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Desorption Electrospray Ionization Mass Spectrometry of Glycosaminoglycans and Their Protein Noncovalent Complex C. Przybylski,† F. Gonnet,† Y. Hersant,‡ D. Bonnaffe´,‡ H. Lortat-Jacob,§ and R. Daniel*,† CNRS UMR 8587, Universite´ d’Evry-Val-d’Essonne, Laboratoire Analyse et Mode´lisation pour la Biologie et l’Environnement, F-91025 Evry, France, CNRS UMR 8182, Universite´ d’Orsay, Laboratoire de Chimie Organique Multifonctionnelle, ICMO, 91405 Orsay Cedex, France, and Institut de Biologie Structurale, CNRS, CEA, Universite´ Joseph Fourier, UMR 5075, 38027 Grenoble Cedex, France Glycosaminoglycans heparin and heparan sulfate are biologically active polysulfated carbohydrates that are among the most challenging biopolymers with regards to their structural analysis and functional assessment. Fragmentation of oligosaccharides and sulfate loss are important hindrance to their analysis by mass spectrometry (MS), requiring thus soft ionization methods. The recently introduced soft ionization method desorption electrospray ionization (DESI) has been applied here to heparin and heparan sulfate oligosaccharides, showing that DESI-MS is well suited for the detection of such fragile biomolecules in their intact form. Characterization of complicated oligosaccharides such as synthetic heparin octadecasulfated dodecasaccharide was successfully achieved. The use of water for a spray solvent instead of denaturing organic solvents allowed the first DESI-MS detection of noncovalent biomolecular complexes between heparin oligosaccharides and the chemokine Stromal Cell-derived Factor-1. The hyphenation of the DESI ion source with the high-resolution LTQ-Orbitrap MS analyzer led to high accuracy of mass measurement and enabled unambiguous determination of the protein-bound sulfated oligosaccharide. Glycosaminoglycans (GAGs), expressed at the cell surface and in the extra-cellular matrix, mediate cell-cell and cell-matrix interactions at the origin of a variety of physiological and pathological functions such as in embryonic development, cell growth and differentiation, homeostasis, inflammatory response, tumor growth, and microbial infection.1 The emblematic GAGs heparan sulfate (HS) is a highly anionic linear polysaccharide made of about 20-200 disaccharide repeating units being comprised of an hexuronic acid and a N-acetyl glucosamine (GlcNAc) substituted with sulfate groups in various positions. During the HS biosynthesis, the hexuronic acid, initially D-glucuronic acid (GlcA), can be epimerized at the C-5 position to give L-iduronic acid (IdoA), and both of them may be sulfated at the C-2 position * To whom correspondence should be addressed. Fax: +33-1-69-47-76-55. E-mail:
[email protected]. † Universite´ d’Evry-Val-d’Essonne. ‡ Universite´ d’Orsay. § Universite´ Joseph Fourier. (1) Bishop, J. R.; Schuksz, M.; Esko, J. D. Nature 2007, 446, 1030–1037. 10.1021/ac1016198 2010 American Chemical Society Published on Web 10/28/2010
