Separation of partially methylated mannitols by liquid chromatography

ANALYTICAL

BIOCHEMISTRY

Separation

151,554-560

(1985)

of Partially Methylated STEVEN

Mannitols by Liquid Chromatography’

R.HULL*ANDSALVATOREJ.

TURCO~

Department of Biochemistry, University of Kentucky Medical Center, Lexington, Kentucky 40536 Received July 11, 1985 To facilitate the methylation linkage analysis of complex carbohydrates containing radioactively labeled mannose residues, an HPLC micromethylation technique has been developed which effectively resolves all of the partially methylated mannitol standards prepared using an undermethylation protocol. The method was applied to the linkage analysis of the nine mannose residues of the dolichol-derived oligosaccharide GlcSMan9GlcNAc2 isolated from BHK-2 1 fibroblasts. This technique should prove widely applicable to the methylation linkage analysis of radiolabeled complex carbohydrates. 0 1985 Academic PFZSS,h.

Methylation linkage analysis is an important technique necessary for structuraJ analysis of carbohydrate moieties found on biological macromolecules. Gas-liquid chromatography/mass spectrometry (GLC-MS) is the best method of choice for the separation and identification of the partially methylated monosaccharide derivatives generated from the analysis (l-3). The sensitivity of GLC-MS is presently in the lOO- to 200~pg range (l-3), although methods have been recently reported which demonstrate improved sensitivity in the l- to 5-pg range of carbohydrate (4). Development of a method that rapidly and effectively separates radiolabeled monosaccharides subjected to methylation linkage analysis would have application to a wide variety of complex carbohydrate structural studies. TLC separation of partially methylated Abbreviations used: GLC-MS, gas-liquid chromatography-mass spectrometry; DAB, 1,4-diaminobutane; TFA, trifluoroacetic acid; EI, electron impact; CI, chemical ionization; DMSO, dimethyl sulfoxide. ’ This investigation was supported by NIH Grant AM26983 and by an NIH Research Career Development Award AM01087 to S.J.T. 2 Recipient of a Physician Service Award from the University of Kentucky. Present address: Department of Anatomy and Cell Biology, University of Miami, Miami, Ra. 33101. 3 To whom correspondence should be sent. QOO3-2697185 $3.00 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

554

mannose residues has been applied to the analysis of radioactively labeled carbohydrate chains obtained from cells in culture (5); however, this method is relatively tedious. HPLC would be the ideal chromatographic medium by which to develop a radioactive methylation linkage analysis system. Recently Saadat and Ballou reported a reverse-phase HPLC system which they applied-to the methylation linkage analysis of an acidic oligosaccharide isolated from a glycolipid of Mycobacterium smegmatis (6). Methylation standards used for this study were isolated from various sources that may not be conveniently available for most investigators. In addition, a complete analysis in one chromatographic run was not possible. Another group reported preparation of most of the methylated derivatives of mannose and galactose with subsequent separation by HPLC on TSK gel LS-4 10 (7), although the utility of this HPLC system in analyzing radioactive biological samples was not demonstrated. In this report, we describe a convenient preparation of chemical and radioactive amounts of partially methylated mannitols, their separation by HPLC in one chromatographic run, and application of this HPLC micromethylation analysis to the linkage determinations of the nine mannose derivatives of the dolichol-derived [3H-Man]ohgosac-

