Anal. Chem. 1999, 71, 1479-1482
On-Target Exoglycosidase Digestions/MALDI-MS for Determining the Primary Structures of Carbohydrate Chains Jennifer Colangelo and Ron Orlando*
Complex Carbohydrate Research Center and Departments of Biochemistry and Molecular Biology and Chemistry, University of Georgia, 220 Riverbend Road, Athens, Georgia 30602-4712
One method used to determine the primary sequence of oligosaccharides is to digest them with exoglycosidases and analyze the resulting digestion products by matrixassisted laser desorption/ionization mass spectrometry (MALDI-MS). Previous research has demonstrated that these digestions can be performed on the MALDI target. However, the procedure requires the sample to be incubated at elevated temperatures, and complete digestion requires a few hours. We demonstrate new conditions that permit exoglycosidase digestions to be performed on the MALDI target at room temperature within 30 min. Oligosaccharide standards were digested with one or more exoglycosidases to show that the enzymes retain their activity and specificity under these new reaction conditions. Using this method, the primary sequences of carbohydrate chains can be determined in a relatively short amount of time. Exoglycosidases are often used as aids in sequencing carbohydrate chains.1,2 These enzymes remove monosaccharides from the nonreducing termini of carbohydrate chains. Many of these enzymes are specific to the stereochemistry and anomeric configuration of the monosaccharide being released and its linkage site to the remainder of the carbohydrate chain. Once the exoglycosidase digestion is complete, the mixture is analyzed to determine the number of monosaccharides released by the enzyme. Analysis of the digestion mixture traditionally is carried out by liquid chromatographic or gel electrophoretic methods, although mass spectrometry (MS) can also be used.3-6 The digestion products are analyzed for any changes in the molecular mass of the substrate, which in turn provides the number of monosaccharides cleaved from the chain during the digestion. * Corresponding author: (tel) 706-542-4401; (fax) 706-542-4412; (e-mail)
[email protected]. (1) Settineri, C. A.; Burlingame, A. L. In Techniques in Protein Chemistry V; Crabb, J. W., Ed.; Academic Press, Inc.: San Diego, 1994; pp 97-104. (2) Prime, S.; Dearnley, J.; Ventom, A. M.; Parekh, R. B.; Edge, C. J. J. Chromatogr., A 1996, 720, 263-274. (3) Harvey, D. J.; Rudd, P. M.; Bateman, R. H.; Bordoli, R. S.; Howes, K.; Hoyes, J. B.; Vickers, R. G. Org. Mass Spectrom. 1994, 29, 753-765. (4) Sutton, C. W.; O’Neill, J. A.; Cottrell; J. S. Anal. Biochem. 1994, 218, 3446. (5) Yang, Y.; Bergmann, C.; Benen, J.; Orlando, R. Rapid Commun. Mass Spectrom. 1997, 11, 1257-1262. (6) Tyagarajan, K.; Lipniunas, P. H.; Townsend, R. R.; Forte, J. G. Biochemistry 1997, 36, 10200-10212. 10.1021/ac980980u CCC: $18.00 Published on Web 02/11/1999
© 1999 American Chemical Society
One problem encountered in the analysis of exoglycosidase digestion products by MS is that the digestion buffers may interfere with MS analysis; consequently, sample cleanup procedures may be required. With its higher tolerance for salts, matrixassisted laser desorption/ionization (MALDI) is often the ionization method of choice for this type of analysis. MALDI-MS has proven to be an effective technique in the sequencing process because of its molecular mass accuracy, speed, and ease of use. Recent research in this area has focused on optimizing exoglycosidase digestions so that the digestion mixtures are directly compatable with MALDI-MS analysis.5,7-9 For instance, digestions were traditionally performed in buffered solutions, some of which are not ideal for use with MALDI-MS. This problem has been remedied by using volatile buffers and lower salt concentrations.8,9 Digestions have also been performed on the MALDI target, reducing the amount of sample required. However, these digestions require incubation of the MALDI target at elevated temperatures (37 °C), and 1 h or more are required for complete digestion.7,8 The digestion procedure could be improved further by eliminating the need for sample incubation and by reducing the time required for complete digestion. We have demonstrated that exoglycosidase digestions can be performed on a MALDI target at room temperature in 5-30 min, by performing digestions on N-linked oligosaccharides from asialofetuin and thyroglobulin. The enzymes retain their activity and specificity under these new reaction conditions. Digestions using multiple enzymes can also be performed using this technique to sequence oligosaccharide chains. Unknown carbohydrate sequences have been determined in this manner. Determining the primary structure of carbohydrate chains using exoglycosidase digestions analyzed by MALDI-MS is now easier and faster. EXPERIMENTAL SECTION Materials. Oligosaccharides from asialofetuin and thyroglobulin were removed previously by N-glycanase. The oligosaccharides from asialofetuin were separated by size exclusion chromotography,10 and the oligosaccharides from thyroglobulin were (7) Mechref, Y.; Novotny, M. V. Anal. Chem. 1998, 70, 455-463. (8) Kuster, B.; Naven, T. J. P.; Harvey, D. J. J. Mass Spectrom. 1996, 31, 11311140. (9) Yang, Y.; Orlando, R. Anal. Chem. 1996, 68, 570-572. (10) Mellis, S. J.; Baenziger, J. U. Anal. Biochem. 1983, 134, 442-449.