while the N-acetyl glucosamine residues may be O-sulfated (at the C-6 and more rarely at the C-3 position) and N-sulfated after de-N-acetylation. In some cases, deacetylation is not followed by re-N-sulfation, and the amine group remains unsubstituted. In addition to this variable pattern of sulfate substitution, clusters of N-sulfated disaccharides rich in IdoA and O-sulfo groups organize the polysaccharide chain in highly sulfated domains (NS), usually 3 to 8 disaccharide repeating units, flanked with intermediate sulfated domains made of N-acetylated and N-sulfated disaccharides (NA/NS domains) and N-acetylated domains lacking sulfated residues (NA). It results in a highly heterogeneous molecular chain exhibiting variable length, sulfation pattern, and domain organization, thus making HS as one of the most challenging biopolymer with regard to structural analysis and functional assessment.2 Heparin (HP), a widely used anticoagulant, is often studied as a HS variant featuring a more homogeneous and highly sulfated structure than HS, thus representative of the highly sulfated NS domain.3 Most of the GAGs functions are mediated by the binding to target proteins through interactions driven by the specific recognition of structural determinants like the monosaccharide sequence and sulfation pattern.4,5 A better understanding of the structural and binding properties of GAGs is required for the development of GAGs mimetics as possible new glycotherapeutics. Therefore, the increasing interest in protein-carbohydrate interactions is accompanied by a challenging demand for viable, accurate, and high-throughput methodologies for their characterization.6,7 During the past decade, mass spectrometry (MS) has been widely recognized as a powerful and highly sensitive method for the structural analysis of carbohydrates, and decisive progresses are expected from recent MS developments for deciphering the huge informational content of HS.8 To date, electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) (2) Sasisekharan, R.; Raman, R.; Prabhakar, V. Annu. Rev. Biomed. Eng. 2006, 8, 181–231. (3) Rabenstein, D. Nat. Prod. Rep. 2002, 19, 312–331. (4) Gandhi, N. S.; Mancera, R. L. Chem. Biol. Drug Des. 2008, 72, 455–482. (5) Skidmore, M.; Guimond, S.; Rudd, T.; Fernig, D.; Turnbull, J.; Yates, E. Connect. Tissue Res. 2008, 49, 140–144. (6) Fermas, S.; Gonnet, F.; Varenne, A.; Gareil, P.; Daniel, R. Anal. Chem. 2007, 79, 4987–4993. (7) Ratner, D. M.; Adams, E. W.; Disney, M. D.; Seeberger, P. H. ChemBioChem 2004, 5, 1375–1383. (8) Zaia, J. Mass Spectrom. Rev. 2004, 23, 161–227.
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have been the most employed ionization techniques for the characterization of oligosaccharides.8 These methods require the samples to be prepared prior to analysis, i.e., dissolved in solvent and electrosprayed for ESI or mixed with well-suited matrix and usually placed into a vacuum system for MALDI. Furthermore, poor ionization efficiency of the sulfated oligosaccharides are often observed upon MALDI-MS analysis with the commonly used matrixes, despite improvements provided by the emerging ionic liquid matrixes.9-12 Soft ionization methods are required for the MS analysis of GAGs oligosaccharides because of the labile feature of their