan analysis, accelerating the enzymecatalyzed deglycosylation is the first hurdle to overcome. A recent experiment demonstrated that rapid removal of N-linked glycans can be achieved by microwave-assisted enzymatic reaction with peptide N-glycosidase F (PNGase F).6 Complete deglycosylation is achievable within 30 min, much faster than conventional deglycosylation methods. Such a technological advancement, together with microwave-enhanced tryptic digestion,7,8 makes it possible to shorten substantially the total analysis time for glycans if the accompanied sample isolation and purification procedures can also be simplified. We have previously developed a new solidphase extraction (SPE) platform based on diamond nanocrystallites surface-functionalized with carboxyl and other oxygen-containing groups produced by strong oxidative acid treatments.9-13 These particles (typically 100 nm in diameter) show an exceptionally high affinity for proteins and their enzymatic digests even in highly contaminated environments at low solution pHs. This high affinity for proteins and peptides is established by the interplay of electrostatic forces, hydrogen bonding, and hydrophobic interactions between adsorbent and adsorbate. Contrary to porous graphitized carbon columns, which have been widely used to isolate and purify glycans released from glycoproteins,14-19 the (4) Dell, A.; Morris, H. R. Science 2001, 291, 2351–2356. (5) Turnbull, J. E.; Field, R. A. Nat. Chem. Biol. 2007, 3, 74–77. (6) Sandoval, W. N.; Arellano, F.; Arnott, D.; Raab, H.; Vandlen, R.; Lill, J. R. Int. J. Mass Spectrom. 2007, 259, 117–123. (7) Pramanik, B. N.; Mirza, U. A.; Ing, Y. H.; Liu, Y. H.; Bartner, P. L.; Weber, P. C.; Bose, M. K. Protein Sci. 2002, 11, 2676–2687. (8) Lill, J. R.; Ingle, E. S.; Liu, P. S.; Pham, V.; Sandoval, W. N. Mass Spectrom. Rev. 2007, 26, 657–671. (9) Huang, L. C. L.; Chang, H.-C. Langmuir 2004, 20, 5879–5884. (10) Kong, X. L.; Huang, L. C. L.; Hsu, C.-M.; Chen, W.-H.; Han, C.-C.; Chang, H.-C. Anal. Chem. 2005, 77, 259–265. (11) Chen, W.-H.; Lee, S.-C.; Sabu, S.; Fang, H.-C.; Chung, S.-C.; Han, C.-C.; Chang, H.-C. Anal. Chem. 2006, 78, 4228–4234. (12) Sabu, S.; Yang, F.-C.; Wang, Y.-S.; Chen, W.-H.; Chou, M.-I.; Chang, H.-C.; Han, C.-C. Anal. Biochem. 2007, 367, 190–200. (13) Ngyuen, T. T. B.; Chang, H.-C.; Wu, V. W.-K. Diamond Relat. Mater. 2007, 16, 872–876. (14) Knox, J. H.; Kaur, B.; Millward, G. R. J. Chromatogr. 1986, 352, 3–25. (15) Koizumi, K.; Okada, Y.; Fukuda, M. Carbohydr. Res. 1991, 215, 67–80. (16) Fan, J. Q.; Kondo, A.; Kato, I.; Lee, Y. C. Anal. Biochem. 1994, 219, 224– 229.