CHROMATOGRAPHIC

charide Glc3Man9GlcNAc2 BHK-2 1 fibroblasts. MATERIALS

AND

SEPARATION

isolated

from

METHODS

Materials. cY-Methylmannoside, 1,4-diaminobutane, and NaBH4 were obtained from Sigma Chemical Company; [2-3H]mannose (25 Ci/mmol) was from New England Nuclear; acetonitrile (HPLC grade) was from Fischer Scientific; Ag-l(acetate) and Ag-SO(H+) were from Bio-Rad; Silica gel G plates (20 X 20 cm) and 5 pm Lichrosorb Si-60 were from E. Merck; and NaBD4 was from Stohler Isotope Chemicals (Waltham, Mass.). All other reagents were of the highest purity commercially available. Preparation of radiolabeled partially methylated mannitol standards. To prepare [(Y& methyl-3H]mannoside, 0.5 mCi of [2-3H]mannose was reacted at 100°C for 3 h with 1 N MeOH/HCl. The radioactive mixture after this treatment was applied to a silica gel G thin-layer plate and developed in Solvent A, CH3CN:H20 (8: 1), to separate the radioactive product from any unreacted [3H]mannose. The region containing [a,fl-methyl-3H]mannoside was scraped into a 12-ml conical centrifuge tube and the radioactive methyl glycoside was recovered by repeatedly washing the gel particles with methanol:water (1: 1) until no further radioactivity was extracted. (YMethyl mannoside (80.0 mg) was mixed with the freshly prepared [a,fi-methy&3H]mannoside and undermethylated using one-half the amount of methylsulfinylcarbanion necessary to fully methylate the sugar (7). Briefly, the sugar sample was dried under N2 and repeatedly dried with absolute ethanol to ensure anhydrous conditions. The dried residue was suspended in 2.0 ml redistilled DMSO and 400 ~1 of 2 N methylsulfinylcarbanion was added. The sample was left at room temperature for 18 h at which time 1.5 ml of methyl iodide was added followed by sonication for 1 h in a sonic water bath. The undermethylated sample was diluted with 4.0 ml of CHC13, dried under N2, and percolated over a column consisting of 10 ml of Ag- 1(acetate) in the upper

OF

METHYLATED

MANNITOLS

555

half and 10 ml of Ag-SO(H+) in the lower half of the column. The column was washed with 5 column vol of methanol:water (1: 1). The effluent was concentrated under reduced pressure and preparatively applied to a Silica gel G plate which was developed in Solvent B, benzene:acetone:water:ammonium hydroxide (50:200:3: 1.5) (5). Six regions were apparent by orcinol staining (8) and were extracted from the gel and rechromatographed on silica gel G for additional purification. Samples of partially methylated mannosides separated by TLC were hydrolyzed, reduced, acetylated, and analyzed by GLC-MS to ascertain their compositions. For HPLC analysis, acetylation was not necessary and mannitols after reduction were simply desalted and injected on HPLC (see below). Hydrolysis of the TLC-purified mannosides was achieved with 2 N TFA at 120°C for 3 h. Reduction after hydrolysis was performed by drying the hydrolysate under a stream of N2, repeatedly drying under NZ using methanol to remove traces of acid, resuspending in 0.3- 1.O ml of water, and adding 10 mg of NaBH4 (NaBD, when GLC-MS analysis was performed). Reduction was stopped by addition of l-2 drops of glacial acetic acid. Samples were dried under N2 and boric acid was removed by repeated evaporations with 10% acetic acid in methanol. Finally the sample was eluted over an ionexchange resin consisting of 5 ml of Ag-SO(H+) in the lower part of the column and 1 ml of Ag- 1(acetate) in the upper part of the column. The effluent was evaporated to dryness and either brought up in a small volume for HPLC analysis or acetylated for GLC-MS analysis. GLC-MS analysis of partially methylated alditot acetates of mannose. Samples after methylation, hydrolysis, and reduction were acetylated by resuspending the sample residue after reduction in pyridine:acetic anhydride (20:80) and heating at 100°C for 2 h. After the mixture was cooled the sample was dried under N2 and repeatedly dried with toluene. The partially methylated mannitol acetates were resuspended in 1 ml of CHC13 and extracted three to five times with 3 ml of water.

