Oligosaccharide Profiling of O-linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2-AA)

Transcription

1 Oligosaccharide Profiling of O-linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2-AA) Elisabeth A. Kast and Elizabeth A. Higgins GlycoSolutions Corporation, Worcester, MA Data originally presented at the 2007 Society for Glycobiology annual meeting ABSTRACT The analysis of O-linked oligosaccharides has been complicated by the absence of an enzyme that removes all O-linked oligosaccharides from the glycoprotein. O-linked oligosaccharides must be removed from the glycoprotein using chemical methods and the best chemical method (Carlson β-elimination) destroys the reducing end of the glycans, preventing labeling of the glycans by reductive amination. Our goal was to find a method for chemically releasing O-glycans without destroying the reducing end so we could develop an oligosaccharide profiling method with fluorescentlylabeled O-linked oligosaccharides. Using the Carlson β-elimination method we had been limited to using high ph anion exchange chromatography (HPAEC) separation methods with pulsed amperometric detection (PAD) of the oligosaccharides. The ammonia-based β-elimination method published by Huang et al. (Anal. Chem. (2001) 73:6063) releases both O-linked and N-linked oligosaccharides from the glycoprotein. We have demonstrated that the N-links are not released quantitatively by this method. We are however, able to efficiently release O-linked oligosaccharides from the glycoprotein, label them with 2-AA and show a good separation using an amino column. The residual N-linked oligosaccharides pose a problem and the optimization of this method for maximal recovery of O-linked oligosaccharides and minimal interference from the residual N-linked oligosaccharides will be discussed. INTRODUCTION N-linked oligosaccharides can be removed from glycoproteins using PNGase F and then labeled fluorescently by reductive amination. There are now several good chromatographic separations available for these fluorescently labeled oligosaccharides. Unfortunately, there is no enzyme available for removal of all O- linked oligosaccharides thus chemical methods are employed to remove O-linked oligosaccharides. And the standard protocol for O-linked oligosaccharide release (Carlson β-elimination) yields reduced sugars, which cannot be labeled fluorescently by reductive amination. However, Huang et al. (Anal. Chem., 2001) have created a protocol for nonreductive removal of O-linked oligosaccharides, which provides the ability to fluorescently label the O-linked oligosaccharides. This protocol utilizes ammonium hydroxide and ammonium carbonate to remove the sugars from the protein. Treatment with boric acid ensures the presence of a reducing end. This procedure also removes N-linked oligosaccharides, which leads to challenges in identification and separation of N-linked and O-linked structures. We have been attempting to determine whether this protocol can be made more specific to removal of O-linked oligosaccharides by minimizing removal of N-linked oligosaccharides. Fetuin is a well-characterized glycoprotein containing both N-linked oligosaccharides and O-linked oligosaccharides. There are three sites with N-linked glycosylation. The structures on these sites are either biantennary or triantennary complex oligosaccharides. Sialyation (both α2-3 and α2-6) with NAcetylneuraminic Acid (NeuAc) varies from monosialyated to tetrasialyated and is typically attached to galactose (Green et al., JBC, 1988). There are also three O- Linked oligosaccharide sites on fetuin. Core 1 (with galactose linked B1-4 to the GalNAc) and Core 2 (with GlcNAc linked B1-6 to the GalNAc) have both been found on fetuin (Spiro and Bhoyroo, JBC, 1974; Edge and Spiro, JBC, 1987).

2 RESULTS Figure 1. Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia-based B-elimination at both 40C and 60C. The pattern of the charged N-linked oligosaccharides is similar, and at 60C ammonia-based elimination appears to fully remove the N-linked oligosaccharides. In the neutral region, O-linked oligosaccharides are visible in the ammonia-based β elimination chromatograms. Fetuin digested with PNGaseF Biantennary Triantennary Tetraantennary Fetuin prepared by ammonia-based β-elimination (40C incubation temperature) O-linked Glycans N-Linked glycans Fetuin prepared by ammonia-based β-elimination (60C incubation temperature)

3 RESULTS (continued) Figure 2. Representative chromatograms of a time course of fetuin at 18 hours, 24 hours and 48 hours of incubation with ammonium hydroxide and ammonium carbonate. At 24 hours maximal recoveries of both N-linked oligosaccharides and O-linked oligosaccharides appears to occur. Because of this, length of incubation cannot be altered to selectively remove O-linked oligosaccharides. 18 Hour Incubation 24 Hour Incubation 48 Hour Incubation

4 RESULTS (continued) Figure 3. Fetuin incubated in ammonium hydroxide and ammonium carbonate at ambient temperature, 30C, 40C and 60C. While temperature appears not to play a role in selectivity between N-linked and O-linked oligosaccharides, at 60C N-linked oligosaccharides appear to be removed quantitatively. Room Temperature 30C 40C 60C