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carboxylated/oxidized diamond nanoparticles exhibit a much lower affinity for oligosaccharides (both neutral and acidic) than for proteinaceous compounds in aqueous solutions. Such a unique property permits utilization of these particles as SPE substrates to achieve selective and rapid removal of proteins and peptides in the deglycosylation mixture prior to MS analysis (vide infra). A complication inherent in MALDI-MS analysis of neutral underivatized oligosaccharides is that ionic complexes of Na+ and K+ are concurrently observed in the mass spectra due to the unavoidable presence of these two ions in sample, solution, and matrix. Their appearance not only complicates interpretation of the recorded spectra but also decreases the intensities of the individual peaks. Similar situations occurred when inorganic substrates such as TiO2 sol-gels20 and gold nanoparticles21 were used as the matrixes. A common practice to alleviate this problem is to add an excess amount of alkaline metal salts such as NaCl and LiCl to promote the formation of the adduct ions of interest, while suppressing unwanted species.22-25 However, because of the presence of the excess amount of salts in the matrix, the sensitivity of the MS analysis was limited and a sample loading of up to 1 nmol was often required. This work explores the feasibility of using NaOH-doped matrixes, such as NaOH in 2,5-dihydroxybenzoic acid (DHBA), to achieve spectral simplification by suppressing K+ adduct ion formation. DHBA was chosen in this work because the matrix has been shown to yield higher signal-to-noise ratios and better signal reproducibility than sinapinic acid and other acidic matrixes.26,27 The matrix salt resulted from NaOH titration, on the other hand, was employed for the first time in this study. A previous work has examined the possibility of using matrix composed of DHBA and its potassium salt (denoted as DHB-K) for MALDI-MS of synthetic polar polymers. Specifically, Dogruel et al.28 investigated the effects of pH and cation availability on desorption/ionization of poly(methyl methacrylate) through titration of the organic acid matrix with KOH. They found that DHBA titrated with KOH up to pH 4 could yield good signals of K+ adduct ions, whereas the desorption and/or ionization ability of the mixed matrix was lost when pure DHB-K was used. Herein, we report our experimental results obtained using NaOH-doped DHBA as matrix for MALDI-TOF-MS of glycans released from glycoproteins. Three salient features of the doped (17) Packer, N. H.; Lawson, M. A.; Jardine, D. R.; Redmond, J. W. Glycoconjugate J. 1998, 15, 737–747. (18) Cheng, H.-L.; Pai, P.-J.; Her, G.-R. J. Am. Soc. Mass Spectrom. 2007, 18, 248–259. (19) Liu, X.; Li, X.; Chan, K.; Zou, W.; Pribil, P.; Li, X.-F.; Sawyer, M. B.; Li, J. Anal. Chem. 2007, 79, 3894–3900. (20) Chen, C.-T.; Chen, Y.-C. Anal. Chem. 2004, 76, 1453–1457. (21) Wu, H.-P.; Su, C.-L.; Chang, H.-C.; Tseng, W.-L. Anal. Chem. 2007, 79, 6215–6221. (22) Stahl, B.; Steup, M.; Karas, M.; Hillenkamp, F. Anal. Chem. 1991, 63, 1463– 1466. (23) Bartsch, H.; Ko ¨nig, W. A.; Strassner, M.; Hintze, U. Carbohydr. Res. 1996, 286, 41–53. (24) North, S.; Okafo, G.; Birrell, H.; Haskins, N.; Camilleri, P. Rapid Commun. Mass Spectrom. 1997, 11, 1635–1642. (25) Pfenninger, A.; Karas, M.; Finke, B.; Stahl, B.; Sawatzki, G. J. Mass Spectrom. 1999, 34, 98–104. (26) Strupat, K.; Karas, M.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1991, 111, 89–102. (27) Hao, C. Y.; Ma, X. L.; Fang, S. P.; Liu, Z. Q.; Liu, S. Y.; Song, F. R.; Liu, J. Z. Rapid Commun. Mass Spectrom. 1998, 12, 345–348. (28) Dogruel, D.; Nelson, R. W.; Williams, P. Rapid Commun. Mass Spectrom. 1996, 10, 801–804.
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matrix are presented. First, we show that DHB-Na is a useful component of MALDI matrix in the analysis of neutral oligosaccharides. The addition of ∼2% (mole percentage) of NaOH in DHBA, yielding DHB-Na, significantly enhances the detection sensitivity. Second, we demonstrate that the undesirable K+ adduct ions of neutral oligosaccharides can be suppressed nearly completely in the mass spectra had the analytes first been treated with NaOH before being mixed with the pure DHBA matrix. Third, we show that doping the MALDI matrix with NaOH effectively diminishes the interfering signals arising from proteins and peptide fragments coexisting in the samples. This last feature has a significant impact on MS analysis of glycans released from glycoproteins, since these analytes are often contaminated with residual proteins and protein digests. The protocol developed in this work consists of three parts: (1) microwave-assisted digestion of glycoproteins with trypsin, followed by microwave-assisted glycan release with PNGase F, (2) rapid removal of proteins and resulting tryptic digests with carboxylated/oxidized diamond nanoparticles, and (3) suppression of peptide and potassiated oligosaccharide ions by use of NaOHdoped matrixes. The protocol is designed to be simple to implement and can be completed in a tube in 2 h prior to MS analysis. The oligosaccharides, peptides, and glycoproteins employed for development and testing of this protocol include β-cyclodextrin (β-CD), maltoheptaose (G7), angiotensin I (A-I), ribonuclease B (RNase B), and ovalbumin. EXPERIMENTAL SECTION Chemicals and Materials. Synthetic diamond powders with a nominal size of 100 nm (Micron+, MDA) were