Analytical Chemistry, Vol. 71, No. 7, April 1, 1999 1479
Table 1. Exoglycosidases Tested Using the New Digestion Protocol enzyme
source
specificity
enzyme/subtrate ratioa
R-galactosidase β-galactosidase β-galactosidase R-mannosidase β-N-acetylhexosaminidase β-N-acetylhexosaminidase sialidase sialidase sialidase R-fucosidase
green coffee bean bovine testes Streptococcus pneumoniae Canavalia ensiformis chicken liver Streptococcus pneumoniae Arthrobacter ureafaciens Clostridium perfringens Streptococcus pneumoniae bovine epididymis
R(1-3,4,6)-galactose β(1-3,4)-galactose β(1-4)-galactose R-linked mannose R(1-2) > R(1-3,6) β(1-3,4)-acetylhexosamine β(1-2)-acetylglucosamine R(2-3,6,8) sialic acid R(2-3,6) unbranched sialic acid R(2-3) unbranched sialic acid R(1-6)-fucose
125 200 8 250 100 1 5 10 5 5
a
Enzyme/substrate ratios are reported in microunits of enzyme used for 1 pmol of substrate.
separated by affinity chromatography with the use of a concanavalin A column.11 Fractions containing oligosaccharides were dried before use. Samples were reconstituted with water to a concentration of ∼20 µM. Pectate lyase from Aspergillus niger was a gift from Jaap Visser at Wageningen Agricultural University, Wageningen, The Netherlands. The glycopeptide from trypsin-digested pectate lyase was previously isolated and identified using liquid chromatography/mass spectrometry (LC/MS).12 The fraction containing the glycopeptide was dried and reconstituted with water to a concentration of ∼10 µM. R-Mannosidase from Canavalia ensiformis and sialidase from Newcastle disease virus were purchased from Boehringer Mannheim (Indianapolis, IN), and all other sialidases were obtained from Prozyme, Inc. (San Leandro, CA). The remaining exoglycosidases were purchased from Oxford GlycoSciences, Inc. (Bedford, MA). See Table 1 for a complete list of enzymes tested. Dry enzymes were reconstituted with water using the concentration recommended by the manufacturer. 2,5-Dihydroxybenzoic acid (DHB) was purchased from HewlettPackard (Palo Alto, CA), and 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid) was from Sigma. Sinapinic acid was dissolved in a solution of 80% (v/v) acetonitrile and 0.08% (v/v) trifluoroacetic acid to a concentration of 100 mM. Digestion. On a sample region of the MALDI target, 0.5 µL of enzyme was added to 0.5 µL of sample. The digestion proceeded at room temperature (22 °C) for 15 min, with some enzyme substrate combinations requiring shorter or longer reaction times. When multiple enzymes were used, 0.5 µL of each enzyme was added to 0.5 µL of sample, and the reaction time was typically longer, requiring 30 min. The sample region should remain solvated during the entire digestion period. Sometimes evaporation of the mixture occurred before the incubation time expired, and water was added as needed. After incubation, 0.5 µL of matrix was added to the digestion, and it was mixed and dried so that MALDI-MS analysis could be completed. DHB was used as the matrix for the oligosaccharide samples, while sinapinic acid was used for the glycopeptides. MALDI-TOF Mass Spectrometry. MALDI analyses were carried out on a Hewlett-Packard (Palo Alto, CA) G2025A timeof-flight mass spectrometer. Samples were ionized with a nitrogen (11) Endo, T. J. Chromatogr., A 1996, 720, 251-261. (12) Colangelo, J. L.; Ziehl, V.; Benen, J.; Bergmann, C.; Orlando, R. Presented at Pittsburgh Conference, New Orleans, LA, March 1-5, 1998; Abstract 571.
