THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 268, No. 7, Issue of March 5, pp. 5121-5130,1933 Printed in U.S.A.

Monosaccharide and Oligosaccharide Analysis of Proteins Transferred to Polyvinylidene Fluoride Membranes after Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis*

(Received for publication, August 4, 1992)

Michael Weitzhandler, Douglas Kadlecek, Nebojsa AvdalovicS, John G . Forte$, Dar Chow$, and R. Reid Townsendn 11 From the Dionex Corporation, Sunnyvale, California 94088, §Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, and the TDepartment of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446

We have developed an intermediate method toward the complete carbohydrate analysis of proteins, which should be universally applicable to all proteins and independent of sample matrix. Using only Coomassie Blue-stained proteins which have been electroblotted onto polyvinylidene fluoride membranes, we report a strategy for: (i) determining unequivocally whether a protein is glycosylated; (ii) obtaining a complete mono- saccharide composition; (iii) oligosaccharide mapping which separates most forms according to size, charge and isomerity; and (iv) sequentially releasing and ana- lyzing specific classes of oligosaccharides with endog- lycosidases. The method was shown to be applicable to a variety of well characterized soluble glycoproteins and to the membrane-bound protein, the gastric H+,K+- ATPase. The monosaccharide composition of the H+,K+-ATPase revealed the absence of N-acetylneu- raminic or N-glycolylneuraminic acids and a monosac- charide composition which indicated 0-linked sugar chains. Oligomannosidic/hybrid and biantennary oli- gosaccharides were sequentially released and analyzed from one electroblotted band of recombinant tissue plasminogen activator using endo-8-N-acetylglucosa- minidase H and endo-B-N-acetylglucosaminidase F2, respectively. Sialylated polylactosamine structures were identified and quantified by analyzing high per- formance liquid chromatography profiles of oligosac- charides first released by peptide-N4-(N-acetyl-&o- glucosaminy1)asparagine amidase and then treated with endo-&galactosidase, using a single, stained band of recombinant erythropoietin. This recombinant erythropoietin was found to contain eight times more tetrasialylated oligosaccharides than previously re- ported (Sasaki, H., Bothner, B., Dell, A., and Fukuda, M. (1987) J. Biol. Chem. 262,12059-12076); 47% of released oligosaccharides were identified as polylac- tosamine structures.

* This work was supported by National Institutes of Health Re- search Grants DDK31376 (to R. R. T.) and DDK38972 (to J. G. F). A preliminary report has been presented at the Keystone Symposium on Glycobiology (J . Cell. Biochem. Suppl. 16D (Abstr, 126). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “uduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ T o whom correspondence should be addressed Dionex Corp., 1228 Titan Way, Sunnyvale, CA 94088,

I( To whom correspondence should be addressed: Dept. of Phar- maceutical Chemistry, University of California, San Francisco, CA 94143-0446. Tel.: 415-476-5189; Fax: 415-476-0688.

Protein glycosylation is a major co- and post-translational modification which structurally diversifies a single polypep- tide chain into an ensemble of glycoforms (1). Over 50% of catalogued protein sequences contain the requisite sequon for N-glycosylation (Asn-Xaa-Ser(Thr); Xaa # Pro) (2), and any Ser or Thr can be potentially modified with 0-linked carbo- hydrate (3). Although it is well established that covalently linked oligosaccharide chains can be involved in such funda- mental biological processes as cellular adhesion (4) and recep- tor-mediated endocytosis (5), their role in the case of an individual glycoprotein is usually enigmatic. Most glycopro- teins are available in limited quantities, and since glycosyla- tion is cell type-specific (6), obtaining sufficient material for structural elucidation of the oligosaccharides on recombinant counterparts may not address the function of native glycosyl- ation. Thus, sensitive, universally applicable analytical ap- proaches are imperative to understand further the biological role of glycosylation.

Gel electrophoresis and replica transfer of proteins to thin sheets of nitrocellulose (7, 8), nylon (9), activated glass (10, l l ) , or PVDF’ (12) are a universally employed approach for the analysis of picomole quantities of proteins. These methods serve to remove interfering substances, minimize preparative losses, and facilitate sample handling. Immunological identi- fication of proteins has been readily accomplished after elec- trotransfer to nitrocellulose sheets (7, 8). Amino acid com- positions have been determined after hydrolysis of proteins immobilized onto PVDF (13, 14). Methods to obtain amino- terminal sequences from as little as 5 pmol of protein trans- ferred to either coated (10) or activated glass (11) or PVDF (12) have been developed. Carbohydrate analysis of immobi- lized proteins, which have been separated by SDS-PAGE, has largely been limited to the use of lectin probes (15-17) or methods based on the relative specificity of periodate for cleaving carbohydrate (18, 19). The former approach requires that the glycoprotein possess epitopes which bind tightly to available lectins while the latter can give ambiguous results from nonspecific staining (20).

We report herein a sensitive (low picomole) strategy to

The abbreviations used are: PVDF, polyvinylidene fluoride; CHO, Chinese hamster ovary; Endo, endo-P-N-acetylglucosaminidase; EPO, erythropoietin; rEPO, recombinant EPO; HPAEC/PAD, high pH anion-exchange chromatography with pulsed amperometric de- tection; Neu5Ac, N-acetylneuraminic acid; NeuGc, N-glycolylneura- minic acid; PNGase, peptide-N4-(N-acetyl-P-D-glucosami- nyllasparagine amidase; PAGE, polyacrylamide gel electrophoresis; rt-PA, recombinant tissue-type plasminogen activator; HPLC, high performance liquid chromatography; Pipes, 1,4-piperazinediethane- sulfonic acid.

5121

5122 Analysis of Proteins Transferred to PVDF Membranes

analyze the carbohydrate of glycoproteins which have been separated by SDS-PAGE and electroblotted onto PVDF mem- branes. Complete monosaccharide composition was deter- mined using high pH anion-exchange chromatography with pulsed amperometric detection (HPAEC/PAD) (21), after acid hydrolysis of the stained protein band on the PVDF membrane. Using this approach, we found that the (Y subunit band of the gastric H+,K+-ATPase (22) was not glycosylated, containing only trace amounts of monosaccharides. The p subunit band contained Man, GlcNAc, Gal, Fuc, and GalNAc, but was devoid of sialic acid. Obtaining additional structural information from single, stained bands was demonstrated using HPLC-based oligosaccharide mapping with standard compounds (23) and release of oligosaccharides with a series of specific endoglycosidases. Tandem endoglycosidase and amidase digestion was used to analyze the oligosaccharides of electroblotted recombinant erythropoietin (rEP0) and tissue- type plasminogen activator (rt-PA). Different classes of oli- gosaccharides could be sequentially released and analyzed from the same immobilized protein bands.