sulfate groups. The recently introduced soft ionization method desorption electrospray ionization (DESI)13 allows the in situ analysis of samples in ambient atmosphere with minimal preparation and exhibits high salt tolerance as compared to usual (nano)electrospray.14 In the DESI experiment, the sample is placed onto a surface without chemical or physical modification and is scanned at atmospheric pressure by a high-velocity gas jet containing electrically charged droplets. Multiply charged ions are then generated from the analytes contained in the sample, which can be analyzed by different mass spectrometer analyzers, such as linear ion trap (LIT),15-19 quadrupole ion trap (QIT),20-22 Fourier transform ion cyclotron resonance (FTICR),23,24 or ion mobility-time of flight module (IM-TOF).25 Most of the DESI experiments reported to date concern the analysis of explosives,26,27 pharmaceutical formulations,17,28 and the identification of metabo-
(9) Laremore, T. N.; Murugesan, S.; Park, T.-J.; Avci, F. Y.; Zagorevski, D. V.; Linhardt, R. J. Anal. Chem. 2006, 78, 1774–1779. (10) Laremore, T. N.; Zhang, F.; Linhardt, R. J. Anal. Chem. 2007, 79, 1604– 1610. (11) Przybylski, C.; Gonnet, F.; Bonnaffe´, D.; Hersant, Y.; Lortat-Jacob, H.; Daniel, R. Glycobiology 2010, 20, 224–234. (12) Tissot, B.; Gasiunas, N.; Powell, A. K.; Ahmed, Y.; Zhi, Z.-l.; Haslam, S. M.; Morris, H. R.; Turnbull, J. E.; Gallagher, J. T.; Dell, A. Glycobiology 2007, 17, 972–982. (13) Taka´ts, Z.; Wiseman, J. M.; Gologan, B.; Cooks, R. G. Science 2004, 306, 471–473. (14) Jackson, A. U.; Talaty, N.; Cooks, R. G.; Van Berkel, G. J. J. Am. Soc. Mass Spectrom. 2007, 18, 2218–2225. (15) Taka´ts, Z.; Wiseman, J. M.; Cooks, R. G. J. Mass Spectrom. 2005, 40, 1261– 1275. (16) Nyadong, L.; Green, M. D.; De Jesus, V. R.; Newton, P. N.; Fernandez, F. M. Anal. Chem. 2007, 79, 2150–2157. (17) Huang, G.; Chen, H.; Zhang, X.; Cooks, R. G.; Ouyang, Z. Anal. Chem. 2007, 79, 8327–8332. (18) Cotte-Rodriguez, I.; Cooks, R. G. Chem. Commun. 2006, 2968–2970. (19) Kauppila, T. J.; Talaty, N.; Jackson, A. U.; Kotiaho, T.; Kostiainen, R.; Cooks, R. G. Chem. Commun. 2008, 23, 2674–2676. (20) Shin, Y.-S.; Drolet, B.; Mayer, R.; Dolence, K.; Basile, F. Anal. Chem. 2007, 79, 3514–3518. (21) Lane, A. L.; Nyadong, L.; Galhena, A. S.; Shearer, T. L.; Stout, E. P.; Parry, R. M.; Kwasnik, M.; Wang, M. D.; Hay, M. E.; Fernandez, F. M.; Kubanek, J. Proc. Natl. Acad. Sci. U.S.A 2009, 106, 7314–7319. (22) Zhang, Y.; Chen, H. Int. J. Mass Spectrom. 2010, 289, 98–107. (23) Bereman, M. S.; Nyadong, L.; Fernandez, F. M.; Muddiman, D. C. Rapid Commun. Mass Spectrom. 2006, 20, 3409–3411. (24) Bereman, M. S.; Williams, T. I.; Muddiman, D. C. Anal. Chem. 2007, 79, 8812–8815. (25) Myung, S.; Wiseman, J. M.; Valentine, S. J.; Taka´ts, Z.; Cooks, R. G.; Clemmer, D. E. J. Phys. Chem. B 2006, 110, 5045–5051. (26) Cooks, R. G.; Ouyang, Z.; Taka´ts, Z.; Wiseman, J. M. Science 2006, 311, 1566–1570. (27) Taka´ts, Z.; Cotte-Rodriguez, I.; Talaty, N.; Chen, H. W.; Cooks, R. G. Chem. Commun. 2005, 1950–1952. (28) Chen, H.; Talaty, N. N.; Taka´ts, Z. n.; Cooks, R. G. Anal. Chem. 2005, 77, 6915–6927.