5 CONCLUSIONS Nonreductive β-elimination successfully removes both N- and O-linked oligosaccharides from fetuin. In an effort to optimize the reaction for O-linked oligosaccharides, we explored the variables of time of incubation and temperature of incubation as a means to selectively remove O-linked oligosaccharides. For both these variables, there is no significant selectivity seen between release of N-linked and O-linked oligosaccharides. Although initially we were performing these experiments at 40C (similar to the Carlson method) where we did not quantitatively release the N-links from the protein (judged relative the PNGase F release) once we raised the temperature to 60C we do see quantitative release of the N-links (Figure 1). This allows for the possibility of having a profiling method for analysis of both N-links and O-links at the same time. For fetuin, the separation between the O-links and N-links is quite good using this method for the chromatography. However, fetuin has almost no uncharged N-linked oligosaccharides and very little monosialylated oligosaccharides. Uncharged and monosialylated oligosaccharides elute earlier and would migrate with the O-links. Fetuin s O-links are smaller, simpler structures and we would expect that larger O-links would also elute with the charged N-links. However, this method could still be preferable to having two separate methods for N-linked and O-linked oligosaccharides. Materials All chemicals used were obtained from VWR, unless otherwise specified. The LudgerClean Glycan Cartridge desalting columns were obtained from QA-Bio. The HPLC used was an Agilent HP1100 system with fluorescent detector, run using the Chromeleon software (Dionex). The column used for chromatography was a Shodex Asahipak NH2P-50 2D 2x150mm. Ammonia-based β-elimination: 25 μg of fetuin (Sigma) was dissolved in 250 μl 28% ammonium hydroxide saturated with ammonium carbonate. 25 mg of ammonium carbonate was added, and the tubes were incubated at 40C for hours, unless otherwise indicated. After the incubation, samples were desalted on LudgerClean Glycan Cartridge desalting columns. After washing with 700 μl DI water two times, oligosaccharides were eluted using 200 μl of a solution containing 50% acetonitrile and 0.1% trifluoroacetic acid four times. The samples were dried under vacuum, and 10 μl of 0.5M boric acid was added and incubated at 37C for 30 minutes. Then, 100 μl of methanol was added and the samples were dried under vacuum. 2-AA Labeling: Samples were labeled with 2-AA using 2-AA Labeling Kits (QA-Bio), following the directions provided. After labeling, 450 μl DI water was added to the samples, and the samples were dialyzed against 400 ml DI water overnight using 500 Da MWCO cellulose acetate membranes (The Nest Group). After dialysis, samples were dried under vacuum and resuspended in 200 μl DI water. Chromatography: Buffer A was 2% glacial acetic acid and 1% diethylamine (Sigma) in acetonitrile. Buffer B was 5% glacial acetic acid and 4% diethylamine in HPLC grade water, adopted from Anumula and Dhume (Glycobiology, 1998). A multistep gradient was used: from 0 to 2 minutes, Buffer A was held at 70%, from 2 to 80 minutes, Buffer A decreased linearly to 5%, from 80 to 95 minutes, Buffer A was held at 5%. The flow rate was held constant at 0.2 ml/min. Fluorescent data was acquired over the entire period, and chromatograms were created using Chromeleon software. REFERENCES Kalyan Rao Anumula and Shirish T.Dhume. High resolution and high sensitivity methods for oligosaccharide mapping and characterization by normal phase high performance liquid chromatography following derivatization with highly fluorescent anthranilic acid. Glycobiology. Vol 8, No. 7. January Pp Albert S.B. Edge and Robert G. Spiro. Presence of an O-Glycosidically Linked Hexasaccharide in Fetuin. The Journal of Biological Chemistry. Vol. 262, No. 33, November pp (continued)

6 Eric D. Green, Gabriela Adelt, Jacques U. Baeziger, Susanne Wilson, Herman Van Halbeek. The Asparagine-linked Oligosacchardies on Bovine Fetuin. The Journal of Biological Chemistry. Vol. 263, No. 34, December 5, Pp Yunping Huang, Yehia Mechref, Milos V. Novotny. Microscale Nonreductive Release of O-Linked Glycans for Subsequent Analysis throught MALDI Mass Spectrometry and Capillary Electrophoresis. Analytical Chemistry. Vol 73, No. 24, December 15, Pp Robert G. Spiro and Vishnu D. Bhoyroo. Structure of the O-Glycosidically Linked Carbohydrate Units of Fetuin. The Journal of Biological Chemistry. Vol. 249, No. 18, September 25, Pp ACKNOWLEDGEMENT The authors would like to thank Dr. Richard Cummings for suggesting we look at the Huang method.