MATERIALS AND METHODS

Reagents-Glass-distilled water was used for the preparation of all buffers, eluents and electrophoresis solutions. The storage flask and spigot were glass throughout to prevent contact with tubings which might support microbial growth and yield artifactual sugars during monosaccharide analysis. Sodium hydroxide (50% solution) and ace- tic acid were from Fisher. Methanol was from VWR Scientific Corp. To avoid potential contamination with cornstarch, powderless gloves from Tagg Industries (Laguna Hills, CA) were used. PVDF sheets (Immobilon PSQ, 15 X 15 cm, 0.1-pm pore size) were from Millipore (Bedford, MA). Microcentrifuge tubes (part 72-694007) were from Sarstedt (Newton, NC) and autosampler vials (part 500-116) were from Sun Brokers (Wilmington, NC). Trifluoroacetic acid was from Pierce (Rockford, IL), and reduced Triton X-100 was from Calbi- ochem. All reagents for electrophoresis (Tris-HC1, acrylamide, bis- acrylamide, SDS, Coomassie Blue, glycine, ammonium persulfate) were from British Drug House. Molecular weight standards were from Pharmacia LKB Biotechnology Inc. (Uppsala, Sweden). Monosac- charide and oligosaccharide standards were from Dionex Corporation (Sunnyvale, CA).

Ribonuclease B was obtained from Sigma. Bovine fetuin (lot 22860) was purchased from GIBCO. PNGase, endo-0-N-acetylglucosamini- dase H (Endo H), and rEPO, expressed in Chinese hamster ovary cells, were supplied by Boehringer Mannheim. Endo-0-N-acetylglu- cosaminidase F2 (Endo F2) from Flavobacterium meningosepticurn was a gift from Dr. Tony Tarentino, New York State Department of Health, Albany, NY. Endo-0-galactosidase from Escherichia freundii was kindly provided by Drs. Minoru and Michiko Fukuda, LaJolla Cancer Research Foundation, Cancer Research Center, LaJolla, CA.

Preparation of the H+,K+-ATPase-Gastric microsomes were pre- pared from the nonsecreting stomachs of New Zealand White rabbits using a general procedure previously described (24) with several modifications in the centrifugation procedures, as indicated below. The gastric mucosa was scraped and homogenized in 10-15 volumes (v/w) of homogenizing buffer (113 mM mannitol, 37 mM sucrose, 5 mM Pipes, pH 6.7, and 0.4 mM EDTA) in a Potter-Elvehjem homog- enizer. The homogenate was centrifuged at 11,000 rpm in a Beckman JA20 rotor for 10 min, and the supernatant was centrifuged again at 33,000 rpm for 1 h in a Beckman type 35 rotor. The pellet of crude microsomes was resuspended in 10% sucrose and then brought to 45% sucrose. This crude microsomal suspension was overlaid with 30 and 10% sucrose layers and centrifuged at 26,000 rpm for 4 h with a Beckman SW 27 rotor (all sucrose media contain 5 mM Tris, pH 7.3, and 0.4 mM EDTA). The material that floated up to the interface between 30 and 10% sucrose was collected and used as gradient purified gastric microsomes. The purified gastric microsomes were diluted 3-fold and pelleted by centrifuging at 100,000 X g for 1 h. SDS-PAGE analysis showed over 90% of the protein in the purified microsomes can be accounted as a and p subunits of the H+,K+- ATPase. The specific activity of K+-stimulated p-nitrophenylphos- phatase for the preparation was above 1.8 pmollmg protein/min.

For deglycosylation of the 0 subunit prior to SDS-PAGE, 500 pg of gastric microsomal protein were solubilized in 0.3% SDS then

adjusted to 0.12% SDS with 200 mM sodium phosphate, pH 8.1, 10 mM EDTA, 10 mM 2-mercaptoethanol, and 0.8% Nonidet P-40. The solubilized material was treated with 3-5 milliunits of PNGase and incubated at 37 "C overnight. The PNGase-treated samples were precipitated with 75% methanol containing 200 mM NaCl at -20 "C. The precipitate was pelleted by brief centrifugation. For running SDS-PAGE the pellets were resuspended in 3% SDS.

Electrophoresis and Transfer to PVDF Membranes-Electropho- resis was performed by the method of Laemmli (25) with 12.5% polyacrylamide and 0.1% SDS cast into mini-gels (12 X 10 cm, 1.5- mm thickness). The gels were run in a Tall Mighty Small unit (Hoefer Scientific) at constant amperage (20 mA) using an LKB Multidrive 3.5-kV power supply. The glycoproteins were transferred to a single sheet of PVDF membrane using a constant current (400 mA for 5 h) in a Hoefer TE 42 transfer unit which was cooled to 15 "C by an LKB Multitemp I1 Recirculator. The transfer buffer consisted of 20% methanol in Tris (25 mM)-glycine (190 mM) buffer, pH 8.8. The proteins were stained on the PVDF membrane using 0.1% Coomassie Brilliant Blue R-250 in water/methanol/acetic acid (5:4:1) for 10 min and then de-stained with 90% methanol and finally washed in distilled water. The PVDF membranes were allowed to dry at room tempera- ture and stored at 4 "C until further analysis. Our strategy for carbohydrate analysis of stained protein bands excised from PVDF membranes is shown in Fig. 1.

Monosaccharide Composition Analysis-To determine if a stained band on the PVDF membrane was glycosylated, neutral and amino monosaccharides were released by acid hydrolysis, as previously de- scribed (21) (Fig. 1, step l ) . For analysis of neutral and amino sugars, stained bands were excised from PVDF membranes, which were wetted with methanol, placed into 1.5-ml microcentrifuge tubes, and submerged with 340 pl of distilled, deionized water and 60 pl of trifluoroacetic acid (2 M, final concentration). If sample was not limiting, approximately 20% more of the amino sugars can be released by hydrolyzing with 6 N HCI (21). Care was taken to ensure that the stained bands were completely immersed and remained submerged during the course of the hydrolysis. All tubes were capped and subjected to hydrolysis at 100 "C for the indicated times. At indicated times, the hydrolysates were centrifuged for 2 min in a bench-top microcentrifuge (Fisher, model 235C) to unite the condensate with the bulk fluid. The hydrolysates were then dried in a Speed Vac centrifuge (Savant, model SVClOO). The dried samples were recon- stituted and transferred to 200 p1 of water in 0.2-ml autosampler vials. Sialic acids were released in a separate, milder hydrolysis (26). Stained, excised bands were prewetted in methanol and submerged in 400 pl of 0.1 N HCl. Hydrolysis was performed at 80 "C for 30 min. The samples were then processed for HPAEC/PAD as described above.