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Figure 1. (A) N-Linked carbohydrate structures isolated from asialofetuin having masses 1640 (F1) and 2005 Da (F2 and F3). (B) N-Linked carbohydrate structures isolated from thyroglobulin having masses 1786 (T1) and 1948 Da (T2).
laser (λ ) 337nm) having a pulse width of 3 ns. The instrument operated with an accelerating voltage of 28 kV, an extractor voltage of 7 kV, and a pressure of ∼1 × 10-6 Torr. A known mixture of proteins was used to calibrate the instrument externally. RESULTS AND DISCUSSION Most of the discussion in this paper will focus on the digestion of N-linked oligosaccharides removed from asialofetuin and thyroglobulin by N-glycanase. These carbohydrate chains are well characterized and have a diverse carbohydrate composition, making them excellent candidates as standards. The oligosaccharide fraction isolated from asialofetuin contained three N-linked structures: one biantennary structure and two triantennary structures that differ by a single linkage (Figure 1A). The oligosaccharide fraction isolated from thyroglobulin contained two similar structures, the only difference being an additional R-galactose residue attached (Figure 1B). MALDI-MS was used to monitor the time needed for the exoglycosidases to cleave monosaccharides from the oligosaccharides. The oligosaccharides (10 pmol) isolated from asialofetuin
Figure 2. MALDI-MS spectra of (A) the oligosaccharides from asialofetuin, (B) after a 10-min digestion using β(1-4)-galactosidase, and (C) after a 1-h digestion using β(1-4)-galactosidase. Peaks seen in the spectra represent the (M + Na)+ species.
were digested with 0.08 munit of β(1-4)-galactosidase followed by MALDI-MS analysis of the digestion mixture. Complete digestion occurs once two galactose residues are released from the biantennary structure and one of the triantennary structures, and three galactose residues are released from the other triantennary structure. The MALDI-MS spectrum of the reaction that proceeded for 1 min contained molecular ions representing cleaved sample, indicating that digestion starts immediately. Spectra obtained after each additional minute of digestion revealed further cleavage taking place. After 10 min of digestion, complete cleavage of all galactose residues was accomplished with no intermediates (Figure 2). The same spectrum was also obtained for a digestion that proceeded for 1 h (Figure 2), confirming that the digestion was finished after 10 min. Retention of enzyme specificity is one of the main concerns when exoglycosidase digestions are performed under new conditions. In the digestion described above, two factors illustrate that β(1-4)-galactosidase retained its specificity. First, one of the triantennary structures contains a terminal β(1-3)-galactose residue that was not cleaved during digestion. If the enzyme specificity for a (1-4) linkage had been lost, this residue would have been cleaved. Second, the enzyme was specific for only galactose residues, which produces mass shifts of 162 Da, the only mass shift seen. The next residue is N-acetylglucosamine, which has an incremental mass of 203 Da; however, no losses of 203 Da were observed. Digestion of the oligosaccharides isolated from thyroglobulin with β(1-4)-galactosidase also demonstrates retention of enzyme specificity (Figure 3). The enzyme cleaves two galactose residues from the first structure, but only the one terminal galactose residue from the second. Note that the nonreducing termini of the second structure contains two galactose residues, one having a β(1-4) linkage that should be cleaved using β-galactosidase and the other having an R(1-3) linkage that should not be cleaved. These experiments show that digestion with β-galactosidase occurs quickly with no loss of specificity. Digestions using other exoglycosidases were also performed at room temperature in 1015 min. Many different substrates with known structures were used while the exoglycosidases were tested to ensure the procedure worked for different samples, but these experiments
Figure 3. MALDI-MS spectra of the oligosaccharides from thyroglobulin (A) before and (B) after digestion with β(1-4)-galactosidase. Peaks seen in the spectra represent the (M + Na)+ species.