556

HULL AND TURCO

The chloroform phase was dried under NZ, resuspended in 50-200 ~1 of CHC&, of which l-2 ~1 was analyzed by EI and/or CI capillary GC-MS. Capillary gas chromatography was on DB-5 (J&I) (30 m X 0.25 mm), with helium carrier gas at 35 ml/min, using a programmed temperature of 160°C held isocratically for the first 3.0 min and then elevated to 240°C at 8”C/min with injectors and transfer lines at 250°C. Chemical ionization mass fragmentography was performed essentially as described by Laine (9,lO). CI spectra were obtained with a Finnigan 3300-6 110 mass spectrometer coupled to a Technivent interactive GC-MS data system. Methane in the ion source was at 1 Torr. Ionizing electron energy was 150 eV. Electron impact mass fragmentography was performed essentially as described by Bjorndal et al. (11) and Stellner et al. ( 12). Gas chromatographic conditions were identical to those used for CI mass fragmentography. Ionizing electron energy was 70 eV. HPLC analysis of partially methylated mannitols. Samples which were methylated, hydrolyzed, reduced, and desalted as described above were resuspended in 50-200 ~1 of methanol:water (1: l), of which 5-20 ~1 was injected for HPLC analysis. The HPLC method described by Turco (13) was adapted to separate the partially methylated mannitols. A column of 5 pm Lichrosorb Si-60 (0.46 X 25 cm) was employed and a solvent program that proportioned Solvent C to Solvent D in a linear gradient was used and is described in the legend to Fig. 2. Solvent C (freshly prepared and discarded after 1 week) consisted of 99% CH3CN containing 0.025% DAB (v/w) and Solvent D was 0.025% DAB in H20. A Varian 5000 liquid chromatograph with a dual-pump system was used and was equipped with a Rheodyne 7 126 injector. A flow rate of 1.O ml/min was used and l.O-ml fractions were collected with an LKB Redi-Rat fraction collector. Sample fractions were allowed to evaporate to dryness at room temperature in a fume hood, resuspended in 0.2 ml of 1% sodium dodecyl sulfate, and 1.8 ml of scintillation cocktail (14), and analyzed for radioactivity.

Micromethylation analysis of Glc3A4an9GlcNAc2. For micromethylation analysis of oligosaccharides, samples were dried under N2 and kept under a constant N2 atmosphere throughout methylation. The sample residue was brought up in 2.0 ml of redistilled DMSO and sonicated for 30-45 min. Dimethylsulfinylcarbanion ( 1.O ml) was then added and the sample was sonicated for 4-5 h. Subsequently, two additions of methyl iodide (1.5 ml) were made during l-h intervals with continual sonication. After sonication, the sample was left at room temperature for 18 h, at which time 5 ml of water was added to terminate methylation. Chloroform (5 ml) was added to the sample to extract the fully methylated species. The CHC13 fraction was washed three to four times with an equal volume of water and then dried under N2. The sample was resuspended in 2-5 ml of 2 N TFA and the reaction vessel sealed under vacuum and hydrolyzed at 120°C for 4 h. The hydrolyzate was cooled, dried under NZ, evaporated in the presence of methanol three to four times to remove traces of acid, and reduced with a freshly prepared 1% NaBH4 solution in 1 N NH40H made up in ethanol:water ( 1: 1). The reduction was stopped by addition of l-2 drops of glacial acetic acid, dried under N2, and desalted as described above. The desalted sample residue after reduction was resuspended in a small volume of methanol:water (1: 1) and analyzed by HPLC as described above. RESULTS

Preparation of partially methylated mannitols. To evaluate the effectiveness of HPLC in separating partially methylated mannitols, tritium-labeled standards were prepared by an undermethylation protocol described under Materials and Methods and analyzed by GLCMS. All but two of the possible partially methylated mannitols were prepared in adequate amounts from [a,p-methyl-3H]mannoside using one half the amount of dimethylsulfinylcarbanion necessary for complete melhylation (7). The variously methylated mannosides