Endoglycosidase and Amidase Digestions-To release and classify oligosaccharides, a stained glycoprotein bands was either treated singly or sequentially with endoglycosidases (Endo H, Endo F2, or endo-0-galactosidase) or the amidase PNGase (Fig. 1, step 2 ) . For PNGase digestions, stained bands on PVDF, after excising and wet- ting in 90% methanol, were immersed in 150 p1 of sodium phosphate buffer (5 mM, pH 7.6) containing 0.1% reduced Triton X-100. PNGase (1 milliunit) was added, followed by incubation at 37 "C for 16 h. To release and analyze sequentially oligomannosidic/hybrid and bianten- nary type oligosaccharides, a stained blot was first immersed in 150 pl of 25 mM sodium acetate buffer, pH 5.5. Endo H (1 milliunit) was added, and the tube was incubated at 37 "C for 16 h. The supernatant was removed and analyzed using HPAEC/PAD, and the blot was then submerged in the same buffer containing Endo F2 (2 milliunits). After 16 h at 37 "C, the supernatant was analyzed for oligosaccharides. To detect polylactosamine oligosaccharides, endo-0-galactosidase (1 milliunit) was added to PNGase digests after the addition of 8 pl of 200 mM sodium acetate (pH 5.0). Detergent (reduced, 0.1% Triton X-100) was included in all amidase and endoglycosidase digestions.

Monosaccharide and Oligosaccharide Analyses-Carbohydrates, which were released from stained glycoprotein bands, were analyzed using HPAEC/PAD (27) on a Dionex GlycoStation. Neutral and amino monosaccharides were separated isocratically (16 mM NaOH) using a CarboPac PA-1 column (4 X 250 mm) equipped with a PA1 guard column at a flow rate of 1 ml/min as previously described (21). Elution conditions were produced using water (eluent 1) and 200 mM NaOH (eluent 2) (prepared from a 50% NaOH solution). The column was regenerated, after 25 min, with 100% of eluent 2 for 10 min, followed by return to 16 mM NaOH. Sample injections were separated by 50 min. Sialic acids were separated with a gradient of sodium acetate (28), using eluent 3 (100 mM NaOH) and eluent 4 (100 mM

Analysis of Proteins Transferred to PVDF Membranes 5123

FIG. 1. Carbohydrate analysis of proteins on PVDF membranes.

I 1

I Glycoprotein

Endo H

NaOH, 1 M sodium acetate). The same column was equilibrated with 95% of eluent 3 and 5% of eluent 4. A gradient from 50 to 180 mM sodium acetate over 20 min was developed.

Oligosaccharides were separated on a Dionex GlycoStation using a CarboPac PA100 column (4 X 250 mm) and guard column (4 X 50 mm) at a flow rate of 1 ml/min. The column was equilibrated in 100 mM NaOH (98% of eluent 3) and 20 mM sodium acetate (2% of eluent 4). After 5 min, an acetate gradient was developed over 60 min to a limit of 200 mM while the concentration of sodium hydroxide re- mained at 100 mM.

Monosaccharides and oligosaccharides were detected, without the addition of post-column base, using the Dionex GlycoStation Pulsed Amperometric Detector. The pulse sequence, which was used for both monosaccharides (300 nA, full scale) and oligosaccharides (100 nA, full scale), was as follows: El = +0.05 V, tl = 480 ms; E2 = +0.6 V, tz = 120 ms; E3 = -0.6 V, t 3 = 60 ms.

Calculation and Interpretation of Monosaccharide and Oligosaccha- ride Chromatographic Data-The chromatographic data for monosac- charide analyses were collected and the peak areas and retention times determined using Dionex GlycoStation software. Monosaccha- rides in experimental samples were identified by comparing retention times (in minutes) as detailed in the Dionen GlycoStation Users' Manuul (Fuc, 5.87; GalN, 12.0; GlcN, 14.3; Gal 15.7; Glc, 17.1; Man, 18.6 min). The recovery of glycoproteins after SDS-PAGE, electro- blotting, and acid hydrolysis was estimated by comparing the amount of each monosaccharide in stock solutions of fetuin, ribonuclease B. and rEPO to the amount detected after hydrolysis of their respective bands on PVDF (Fig. 3, panel A, gel inset), after loading equivalent amounts to the gel.

Oligosaccharide mapping using HPAEC/PAD was performed as recently described (23) using disialylated biantennary oligosaccha- rides from a mixture of oligosaccharide alditols from bovine fetuin as a standard. Its retention time, using the acetate gradient described above for the analysis of oligosaccharides, was 35.7 f 0.6 min (u".~, n = 8) over a period of 24 h. The oligosaccharide designation codes are those previously used (23).

RESULTS

Direct carbohydrate analysis of glycoproteins, which have been immobilized onto PVDF membranes, requires that the released sugars (mono- and oligosaccharides) do not rebind to PVDF after cleavage from the peptide backbone. Table I

I I

TABLE I Binding of carbohydrate to PVDF membranes

Carbohydrate Percent detecteda

Monosaccharides Gal 92 GlcN 94 Fuc 93 Neu5Ac 102

Asialo-triantennary (C3-035300) 98 Asialo-tetra-antennary (C4-046300) 100 Man9 (M3-042900) 100 Sialo-biantennary (C2-224300) 100 Sialo-triantennary (C3-335300) 102

Oligosaccharides*

a Triplicate 32-~1 injections from solutions (with and without a 4 X 2-mm strip of methanol-wetted PVDF) of either monosaccharides (1.25 PM) or oligosaccharides (2.5 NM) were injected into the described chromatograph. The PVDF strips remained totally immersed throughout the experiment and were exposed to sugars for approxi- mately 2 h. The value was calculated as the ratio of the average peak areas, in the absence of membrane, to the average in the presence of the PVDF strip.