are not detailed here for the sake of brevity. These experiments showed that the enzymes listed in Table 1 removed the expected monosaccharides while retaining activity and specificity. We found this technique to be successful with 1-10 pmol of substrate loaded onto the MALDI target. Digestions may be possible with smaller amounts but were not tested. This reaction protocol works for digestion with a single exoglycosidase, but in order to sequence hybrid and complex oligosaccharide structures, more than one enzymatic digestion must be performed on a sample. To evaluate the potential for multiple digestions, the oligosaccharides isolated from thyroglobulin were digested with a total of six enzymes: R-galactosidase, β-galactosidase, β(1-4)-galactosidase, β(1-2)-N-acetylhexosaminidase, R-mannosidase, and R-fucosidase. Multiple digestions were performed by mixing the sample with two or more enzymes on the MALDI sample region. For example, to remove all galactose residues from the structures, this fraction of thyroglobulin's oligosaccharides was mixed with R- and β-galactosidase. Complete digestion occurred when up to four enzymes were used in the mixture (Figure 4). These digestions required a longer time period to complete than the digestions with only a single enzyme, since the first enzyme must release the terminal monosaccharide before the subsequent enzyme(s) can react with the oligosaccharide, since these enzymes only cleave the terminal residues. The only enzyme used that never produced complete digestion products was R-fucosidase. Partial cleavage of the fucose residue was seen, but it was never removed from all chains. Digestions performed under the manufacturer's conditions and under the new protocol using only R-fucosidase also resulted in partial cleavage. One explanation for the incomplete reaction is a possible steric interference that prevents complete cleavage for this particular structure. Sequential digestions were also performed on the oligosaccharides from asialofetuin. β(1-4)-Galactosidase, β(12)-N-acetylhexosaminidase, and R-mannosidase were used to digest the carbohydrate chains. These digestions allowed us to confirm the structure and distinguish between the three structures. These digests have been proven successful for oligosaccharides of known structures, and we wanted to test their success as aids in sequencing carbohydrate chains of unknown structures. LC/MS analysis of trypsin-digested pectate lyase from A. niger identified one fraction that contained a glycopeptide.12 Using mass Analytical Chemistry, Vol. 71, No. 7, April 1, 1999
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Figure 6. MALDI-MS spectra of the glycopeptide isolated from pectate lyase (A) before and (B) after digestion with R-mannosidase. Peaks seen in the spectra represent the (M + Na)+ species. The peaks labeled * represent signals present in the MALDI-MS spectrum of the enzyme solution and thus are thought to be background peaks.
Figure 4. MALDI-MS spectra of the oligosaccharides from thyroglobulin (A) before digestion, (B) after digestion with R-galactosidase, (C) after digestion with R-galactosidase and β(1-4)-galactosidase, (D) after digestion with R-galactosidase, β(1-4)-galactosidase, and β(1-2)-N-acetylhexosaminidase, and (E) after digestion with R-galactosidase, β(1-4)-galactosidase, β(1-2)-N-acetylhexosaminidase, and R-mannosidase. Peaks seen in the spectra represent the (M + Na)+ species.
Figure 5. Man5GlcNAc2 structure identified by searching the carbohydrate database for the mass of this oligosaccharide.
information obtained from the LC/MS experiment and other sequence information, the mass of the carbohydrate was calculated. This mass was then entered into CarbBank (http:// 128.192.9.29/carbbank/default.htm), a carbohydrate database, to search for possible structures having the same mass. The search produced one match, Man5GlcNAc2 (Figure 5). To verify that this was the structure attached to the peptide, the fraction containing the glycopeptide was digested with R-mannosidase on the MALDI sample region. After 15 min, the digestion was complete; i.e., no further decreases in mass were observed at longer digestion
1482 Analytical Chemistry, Vol. 71, No. 7, April 1, 1999
intervals. The spectrum revealed a loss of 650 Da, which is equivalent to the mass of the four R-mannose residues (Figure 6). These data helped to confirm the structure obtained from the database and illustrates how this protocol may be used in sequencing unknowns. We have demonstrated that exoglycosidase digestions can be simplified, especially in preparation for MALDI-MS analysis. By performing the digestions on the MALDI target, the amount of sample and enzyme required is reduced. We have further simplified the digestion procedure by performing the reactions at room temperature, eliminating the need for incubation at elevated temperatures. Most importantly, the time needed for experimental completion has been reduced, allowing analysis to be performed under 30 min. We have shown that commonly used exoglycosidases can be used in this manner and that even multiple digestions can be performed successfully. Digestions were also performed on glycopeptides of unknown carbohydrate structure with various exoglycosidases, providing sequence information for these structures and illustrating that the technique may be successfully used for unknown structures. ACKNOWLEDGMENT We thank Roberta Merkle for supplying oligosaccharide fractions. This research was supported by grants from the National Institutes of Health (P41RR05351) and the National Science Foundation (9626835). Received for review August 31, 1998. Accepted January 5, 1999. AC980980U