produced by General Electric. High-purity DHBA was obtained from BrukerDaltonics. Acetonitrile, sodium hydroxide, DHBA sodium salt, 2-mercaptoethanol, β-cyclodextrin (MW 1135.0 Da), maltoheptaose (MW 1153.0 Da), angiotensin I (MW 1296.5 Da), chicken ovalbumin, and bovine pancreatic ribonuclease B were purchased from Sigma. Trypsin was received from Promega, and PNGase F was from New England BioLabs. All the chemicals were used without further purification. Diamond Surface Functionalization. Diamond powders were purified and surface-functionalized with oxygen-containing groups in concentrated H2SO4-HNO3 solution (3:1, v/v) at 100 °C in a microwave reactor (Discover BenchMate, CEM) for 3 h.9,29 After the microwave treatment, the diamond nanoparticles were recovered by centrifugation, rinsed extensively with deionized water (Millipore), and resuspended in water at a concentration of 20 mg/mL before use. Glycan Release. Glycoproteins (typically 40 µg) were reduced in 50 mM NH4HCO3 solution (60 µL) containing 1.5% 2-mercaptoethanol at 100 °C for 15 min, after which the protein solution was mixed with 40 µL of deionized water containing 4 µg of trypsin. Tryptic digestion occurred at 50 °C for 10 min in a microwave reactor (Discover BenchMate, CEM) with a power setting of 60 W.7,30 The digestion reaction was followed by microwave-assisted deglycosylation using PNGase F (40 units) in the same NH4HCO3 solution at 37 °C for 30 min. A microwave power of 20 W was typically applied in the glycan release.6 (29) Wang, Y.; Iqbal, Z.; Mitra, S. J. Am. Chem. Soc. 2006, 128, 95–99. (30) In cases of tryptic digestion of larger glycoproteins such as transferrin, a microwave power setting of up to 100 W has to be used.
Enzymatic release of glycans was also conducted in parallel using conventional methods.17-19 In this comparative experiment, glycoproteins (40 µg) were first reduced by 2-mercaptoethanol (1.5%) in 50 mM NH4HCO3 solution. After heat treatment at 100 °C for 15 min, the reduced protein was digested by trypsin (4 µg) at 50 °C for 2 h and deglycosylated with PNGase F (40 units) at 37 °C for 19 h in the same NH4HCO3 solution without microwave irradiation. Glycan Purification. Approximately 300 µL of acid-treated diamond nanoparticle suspensions (20 mg/mL) was mixed with the aforementioned glycan solution (100 µL) containing tryptic digests and residual enzymes. The mixture was then incubated with 70 µL of 20% formic acid in an Eppendorf centrifuge tube at room temperature for 10 min with gentle vortexing. After separation by centrifugation at 15 000 rpm for 5 min and careful removal of the diamond pellet, the glycan-containing supernatant was analyzed directly by MALDI-TOF-MS. MALDI Sample Preparation. DHBA and DHB-Na were dissolved separately in a 1:2 (v/v) acetonitrile/water solution and made to a final concentration of 20 mM. To prepare the sample targets for MALDI, 1 µL of the analyte solution and 1 µL of the matrix solution (pure DHBA or DHB-Na/DHBA mixtures at various molar ratios) were mixed directly on a stainless steel MALDI plate and dried rapidly in vacuum within 30 s. In experiments using NaOH-doped DHBA as matrix, 1 µL of the analyte solution (typically 1 µM) was first mixed with 1 µL of NaOH solution (typically 1-2 mM) in an Eppendorf tube at room temperature for 3 min. One-half of the mixture (1 µL) was then spotted together with 1 µL of 20 mM DHBA on the MALDI plate and rapidly vacuum-dried for MS analysis. TOF Mass Spectrometry. MS spectra were obtained with a reflectron TOF mass spectrometer (Microflex, Bruker-Daltonics). The spectrometer was operated in positive ion mode at an extraction voltage of 19 kV and an extraction delay of 340 ns. Desorption and ionization of the analyte were accomplished by using a 337 nm nitrogen laser at a repetition rate of 5 Hz scanning randomly across the sample surface. The typical laser fluence applied was in the range of 300 mJ/cm2, which was about 30% higher than that typically used for peptide analysis. All the reported MS spectra were acquired with the spectrometer operated in the reflectron mode by averaging the ion signals from 150 laser shots. RESULTS AND DISCUSSION The key material that makes this protocol special is the oxidative-acid-treated diamond nanoparticles. These particles are surface-functionalized with carboxyl, carbonyl, ether, and other oxygen-containing groups.9 We have previously characterized these functional groups by using infrared spectroscopy and determined a content of ∼7% for the carboxyl groups by conductometric titration.13 The negative charge state of the carboxylated surface at solution pH above 3 was further confirmed by ζ-potential analysis.13 Containing a rich variety of functional groups, the carboxylated/oxidized diamond nanoparticles exhibit a remarkably high affinity for both proteins and peptides in aqueous solution acidified with 1% (or higher) of formic acid.11 Figure 1 displays MALDI-TOF mass spectra of 1:1 mixtures of β-CD and A-I extracted with the acid-treated diamond nanoparticles. In this experiment, approximately 300 µL of the diamond
Figure 1. MALDI-TOF mass spectra of 1:1 mixtures of β-cyclodextrin (β-CD) and angiotensin I (A-I) extracted with carboxylated/oxidized diamond nanoparticles in aqueous solution containing 3% formic acid. Both the supernatant (a) and the precipitate (b) of the mixtures after separation by centrifugation were analyzed. The loading of each sample on the target plate was ∼1 pmol.