'The designations in parentheses are the codes for the primary structure according to Hermentin et al. (23).

shows that representatives of the four major classes of mono- saccharides found on glycoproteins, neutral (Gal), amino (GlcN), deoxyhexose (Fuc), and anionic (Neu5Ac) sugars remained completely in solution in the presence of strips of PVDF membranes. High mannose (MansGlcNAcn), lactosa- mine-type (asialo-triantennary and tetra-antennary) and an- ionic oligosaccharides (sialo-biantennary and sialo-trianten- nary), in water a t room temperature, also did not bind signif- icantly to PVDF. From these studies, we concluded that both mono- and oligosaccharides, once released from proteins on PVDF membranes, could be quantitatively recovered. To determine if previously optimized hydrolysis conditions (21) were appropriate for PVDF membranes, we examined the recovery of monosaccharides under the conditions used for their release by acid hydrolysis. Fig. 2 shows that the time-

5124 Analysis of Proteins Transferred to PVDF Membranes

TIME ( h ) TIME ( h )

FIG. 2. Release of monosaccharides from glycoproteins before and after SDS-PAGE and electroblotting onto PVDF mem- branes. Aliquots of a solution of bovine fetuin (22 pg) or rEP0 (10 pg) were hydrolyzed at the indicated times with 2 M trifluoroacetic acid and analyzed for monosaccharides as described under “Materials and Methods” (panel A ) . The fetuin hydrolysates were analyzed for Gal (0) and GlcN (O), and the rEPO hydrolysates were analyzed for Fuc (X). The same amount of glycoprotein was purified by SDS-PAGE, electroblotted onto PVDF, hydrolyzed, and similarly analyzed (panel B ) .

dependent increase of neutral (Gal), amino (GlcN), and the 6-deoxy sugar (Fuc) from immobilized glycoproteins (panel E ) (bovine fetuin and rEPO) are the same as their release from the glycoprotein in solution (panel A ) . Similarly, the appearance of Neu5Ac from soluble and immobilized sub- strates were indistinguishable (data not shown). The average recovery of all classes of monosaccharides from immobilized, well characterized glycoproteins (bovine fetuin, ribonuclease B, and rEPO) was 72% and is in agreement with a previous report (57-66%) (29). We interpreted this value to reflect the similar, overall recovery of fetuin, ribonuclease B, and rEPO after SDS-PAGE, electroblotting, and the multiple sampling handling steps associated with acid hydrolysis.

Three well characterized glycoproteins, bovine fetuin (30- 34), ribonuclease B (35), and rEPO (36, 37) were run on the same gel slot, electroblotted, stained, excised, and analyzed for monosaccharides after acid hydrolysis. The bands which were analyzed are those included in the labeled brackets of the gel inset in panel A of Fig. 3 (a , bovine fetuin; b, rEPO; c, ribonuclease B). The acid released amino and neutral mono- saccharides from the major stained bands of bovine fetuin were separated isocratically using HPAEC and detected with pulsed amperometry (Fig. 3, panel A ) . The acidic sugars, Neu5Ac and N-glycolylneuraminic acid (NeuGc) were ana- lyzed using the same HPLC method with an acetate gradient (Fig. 3, panel B ) . Quantification of individual sugars from electroblotted bovine fetuin, bovine ribonuclease B and, rEPO is given in Table 11. Detection of Man, Gal, GlcN, and Neu5Ac confirm the known occurrence of sialylated lactosamine-type structures in bovine fetuin (31, 32), while the occurrence of NeuGc is a new finding. The presence of GalN substantiates 0-glycosylation (33, 34) of fetuin. An approximately equal ratio of Gal to Man suggests the presence of lactosamine-type oligosaccharides (bi- (GakMan = 2:3), tri- (3:3), and tetra- antennary (4:3)). We have previously found that Man cannot be quantitatively released (without destruction) during acid hydrolysis (21), resulting in losses of approximately 35%. With this caveat, the ratio of Ga1:Man of 1.7:1.2 (Table 11) is consistent with the known predominance of triantennary structures on fetuin (31, 32). The ratio of sialic acids to Gal gives information on the degree that chains are terminated with sialic acid, provided that the oligosaccharides do not contain polysialic acids (38). Monosaccharide analysis re- vealed that after SDS-PAGE and electroblotting, fetuin re- mained approximately 90% sialylated (Table 11), the same value obtained from the stock solution of fetuin which was applied to the gel (data not shown). GalN was found in the stained fetuin bands which is in agreement with the known presence of 0-linked glycans (33, 34). Ribonuclease B, which contains only oligomannosidic structures (35), was, accord-

ingly, found to have only Man and GlcN (Table 11). rEPO had a significantly higher ratio of GakMan, 6.2~2.3, and a ratio of Ga1:GlcN = 6.2:8.3, which suggests that this rEPO also contains Galp(l4)GlcNAc repeats (polylactosamine structures) attached to the trimannosyl core of the N-linked chains (36). GalN also was detected, which is from the single 0-glycosylation site on rEPO (37).

We next assessed the presence of any interfering peaks from materials used for SDS-PAGE and electroblotting by analyzing a protein which is known not to be glycosylated (39). The HPAEC/PAD chromatogram of the trifluoroacetic acid hydrolysate of a stained bovine serum albumin band (8 pg) is shown in Fig. 2, panel C. Small peaks at a retention time corresponding to Glc and one eluting slightly later then Man were observed. Although we have found that the size of the Glc peak can vary among samples, these amounts have not interfered with quantification of the other sugars. Glc is a ubiquitous contaminant and can be introduced from sources such as the water, hydrolysis vials, and cornstarch powder from gloves (see caveats under “Materials and Methods.”

We next determined the monosaccharide composition of the individual subunits of the H+,K+-ATPase, a membrane glycoprotein which requires salt and detergent for solubility (40). Using SDS-PAGE, H+,K+-ATPase can be separated into a sharp a subunit band (94 kDa) and a broad ,8 subunit band extending 60-80 kDa (40). The a and intact p subunits from rabbit microsomes were electroblotted onto a PVDF mem- brane and acid hydrolyzed for monosaccharide analysis. Fig. 4 shows the HPAEC/PAD chromatograms of released neutral and amino sugars (panel A ) and sialic acids (panel B ) from the p subunit. The /3 subunit contained Man, Gal, GlcN, and Fuc (Table 111), which suggests the presence of lactosamine type oligosaccharides. However, no sialic acid was detected from the p subunit band (Fig. 4, panel B ) . Only trace amounts of monosaccharides were found associated with the a subunit, confirming that the a subunit is not glycosylated (40). Pre- viously, evidence was lacking for 0-glycosylation (40), but the detection of GalN in the intact p subunit suggested the presence of 0-linked carbohydrate. To obtain further evidence that the @ subunit is 0-glycosylated, the p subunit was N - deglycosylated, prior to SDS-PAGE, using PNGase, to give a p-core (Mr = 34,000), which was then analyzed for monosac- charides (Table 111). GalN, GlcN, and Gal, monosaccharides which are commonly found in 0-linked chains, were detected and the disappearance of Man confirmed complete de-N- glycosylation.