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lites and biomarkers in biological fluids and tissues.18,21,29-31 Only a few papers have reported the successful use of DESI for the analysis of carbohydrates, mostly neutral saccharides, but none of them about sulfated sugars. The MS detection of oligosaccharides was usually achieved through their cation adducts15,19 or using chemical reagents in the spray solvent.15,22,24,30 There are also a few reports on DESI analysis of proteins in nondenaturing conditions.13,15,20,23,25,32 In the study herein, we report the first DESI-MS analysis of heparan sulfate and heparin oligosaccharides as well as their noncovalent complex with the chemokine Stromal Cell-Derived Factor-1 (SDF-1R). The hyphenation of this ambient soft ionization mode with the high-resolution LTQ-Orbitrap analyzer opens the way to an easy method for characterization of biomolecular noncovalent complexes and unambiguous determination of the ligand bound to the complex. EXPERIMENTAL SECTION Chemicals and Biological Samples. Aprotinin (Mw 6511.44 g mol-1) was purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France) and prepared at a concentration range from 1 to 200 µM in water. Recombinant SDF-1 (SDF-1R, residue 1-68, Mw 7963.43 g mol-1) was purchased from PeproTech (Neuilly-Sur-Seine, France) and was diluted to 75 µM in 75 mM ammonium acetate, pH 6.5. The mutant SDF-1 (3/6) (Mw 7698.96 g mol-1) (25 µM in water) in which basic residues Lys,24 His,25 and Lys27 in clusters were substituted with a Ser residue and Lys68 was suppressed, has been synthesized as previously described,33 and was kindly provided by F. Balleux (Institut Pasteur, Paris, France). Synthetic heparin hexasulfated tetrasaccharide and octadecasulfated dodecasaccharide (Figure 1) were obtained by combinatorial34 or conventional chemical synthesis as previously reported.35,36 HP and HS tetra- and octaoligosaccharides were prepared by enzymatic depolymerization using, respectively, heparinase I on porcine mucosal HP and heparinase III on HS as described previously.11,37 Other chemicals and reagents were obtained from Sigma-Aldrich (Saint-Quentin Fallavier, France) at the highest purity available. All buffers were prepared using ultrapure water (Milli-Q, Millipore, Milford) and degassed by filtration through 0.2 µm filter units before use. DESI Mass Spectrometry. DESI-MS experiments were carried out using a LTQ-Orbitrap XL from Thermo Scientific (San Jose, CA) equipped with the DESI ion source Omnispray from Prosolia (Indianapolis, IN). The experimental set included a source (29) Wiseman, J. M.; Ifa, D. R.; Zhu, Y. X.; Kissinger, C. B.; Manicke, N. E.; Kissinger, P. T.; Cooks, R. G. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 18120–18125. (30) Manicke, N. E.; Nefliu, M.; Wu, C.; Woods, J. W.; Reiser, V.; Hendrickson, R. C.; Cooks, R. G. Anal. Chem. 2009, 81, 8702–8707. (31) Wu, C. R.; Ifa, D.; Manicke, N. E.; Cooks, R. G. Analyst 2010, 135, 28–32. (32) Taka´ts, Z.; Wiseman, J. M.; Ifa, D. R.; Cooks, R. G. Cold Spring Harbor Protocols 2008, DOI:, 10.1101/pdb.prot4992. (33) Amara, A.; Lorthioir, O.; Valenzuela, A.; Magerus, A.; Thelen, M.; Montes, M.; Virelizier, J.-L.; Delepierre, M.; Baleux, F. O.; Lortat-Jacob, H.; ArenzanaSeisdedos, F. J. Biol. Chem. 1999, 274, 23916–23925. (34) Dilhas, A.; Lucas, R.; Loureiro-Morais, L.; Hersant, Y. l.; Bonnaffe´, D. J. Comb. Chem. 2008, 10, 166–169. (35) Baleux, F.; Loureiro-Morais, L.; Hersant, Y.; Clayette, P.; ArenzanaSeisdedos, F.; Bonnaffe´, D.; Lortat-Jacob, H. Nat. Chem. Biol. 2009, 5, 743–748. (36) Lubineau, A.; Lortat-Jacob, H.; Gavard, O.; Sarrazin, S.; Bonnaffe´, D. Chem.sEur. J. 2004, 10, 4265–4282. (37) Vives, R. R.; Sadir, R.; Imberty, A.; Rencurosi, A.; Lortat-Jacob, H. Biochemistry 2002, 41, 14779–14789.