CHROMATOGRAPHIC

SbPARA I IUN

Jr

MC

1 HY LA 1 EIJ

MAlYlVl

1 UL3

557

I

40 -

38 36 34 32 . 30 28 26 24 22 _ = 20. 18 16. 14 12

TLC 6 TLC 4

TLC 5

TLC 3

TLC 2

10 8

6 4 2 0

FIG. 1. TLC analysis of partially methylated mannosides. [a,&methyPH]Mannoside

I

was prepared, undermethylated, applied to a preparative TLC plate of sihca gel G, developed in solvent A, and rechromatographed as described under Materials and Methods.

were purified into six resolved regions on a preparative TLC plate of silica gel G (Fig. 1). The composition of rechromatographed TLC regions l-6 (Table 1) was ascertained by hy-

TABLE I

COMPOSITION OFTLC REGIONS l-6 TLC region

Partially methylated mannosides”

1

cu&MethyImannoside

2

2-Methyl-a,&methylmannoside 4-Methyl-cY&methylmannoside 6-Methyl-a$-methylmannoside

3

2,3-Dimethyl-a,&methylmannoside 2,4-Dimethyl-cu,&methyImannoside 2,6-Dimethyl-ol,&methylmannoside 3,6-Dimethyl-a$-methylmannoside 4,6-Dimethyl-ol,&methylmannoside

4

2,3,4-Trimethyl-a$-methylmannoside 2,3,6-Trimethyl-&methylmannoside

5

2,4,6-Trimethyl-a,@-methylmannoside 3,4,6-Trimethyl-cu&methylmannoside

6

2,3,4,6-Tetramethyl-&methylmannoside

a Determined by hydrolysis, reduction, acetylation, and subsequent GLC-MS analysis as described under Materials and Methods.

drolysis (which cleaves the methyl group from the anomeric carbon), reduction, acetylation, and GLC-MS analysis. The only two partially methylated mannitols not made in relatively large amounts by this procedure were 3,4diO-methyl-cY&methylmannoside and 3-0methyl-cY,&methylmannoside. It should be noted that upon acid hydrolysis, 3-O-methylmannitol and 4-0-methylmannitol are identical. Separation of partially methylated mannitols by HPLC. After hydrolysis and reduction of the different methylated derivatives of mannose, a mixture of mannitols exists which differ in the number and stereochemistry of free hydroxyl groups. These components are potentially separable on silica gel modified with 1,Cdiaminobutane ( 11). The partially methylated mannitols were resolved into individual mannitols when applied to a highperformance column of 5-pm silica gel and eluted with an CH&N/H20 solvent gradient containing DAB (Fig. 2). Identity of each peak was confirmed by isolating the derivative eluting from the column, desalting over a column of Ag-SO(H+) to remove DAB, acetylating, and analyzing by GLC-MS (data not shown). It is noteworthy that absolute elution volumes varied slightly from experiment to experiment.

558

HULL

t

0

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10

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30

I

40

I

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AND TURCO

I

80

I

90

I

100

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110

I

120

I

130

I

140

I

150

I

160

I

170

I

180

ELUTION VOLUME

FIG. 2. Separation of partially methylated mannitol standard mixture on 5 pm Lichrosorb Si-60. Partially methylated methylmannosides isolated from TLC regions 1-6 were hydrolyzed, reduced, and desalted as described in the methods section. Individual TLC regions 1-6 were mixed together, and 25 pl was injected for analysis. Solvent program used was To = 100% C; T,w = 94% C, TIJo = 94% C; TIM) = 30% C: T,,,, = 30% C. Peaks were identified by recovery from HPLC, acetylation, and GLC-MS as described under Materials and Methods.