To analyze the oligosaccharides from stained, immobilized glycoprotein bands, we released oligosaccharides using ami- dases and endoglycosidases. To avoid losses from additional sample handling and derivatization, we used incubation con-

Analysis of Protcins ‘I‘ransfrrrpd to I>VI)F Mrmbrancs

FIG. 3. Monosaccharide analysis of proteins on I V D F membranes. The major stained hands from a single lane of either hovine fetuin (hrnchct a of the gel insct in pnnrl A ) or hovine serum alhumin (the M , = 67,000 hand from the adjacent lane of molecular weight mark- ers) were excised from wetted PVDF membranes. Twenty-two and 8 pg of fe- tuin and hovine serum alhumin were ap- plied to each lane, respectively. Individ- ual bands were hydrolyzed with either 2 M trifluoroacetic acid (pone/.$ A and C ) or 0.1 N HCI ( p o n d H ) and analyzed for monosaccharides as descrihed under “Materials and Methods.” The num- bered nrmros indicate the elution posi- tions of the standard monosaccharides: 1, Fuc; 2. GalN; 3 , GlcN; 4 , Gal; 5 , Glc; 6, Man; 7, NeuFiAc; and H, NeuCc. The dashed line in panel H indicates the so- dium acetate gradient which was used to elute sialic acids. Hrnckets h and c of the stained hlot in panelA include the rEPO and rihonuclease H. respectively. which were analyzed (see Tahle 11). The molec- ular weight standards are phosphorylase h ( M , = 94,000), bovine serum alhumin ( M , = 67,000), ovalhumin ( M , = 43,000). carhonic anhydrase ( M , = :10,000), soy- bean tr.ypsin inhihitor ( M , = 20,000) and tr-lactalbumin ( M , = 14,000). respec- tively.

100

50

0 n Q W

c 0 Q m

1 1

Q a

Glycoproteins Monosncrhnridrs“

Mnn Gal GlrN Fuc Neu5Ac NeuGr GnIN

Fetuin 1.2 1.7 2.9 -’ 1.5 0.15 0.46 Ribonuclease R 2.2 - 0.91 - - - - rEPO 2.3 6.2 8.3 1.7 5.6 - 0.31

Expressed as nanomoles of monosaccharide found in the stained hands, which were excised from PVDF memhranes. Twenty, 3 0 , and 8 p g of hovine fetuin, rihonuclease R, and rEPO, respectively. were applied to the same gel slot, separated hy SIX-PAGK, and hlotted onto I’VDF membranes. The hands which were analyzed are indicated hy the labeled hrackets in the stained hlot of Fig. 3. The neutral ( 2 N trifluoroacetic acid). amino sugars (fi N HCI), and sialic acids (0.1 N HCI) were quantified using HI’AEC/l’AD, after acid hydrolysis, as descrihed under “Materials and Methods.” ’ Indicates none detected.

ditions which were also compatible with direct analysis by HPAEC/PAD. Fig. 5, panel A , shows the profile of oligosac- charides that were released with the amidase, PNGase, from the major bands (Fig. 3 , hrnchet a ) of a single lane of electro- blotted bovine fetuin. The proportions of tet,ra-, tri-, hi-, and

monosialylated species and their isomeric forms were ident icnl to those ohserved after digestion of the s:me fc,tuin solution used for SDS-PAGE and electrohlotting (data not shown). T h e large peak detected from 1 t o 10 min of elution time (pnncl A ) is from the glycerol present in this PX(;asr prepa- ration. Fig. :i, pnncl H , shows the chromatogram of oligosac- charides which were released from a single, stained band o f rihonuclease H (Fig. 3 , hrachd c ) after treatment with Endo H. The expected MansGlcNAc, through ManJ;IcNAc, st ruc- tures were ohserved with separation of the Man$lcNAc, in to its two branch isomers (35). T h e oligosaccharide profile was identical to that ohtained from Endo H digestion of rihonu- clease H in solution (data not shown). Peaks eluting prior to 5 min ( p a n r l H ) were detected in incrlhations not containing the glycoprotein substrate. Fig. 5. p o n d (’, shows that oligo- saccharides were not released unless detergent (O.lc; Triton X-100) was present in the Endo H digest. The same negative result was also ohtained if detergent was omitted from I’NGase digestions of either immohilized fetuin or rRl’O. IVr surmised that the detergent blocks the adsorption o f glycosi- dases to the I’VIIF strips, hu t other possihilities. such as “exposing” glvcosylation sites, have not heen investigatrrl.

We investigated whether the detergent, which was rrquirrd

5126 Analysis of Proteins Transferred to PVDF Membranes

FIG. 4. Monosaccharide analysis of the @ subunit of the H+,K+-ATP- ase. The H+,K+-ATPase was prepared

by SDS-PAGE as described under “Ma- from rabbit gastric mucosa and purified

terials and Methods.” The 60,000- 80,000-Da band was excised from the PVDF membrane after electroblotting (20 pg applied to the gel) and analyzed for monosaccharides after acid hydroly- sis with 2 M trifluoroacetic acid (4 h) (panel A). A similar band was excised from the PVDF membrane, treated with 0.1 N HCl, and analyzed for sialic acids as described under “Material and Meth- ods” (panel B ) . The numbered arrows designate the elution positions of stand- ard monosaccharides: 1 , Fuc; 2, GalN; 3, GlcN; 4, Gal; 5, Glc; 6, Man; 7, Neu5Ac; and 8, NeuGc. The dashed line in panel B shows the sodium acetate gradient which was used to elute sialic acids.

TABLE 111 Complete monosaccharide analysis of electroblotted H+/K+ ATPase

subunits onto PVDF membranes

Glycoproteins Monosaccharides“

Man Gal GlcN Fuc Neu5Ac NeuGc GalN

@subunit 400 720 1500 360 -b - 40 @-Core - 240 100 - - - 120 Expressed as pmoles of monosaccharides found in the p subunit

(M, = 60-80,000) and @-core (Mr = 34,000). Twenty pg of p subunit were applied to the gel. To produce the &core, 50 pg of the 0 subunit were digested with PNGase, as described under “Materials and Meth- ods,” prior to SDS-PAGE. The identity of the @core band was confirmed by Western blotting. The neutral (2 N TFA), amino sugars (6 N HCl), and sialic acids (0.1 N HCl) were quantified using HPAEC/ PAD, after acid hydrolysis, as described under “Materials and Methods.”