Figure 1. Structure of the synthetic heparin tetrasaccharide (A) and dodecasaccharide (B) used in this study. The theoretical monoisotopic and average masses of the fully sodium form of hexasulfated tetrasaccharide are 1389.8557 and 1390.8949 g mol-1, respectively. The theoretical monoisotopic and average masses of the fully sodium form of octadecasulfated dodecasaccharide are 4092.4944 and 4095.5624 g mol-1, respectively.
emitter in fused silica (0.05 mm i.d., 0.15 mm o.d., 40 mm length) and an extended ion transfer tube for LTQ apparatus. A 250 µL syringe was used to spray solvent at 2.5 µL min-1 onto the DESI surface. The ESI spray voltage was +4.5 kV in positive ionization mode and -4.5 kV in negative ionization mode. Applied voltages were ±30 and ±100 V for the ion transfer capillary and the tube lens, respectively. The ion transfer capillary was held at 200 °C. The nebulizing gas (N2) pressure was 7.5 bar. The emitter was protruding 0.5 mm outward from the nebulizing device. The angle (R) between the spray and the DESI surface was set to 60°, and the ion uptake angle (β) was set to 5°. The tip-to-surface distance (d1), the tip-to-inlet distance (d2), and the surface-to-inlet distance (d3) were set to 1.5, 4, and 2 mm, respectively.15 The spraying solvents were methanol/water 1/1 for carbohydrate characterization and ultrapure water or ammonium acetate buffer as specified for protein/oligosaccharide complexes. MS analyses were performed in the negative ion mode for the sulfated oligosaccharides and in the positive ion mode for protein/oligosaccharide complexes. The total ion current was recorded in the Orbitrap mass analyzer. The automatic gain control, maximum injection time, and µscans number were set at 1 × 106, 500 ms, and 1, respectively. Resolving power was set to 30 000 (at m/z 400) for all studies, and the m/z ranges were set to 150-2000 (standard mass range) for the detection of saccharides and to 500-4000 in the high mass range for both protein and protein/ sugar complexes detection. Spectra were averaged over at least 0.20 min (12 s, 24 scans), without smoothing and background subtract. DESI mass spectra were analyzed using the Xtract deconvolution module (Thermo Scientific) provided with the acquisition software (XCalibur 2.0.7, Thermo Scientific, San Jose, CA). Sample Preparation. Synthetic heparin hexasulfated tetrasaccharide and octadecasulfated dodecasaccharide were dissolved in ultrapure water at 100 and 244 µM, respectively. HS and HP derived oligosaccharides were dissolved in ultrapure water at 3 and 1 mg/mL, respectively. The concentration of the aqueous oligosaccharide solutions were determined by a modified uronic acid carbazole reaction,38 measuring the absorbance at 530 nm using a nanodrop 1000 spectrophotometer (Thermo Scientific, (38) Bitter, T.; Muir, H. M. Anal. Biochem. 1962, 4, 330–334.
Waltham, MA). For interaction experiments, oligosaccharide (30 µM) was incubated with 15 µM SDF-1R or SDF-1(3/6) (1/2 protein/tetrasaccharide molar ratio) in 75 mM ammonium acetate, pH 6.5, at room temperature for 20 min. The samples were then used directly without further purification or salt elimination steps. Sample solution (1-2 µL) were spotted on the hydrophobic DESI surface HTC Omni Slide (Prosolia, Indianapolis, IN) and allowed to dry at room temperature and atmospheric pressure for 25 min. Choice of the DESI surface and buffer conditions for analyses of protein and protein/oligosaccharide complexes were optimized with Aprotinin protein and described in the Supporting Information, Figure S1. RESULTS AND DISCUSSION Analysis of Sulfated Oligosaccharides. Synthetic Heparin Hexasulfated Tetrasaccharide. The negative ionization DESI analysis of the hexasulfated tetrasaccharide (Figure 1A) led to a mass spectrum with numerous peaks, which was nevertheless unambiguously interpreted owing to the high resolution of the Orbitrap analyzer. A distribution of negative charge states ranging from 2- (m/z 671.9553) to 5- (m/z 246.2021) was assigned to the intact sodiated hexasulfated tetrasaccharide (Figure 2A). Deconvolution of the multiply charged peaks yield an experimental mass of 1389.8968 g mol-1 in agreement with the theoretical mass 1389.8949 (