This is attributed to variations in solvent preparation, age, and condition of the HPLC column, and the performance of the instrument. A mixture of partially methylated mannitols was chromatographed and the Kd values were determined using the elution volume of 2,3,4,6-tetramethylmannitol as V, and the elution volume of mannitol as Vi. It was found that the Kd values for each partially methylated mannitol (Table 2) were consistent from experiment to experiment. Analysis of Glc3MangGlcNAc2 isolatedfrom BHK jibroblasts. To demonstrate the utility

of this micromethylation technique, we examined the linkages of various mannose residues of the major dolichol-derived oligosaccharide Glc3Man9GlcNAc2 isolated from BHK-2 1 fibroblasts. GlcXMan9GlcNAcz was prepared and isolated by HPLC as described in a previous report (13). After methylation, extraction with CHC13, hydrolysis, and reduction as described under Materials and Methods, the partially methylated mannitols were then analyzed by HPLC (Fig. 3). The results from two separate methylation experiments

are summarized in Table 3. Identities of each peak were ascertained by comparison of their Kd values with those obtained from a standard TABLE 2 Kd VALUES~FORTHE

VARIOUSPARTIALLY METHYLATEDMANNITOLS SEPARATEDBY HPLCON 5-pm SILICA GEL MODIFIED WITH DAB

Derivative 2,3,6-Trimethylmannitol 3,4,6-Trimethylmannitol 2,3,4-Trimethylmannitol 2,4,6-Trimethylmannitol 3,6-Dimethylmannitol 2,3-Dimethylmannitol 2,6-Dimethylmannitol 4,6-Dimethylmannitol 2,CDimethylmannitol 6-Methylmannitol 4-Methylmannitol 2-Methylmannitol

0.041 0.134 0.156 0.225 0.265 0.294 0.337 0.384 0.468 0.645 0.691 0.739

a Kd = V, - V,/V, - V,; V, is the elution volume of 2,3,4.6-tetramethylmannitol, Vi is the elution volume of mannitol, and V, is the elution volume of the mannitol derivative. b Average of three experiments.

CHROMATOGRAPHIC

SEPARATION

OF METHYLATED

MANNITOLS

559

2@+----:-240.

: F

b

220.

a

2 t:

200. HO160.

0

20

10

30

50

40

60

70

80

90

100

110

ELUTION VOLUME

FIG. 3. Micromethylation linkage analysis of GlcjMan9GlcNAc2. 35,000 cpm of GlcXMan9GlcNAcz isolated by HPLC from BHK-2 I fibroblasts was methylated, hydrolyzed, reduced, and analyzed by HPLC as described in the text. Solvent program used was that described in the legend to Fig. 2. Areas of peaks were used for quantitation of each derivative, the results of which are summarized in Table 3 for two separate experiments.

mixture run on the HPLC just prior to analysis of the sample. The results from radioactive amounts of Glc3Man9GlcNAc2 using this technique are comparable to results achieved by others (5) using a radioactive TLC method. DISCUSSION

Although methylation linkage analysis has been developed to a sensitivity of l-5 pg of TABLE 3 QUANTITATION MANNITOLS

OF PARTIALLY METHYLATED OF Glc,MangGlcNAc2

Experimental ratio”

Theoretical Derivative

2,3,4,6-Methyl 3.4,6-Methyl 2,4,6-Methyl 2,4-Methyl

Expt I

Expt 2

1.8 4.0b 1.0 2.0

2.1 4.0 1.1 2.2

ratio

2.0 4.0 1.0 2.0

L1Determined by cutting out the area eluting as such derivative and weighing on a Metler balance. Figure 3 is a profile of the derivatives generated from Expt 2. ’ Arbitrarily set to 4.0.

oligosaccharide (4), such sensitivity is highly dependent on sophisticated instrumentation which is not always available. Moreover, the attainment of the necessary standards for proper analysis of unknown samples is always essential. Most investigators have isolated standards from various biological sources and one group has described the preparation of most of the partially methylated standards using aromatic blocking groups for synthetic as well as detection purposes (7). We have described a simple preparation of all the partially methylated radiolabeled mannitols, their separation on HPLC using one chromatographic run, and the application of this HPLC micromethylation technique to the determination of the linkage positions of the nine mannose residues of the dolichol-derived oligosaccharide, Glc3MangGlcNAcz. An important aspect essential to the resolution and reproducibility of the separation of the partially methylated mannitols by this HPLC micromethylation technique was found to be the concentration of the diaminobutane in the mobile phase. A concentration of 0.025% DAB was found to give effective resolution of the mannitol derivatives. As the