Indicates none detected.

for amidase and endoglycosidase release of oligosaccharides, displaced glycoproteins into solution. PVDF blots of fetuin and rEPO were incubated without cleaving enzymes for 24 h at 37 “C in buffer and detergent (0.1% reduced Triton X-100). The supernatants and blots were then analyzed for monosac- charides. Greater than 97% of the monosaccharides remained associated with their respective blots. These results indicated that sufficient quantities of glycoprotein remained associated with the PVDF membranes to enable sequential treatments of the same blot with different endoglycosidases.

rt-PA contains both high mannose and complex-type oli- gosaccharides (42). Fig. 6, panel A , shows the profile of the oligosaccharides that were released by Endo H treatment of electroblotted rt-PA after SDS-PAGE. The retention times of the two major peaks (9.00 and 12.2 min) corresponded to the elution positions of Man5GlcNAc and Man~GlcNAc (from elution times of known oligosaccharides from Endo H diges- tion of ribonuclease B). The arrows indicate the elution po- sitions of standard Man5GlcNAc2 and ManQGlcNAc2. Among the oligomannosidic and hybrid structures from rt-PA, Man5GlcNAc and MansGlcNAc comprise 36 and 34%, respec- tively (42). The areas of these two peaks (Fig. 6, panel A )

TIME (min)

were found to be 41 and 34%. A peak, which comprised 13.7% of the electrochemical response, eluted at the same time (-12.5 min) as MawGlcNAc isomers, structures which accounted for 20% of the oligomannosidic and hybrid struc- tures on rt-PA (42). Minor peaks (<5% of the signal) eluted in positions corresponding to MansGlcNAc and ManQGlcNAc, structures not found in sufficient abundance for structural studies (42). Endo F2 is a recently described endoglycosidase which cleaves predominantly biantennary-type chains (43). Incubation of the Endo H-treated blot of rt-PA with Endo F2 and analysis of the released oligosaccharides gave the profile shown in Fig. 6, panel B . Three major peaks (Rt = 12.2, 26.8, and 41.8 min) with relative areas of 30, 33, and 37% eluted near asialo-, mono- and disialylated biantennary oligosaccha- rides, respectively. Structures which accounted for the minor peaks in the “neutral region” of the chromatogram (5-18 min) have presently not been identified, but given the specificity of Endo F2, may be hybrid and or oligomannosidic structures which are not completely removed by the prior digestion with Endo H. Two peaks which appeared with a Rt - 55 min suggest a newly described biantennary-type structures for t- PA with three formal negative charges.

Endo-@-galactosidase (44) is an invaluable tool for identi- fying and analyzing polylactosamine oligosaccharides on gly- coproteins (for review, see Ref. 45). Since polylactosamine oligosaccharides are often linked to membrane glycoproteins, we determined whether this oligosaccharide class could be analyzed using our protocol. The chromatographic profile of the oligosaccharides which were released from rEPO with glycerol-free PNGase is shown in Fig. 7, panel A. Mono- (4%), di- (16.1%), tri- (43.7%), and tetrasialylated (33%) species were observed. These released oligosaccharides were then treated with endo-P-galactosidase, in the same incubation, and analyzed using HPAEC/PAD (Fig. 7, panel B ) . After enzyme treatment, the four major peaks in the tetrasialylated complex were changed to a major, symmetrical peak (Rt = 58.3 min). The relative area of this peak was 17.5% and 16.6% before and after endo-&galactosidase treatment, respectively. From previous studies of the oligosaccharides from rEPO (361,

Analysis

FIG. 5. Oligosaccharide analysis of electroblotted glycoproteins. Either bovine fetuin (22 pg), panel A ) or bovine ribonuclease B (panels C and D) (30 pg) was electrophoresed, stained, and transferred to a 0.1-pm PVDF mem- brane. The major stained bands of bo- vine fetuin (Fig. 3, bracket a ) were ex- cised and placed in incubation buffer for PNGase digestion as described under "Materials and Methods." After incuba- tion, the digests (80%) were injected di- rectly into the chromatograph. The elu- tion positions of mono-, di-, tri-, and tetrasialylated oligosaccharides are in- dicated by the labeled brackets (panel A). The higher molecular weight band of ribonuclease B (Fig. 3, bracket c ) was digested with Endo H as described under "Materials and Methods" (panel B ) . The numbered arrows in panel B indicate the elution positions of Man6GlcNAcz (51) and MansGlcNAcn (12). The dashed line shows the acetate gradient which was developed with a constant concentration of sodium hydroxide (100 mM). In panel C, ribonuclease B was incubated with Endo H, as described in "Materials and Methods" for panel B, but in the absence of reduced Triton X-100 (0.1%).

of Proteins Transferred to PVDF Membranes

40 Trl-

20 -

n Q o - L 1 " l l l " l ' l / S - 1

30- &

0 . 0

0 0

. Q) v) S 0 Q v) Q)

0

L3L - . ~ o - ' l ' l l l l l ' l l l l ' L1 C Q

20 -

10-

L

O O 1 l l l ' l ' l r t l l l 10 20 30 40 50 60 i TIME (min)

5127

200

z U 0)

0 100 3

6

E 1 -a .- (s:

0

3

200

n I E

W

Q

100 3 Y 9)

.- 5 U v) 0

?

FIG. 6. Tandem endoglycosidase digestion of rt-PA electrotrans- ferred onto PVDF membranes. rt-PA was electrophoresed and electroblotted onto a 0.1-pm PVDF membrane. The major band was excised and treated with Endo H (panel A ) as described under "Materials and Methods." The superna- tant (150 pl) was removed and analyzed for oligosaccharides by HPAEC/PAD. The treated blot was then placed in 150 p1 of 25 mM sodium acetate buffer (pH 5.5), and 2 milliunits of Endo F2 were added and then incubated for 8 h at 37 "C. One hundred and 50 p1 were re- moved and analyzed (panel B ) . The dashed line indicates the acetate gradient which was used for both separations. The elution positions of MansGlcNAcz,- Man9GlcNAc,, are indicated by the num- bered arrows.