particle suspension (1 mg/mL) was mixed with 100 µL of the β-CD/A-I sample solution (5 × 10-6 M each). After incubation for 5 min, the mixture was separated by centrifugation and both the supernatant (Figure 1a) and the precipitate (Figure 1b) were analyzed. In accord with previous findings,11 the majority of A-I was not captured by the particles at neutral or higher pHs where tryptic digestion was typically carried out (see Figure S1 in the Supporting Information). However, adsorption of the peptides to the diamond nanoparticles occurred upon addition of 3% formic acid (pH < 2) into the sample solution. Within the detection limit of the mass spectrometer used, no sign of adsorption was found for the oligosaccharide over the pH range investigated. The result illustrates a unique selective extraction ability of the surfacefunctionalized diamond nanoparticles for peptides or small proteins, but not for neutral oligosaccharides,31 in acidic solution. The diamond-based SPE protocol was first tested with glycans released from ovalbumin through conventional methods. Shown in Figure 2a is the mass spectrum of a solution containing both the glycans released enzymatically and the tryptic digests of ovalbumin using pure DHBA as MALDI matrix. The peaks appearing in the m/z range of 1200-2200 are contributed exclusively by nonglycosylated peptide ions. Upon depletion of the peptides with carboxylated/oxidized diamond nanoparticles, ions arising from glycans became dominant in the mass spectrum (31) The surface-functionalized diamond nanoparticles can also be applied to extract peptides or small proteins selectively in solution containing acidic (sialylated) glycans released from glycoproteins such as transferrin (refs 41 and 42). In this case, no doping of NaOH in the MALDI matrix is required if negative ion mass spectra are to be obtained (see Figure S6 in the Supporting Information for details).
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Figure 2. MALDI-TOF mass spectra of peptide-glycan mixtures resulted from enzymatic digestion and deglycosylation of ovalbumin (a) before and (b) after peptide depletion with carboxylated/oxidized diamond nanoparticles. Asterisks denote peaks derived from glycan ions (ref 32).
(Figure 2b). Although many of the peaks (denoted by asterisks) can be assigned to the Na+ adduct ions of oligosaccharides,32,33 some peaks originating from K+ adduct ions and residual peptide ions are also present. The appearance of these peaks made clearcut identification of the glycan ions very difficult, if not totally impossible. To eliminate both types of interference, enrichment of the DHBA matrix with Na+ by adding either DHB-Na or NaOH was attempted. Figure 3a shows a typical MALDI mass spectrum of 1 pmol of β-CD using pure DHBA as matrix. The peak dominating the mass spectrum is that of the Na+ adduct ion (denoted as [β-CD + Na]+) at m/z 1158, accompanied with K+ adduct ions (denoted as [β-CD + K]+) appearing as a weaker feature at m/z 1174. The latter was significantly attenuated as DHB-Na was included as a minor component (2-5% in molar ratio) in pure DHBA (Figure 3b). Further increase of the salt content up to 50% eliminated the [β-CD + K]+ peak completely; however, the tradeoff was that the ion intensity of the main feature decreased by a factor of ∼3 accordingly (see Figure S2 in the Supporting Information). In order to suppress [β-CD + K]+ completely without sacrificing the ion intensity of [β-CD + Na]+, we treated the analyte first with NaOH, followed by addition of DHBA to neutralize the base to form DHB-Na. A representative result of the MS measurement using this new sample preparation approach for β-CD is shown in Figure 3c. As seen, the mass spectrum was free of K+ adduct ions (