560

HULL

AND TURCO

DAB concentration was increased, elution This micromethylation procedure should volumes of derivatives decreased. A concen- provide the investigator with a convenient, tration of 20.05% in the more nonpolar buffer economical, and sensitive tool for methylation (Solvent D) proved to be less than desirable linkage analysis applicable to the structural due to solubility problems. To overcome vari- analysis of carbohydrate moieties available ations in absolute retention times from ex- only in radioactive form. periment to experiment, calculation and utilization of Kd values for each mannitol derivACKNOWLEDGMENT ative gave reproducible results. Two separate experiments were done to deThe authors are indebted to Dr. Alan Elbein for helpful termine quantitatively and qualitatively the discussions on the methylation protocol. linkages of the nine mannose residues of the HPLC-purified dolichol-derived Glc3Man9REFERENCES GlcNAc2. With 35,000-50,000 cpm of starting material it was possible to determine the linkLindberg, B. (1972) in Methods in Enzymology ages and relative ratios of the nine mannose (Ginsburg, V., ed.), Vol. 28, Part B, pp. 178-195, residues of Glc3Man9GlcNAc2. Academic Press, New York. While this report describes the preparation 2. Bjomdal, H., Hellerqvist, C. G., Lindlxrg, B., and and analysis of partially methylated mannitols, Svensson, S. (1970) Chern. Int. Ed. Engl. 9,610this technique can be potentially applied to 619. 3. Rauvala, H., Finne, J., Krusius, T., Karkkainen, J., analogous derivatives of galactose, glucose, and Jamefelt, J. (1981) Adv. Curbohydr. Chem. N-acetylglucosamine, and N-acetylgalactosB&hem. 38,389-415. amine. Although not done in this study an 4. Waeghe, T. J., Darvill, A. G., McNeil, M., and Aladvantage of this method over conventional bersheim, P. ( 1983) Carbohydr. Res. lt3,28 l-304. analysis is the possibility of adding a 14C-la- 5. Li, E., Tabas, I., and Komfeld, S. (1978) J. Biol. Chem. 253,7762-7770. beled saccharide of known structure as an in6. Saadat, S., and Ballou, C. (1983) Carbohydr. Rex 119, ternal standard, such as [U-‘4C]maltose. Such 248-253. an internal standard would provide an internal I. Mega, T., Nishikawa, A., and Ikenaka, T. (1983) Curmonitoring of the integrity of hydrolysis as well bohydr. Rex 112, 3 13-3 19. as the possible losses of the volatile derivatives 8. Dubois, M., Giles, K. A., Hamilton, P. A., and Smith, F. (1956) Anal. Chem. 28,350-356. during the evaporation steps of the procedure. Laine, R. (1980) in 27th International Congress of 9. As demonstrated by the results obtained with Pure and Applied Chemistry (Varmavuori, A., ed.), Glc3Man9GlcNAc2, with cautious evaporation pp. 193-198, Pergamon, Elmsford, N. Y. technique only at room temperature, it was IO. Laine, R. (1981) Anal. Biochem. 116,383-388. possible to obtain reliable results without an Il. Bjomdal, H., Lindberg, B., Pilotti, A., and Svennsson, S. (1970) Curbohydr. Res. 15,339-349. internal standard. Additionally, an external 12. Stellner, K., Saito, H.. and Hakomori, S. I. (1973) standard of 14C-labeled partially methylated Arch. Biochem. Biophys. 155,464-472. mannitols added to an unknown sample just 13. Turco, S. J. (198 1) Anal. Biochem. 118, 278-283. prior to HPLC analysis can also provide un- 14. Angus, W. W., and Lester, R. L. (1972) Arch. B&hem. ambiguous identification of derivatives. Biophys. 151,483-485.