TIME (min)

5128 Analysis of Proteins Transferred to PVDF Membranes

FIG. 7. Endo-&galactosidase di- gestion of PNGase-released oligo- saccharides from electroblotted rEPO. rEPO, which was electroblotted onto PVDF membranes after SDS- PAGE (10 /.tg applied to the gel), was stained and excised. The band was then incubated with 2.5 milliunits of glycerol- free PNGase in 150 p1 of buffer as de- scribed under “Materials and Methods.” After 16 h, 75 p1 were removed and ana- lyzed using HPAEC/PAD (panel A ) . The pH value was decreased to approx- imately 5.0 using 8 pl of 200 mM sodium acetate buffer, pH 5.0, and then 1 mil- liunit of endo-@-galactosidase was added. After 16 h, the remaining volume was analyzed using HPAEC/PAD (panel B ) . The elution positions of mono-, di-, tri-, and tetrasialylated species are indicated by the like-labeled brackets. The dashed line indicates the sodium acetate gradient.

A n 2 80 u

a, cn C 0 Q 30 VI

$105

n Q [L

30

we assigned this peak as a tetrasialylated tetra-antennary structure (C4-457301.40.00). Based on their disappearance after endo-@-galactosidase treatment, we concluded that three of the four major tetrasialylated species contained polylacto- samine structures (C4-457301.40.1R). Since HPAEC/PAD separates oligosaccharides not only according to linkage dif- ferences but also by branch location of sialic residues (27) and since only a2-3-linkages have been identified in CHO cell- derived recombinant rEPO (36), the three earliest eluting peaks in the tetrasialylated complex likely differ only in the branch location of the sialylated lactosamine repeats. The profile of the trisialylated complex was also significantly changed after endo-@-galactosidase treatment (Fig. 7, panel B) . The four incompletely resolved peaks in the PNGase- released oligosaccharides were replaced by three sharp peaks of approximately equal areas (R, = 46.5-48.3 min). These trisialylated species are either derived from the tetrasialylated forms, containing one sialylated polylactosamine structure, or represent trisialylated oligosaccharides without such units. The overall reduction of the area of the trisialylated complex from 43.7 to 30.4% and the known predominance of tetra- antennary oligosaccharides with one sialylated lactosamine repeat strongly suggests the presence of trisialylated polylact- osamine structures in this rEPO preparation. Only a modest change in the area of the disialylated complex was observed. However, a new peak ( R , = 21.3), which comprised 19.6% of the total electrochemical response, appeared near the mono- sialylated complex (Fig. 7, panel B ) . From the described specificity of endo-@-galactosidase (44) and previous struc- tural studies of rEPO (36), we concluded that this peak (Rt = 21.3 min) was Neu5Aca(2+3)Gal@(l+4)GlcNAc@(l+3)Gal. The smaller amount of sialylated polylactosamine structures with two and three repeats, which have been found in recom- binant rEPO, likely comprise the monosialylated complex which elutes after the above larger, symmetrical peak ( R , = 24 min). New peaks did not appear in the region of the chromatogram where neutral species elute (5-18 min), con- sistent with a conclusion that all the polylactosamine oligo- saccharides, which were released by endo-@-galactosidase, are sialylated.

TIME (min)

DISCUSSION

Determining whether a protein is glycosylated is an impor- tant initial step in the structural characterization of natural or recombinant proteins. A diffuse band after SDS-PAGE suggests glycosylation and a reduction in apparent molecular weight after treatment with an endoglycosidase or amidase is often used to substantiate covalently linked sugar chains. However, negative results may lead to erroneous conclusions, since N-linked oligosaccharide chains on some glycoproteins are not easily released (46) and only one 0-linked sugar structure (Gal@l+3GalNAc+) can be cleaved with the only described “0-glycanase” (endo-a-N-acetyl-D-galactosamini- dase) (47). If a protein is modified with oligosaccharides that are recognized by available lectins, the presence of glycosyla- tion and some structural information can be deduced (15-17). However, a more direct and universal approach is to determine the monosaccharide content of the protein band.

Most “sensitive” methods for monosaccharides analysis require large amounts of starting material due to complicated derivatization schemes and concomitant sample loss (48,49). Further, multiple HPLC methods are often needed to analyze all classes of monosaccharides found on glycoproteins (49). Recently, a sensitive HPLC method (low picomole) has been developed which neither requires derivatization nor multiple separation methods for not only monosaccharide but also oligosaccharide analyses (27). However, application of these advances to the analysis of proteins in commonly encountered sample matrices, which, contain, for example, salt and deter- gent, can be problematic. Separation of proteins by SDS- PAGE and electroblotting to a thin membrane sheet is a simple, generic approach for the purification and removal of such interfering substances from proteins. We showed that neither monosaccharides nor oligosaccharides bind to PVDF membranes, with recoveries approaching 100%. The suitabil- ity of PVDF membranes for releasing amino acids (13, 14) and monosaccharides (29) by acid hydrolysis has been dem- onstrated. We report herein the usefulness of HPAEC/PAD for the carbohydrate analysis of stained proteins on PVDF membranes.

Analysis of Proteins Transferred to PVDF Membranes 5129

We obtained a complete monosaccharide composition (neu- tral, amino and anionic sugars) using only two protein bands from parallel lanes and two chromatographic analyses. Since electroblotting onto PVDF eliminates substances that can interfere with both chromatography and detection (e.g. salts and detergents), we showed that this method was applicable to both soluble and membrane glycoproteins. Because sepa- ration of subunits of membrane glycoproteins is best achieved by SDS-PAGE, our method permits unambiguous monosac- charide and oligosaccharide analysis of individual subunits. In addition to identifying stained protein bands which are glycosylated, quantitative monosaccharide analysis suggests structural features and, in some cases, oligosaccharide classes (e.g. oligomannosidic, lactosamine, or polylactosamine). The monosaccharides composition of bovine fetuin, ribonuclease B, and rEPO blots were determined, exemplary of glycopro- teins that contain lactosamine (complex), oligomannosidic-, or polylactosamine-type structures, respectively.

The utility of our approach for analyzing membrane gly- coproteins was demonstrated with the gastric H+,K'-ATPase, which is responsible for HC1 secretion in the stomach (for review see Ref. 22). The detergent and salt requirements for maintaining solubility of this multi-subunit membrane gly- coprotein during purification have made carbohydrate anal- yses laborious (40). This cation antiporter consists of a and @ subunits with M, = 94,000 and 60,000-80,000 respectively. The cloned rat a subunit consists of 1033 amino acids ( M I = 114,012) and 5 potential glycosylation sites (50), which have been proposed not to be glycosylated on the basis of their suspected location on the cytoplasmic side of the plasma membrane, the inability to bind to lectin-Sepharose columns, and the nonspecific staining of the a subunit with dansyl hydrazine after periodate treatment of gel bands (40). The present studies demonstrated that the a subunit is definitively not glycosylated on the basis of monosaccharide analysis of the electroblotted band. The cloned rat @ subunit consists of 294 amino acids (Mr = 33,689) with 7 potential N-glycosyla- tion sites (41). The rabbit @ subunit stained positively for carbohydrate on SDS gels, bound to wheat germ agglutinin and Ricin A-Sepharose columns, changed mobility after treat- ment with PNGase and was found to contain hexose and hexosamines (40). We found that the (3 subunit contained a ratio of Gal and GlcNAc to Man which suggested the presence of lactosamine-type oligosaccharides. Further, the presence of GalN on the protein after removal of all N-linked oligosac- charides indicated the presence of O-linked structures, a novel finding for the H+,K'-ATPase. The @ subunit was found to be devoid of sialic acids, a noteworthy finding since sialyl transferase activity is region-specific in gastrointestinal tis- sues (51).

Further carbohydrate analysis of a glycoprotein requires release of the oligosaccharides. Using a single, stained band on PVDF, the N-linked oligosaccharides can be released and the charge classes deduced. A portion of this same digest can then be treated with endo-@-galactosidase to test for polylact- osamine structures. Another band can be treated with Endo H and an HPLC profile of the oligomannosidic/hybrid struc- tures can be obtained. The same blot can then be treated with Endo F2 to determine if biantennary oligosaccharides are present. Well characterized, purified enzymes have been used extensively to cleave N-linked sugar chains from glycopro- teins in solution (for review, see Ref. 52). In agreement with previous studies (53), we found that the amidase, PNGase, and endoglycosidases (Endo H and Endo F2) released oligo- saccharides from immobilized glycoproteins only in the pres- ence of detergent.

Most (>go%) of the glycoprotein (fetuin, rEPO, or ribonu- clease B) remained associated with the membrane after in- cubation within detergent. This finding enables the treatment of a single blot with a series of endoglycosidases of different specificity and analysis of oligosaccharide after each digestion. Not only does this approach conserve substrates, but it also classifies oligosaccharide structures and augments the infor- mation obtained from peak elution positions (23). Detection of oligosaccharides after incubation with Endo H is diagnostic of oligomannosidic and hybrid structures (52). This endogly- cosidase requires a Mana( 1+6)Mana(l+3)Man@l4)- consensus sequence and cleaves between the two GlcNAc residues which comprise the chitobiosyl core. After Endo H treatment of rt-PA, primarily oligomannosidic structures were detected. The major peaks eluted at the same time as Man7GlcNAc, ManGGlcNAc, and ManbGlcNAc, the three ma- jor oligomannosidic structures in rt-PA (42). Disialylated (a- 2,3-linked) biantennary with fucose attached a-1-6 to the innermost core GlcNAc (C2-224301.20) comprised 66% and its monosialylated counterpart (C2-124301.20) 20% of the biantennary species (42). After Endo F2 digestion, we found 38 and 37% of the biantennary structures were di- and mon- osialylated, respectively, and 30% were in the asialo form. We attributed this greater percentage of partially sialylated spe- cies to the fact that our rt-PA had lost sialic acid upon prolonged storage.

A major impediment in understanding the role of oligosac- charides using recombinant counterparts has been the cell type-dependent nature of glycosylation (6), requiring compar- isons of oligosaccharides from the natural product. Significant differences between the oligosaccharides of human urinary and rEPO from both CHO and baby hamster kidney cells have been described (36). Urinary EPO was found to have much lower amounts of polylactosamine structures than rEPO, and EPO from different sources vary in the proportion of sialylated species (36). The methods described herein en- able the identification of such structural features from a single, stained band on PVDF. Comparison of the chromato- graphic profiles of EPO oligosaccharides before and after endo-@-galactosidase treatment showed a significant change in the profile of the sialylated complexes and the appearance of a diagnostic enzyme cleavage product (Neu5Aca(2+ 3)Gal@( 14)GlcNAc@(l-3)Gal). Such an approach should prove particularly useful for the analysis of polylactosamine structures, which are commonly encountered on membrane glycoproteins (45). Disialylated EPO is rapidly cleared from the circulation (54) and thus, a sensitive method for assessing sialylation is important for unraveling the effect(s) of glyco- sylation on in vivo activity. Previous studies of oligosaccha- rides from EPO, expressed in CHO cells found the proportion of mono-, di-, tri- and tetrasialylated species to be 7, 41, 48, and 4% (36). The percentage of more highly sialylated species was much greater in our EPO (expressed in the same cell type) with only a small amount of monosialylated oligosac- charides (4%) and 16.1, 43.7, and 33% being di-, tri-, and tetrasialylated forms. Further, in agreement with previous studies, we determined that negligible amounts of the poly- lactosamine units lacked sialic acid.

We have developed a new method for monosaccharide and oligosaccharide analysis of sample-limited proteins which minimizes sample handling and eliminates interfering sample matrices. With two stained, electroblotted protein bands on PVDF membranes a complete monosaccharide composition is determined using HPAEC/PAD. Identification and quan- tification of neutral, amino-, deoxy-, and anionic sugars can be accomplished from two stained bands (10-50 pg of protein)

5130 Analysis of Proteins Transferred to PVDF Membranes

after acid hydrolysis. Glycosylated proteins can be further characterized after the sequential release of oligosaccharides with specific endo-glycosidases (Endo H, Endo F2, and endo- 8-galactosidase) and the amidase, PNGase. The N-linked oligosaccharides, from two stained band can be classified as oligomannosidic/hybrid, polylactosamine, and/or bianten- nary. This strategy represents an obligatory first step toward developing sufficiently sensitive methods for the complete structure elucidation of oligosaccharides from sample-limited glycoproteins. The information obtained from this method should not only identify strategies to pinpoint critical struc- tural features, but also facilitate a better understanding of the function of protein glycosylation.

Acknowledgments-We thank Richard Lahti for his assistance in preparing this manuscript. We appreciate the critical reading of this manuscript by Dr. Tony Tarentino, New York State Department of Health, Albany, NY.

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