Electroblotting onto glass-fiber filter from an analytical isoelectrofocusing gel: A preparative method for isolating proteins for N-terminal microsequencing

ANALYTICAL

BIOCHEMISTRY

170, l-8

(1988)

Electroblotting onto Glass-Fiber Filter from an Analytical lsoelectrofocusing Gel: A Preparative Method for Isolating Proteins for N-Terminal Microsequencing JYH-CHENG

HSIEH,*

Fu-PANG

LIN,t

AND MING F. TAM?'

*Institute of Life Science, National Tsing Hua University, Hsinchu. Taiwan 30043, Republic of China, and tlnstitute of Molecular Biology, Academia Sinica. Nankzng Taipei, Taiwan 11529, Republic of China Received July 20, 1987 A new method has been developed for the isolation of proteins for microsequencing. Proteins were separated by isoelectric focusing on polyacrylamide slab gels. Ampholytes in the gel were washed out with 3.5% (v/v) perchloric acid, and the proteins were electroblotted onto unmodified glass-fiber sheets. The immobilized proteins on the glass-fiber sheet were detected with Coomassie blue dye staining. The protein bands were then excised from the sheet and inserted into a gas phase sequenator for direct sequencing. They could also be extracted with sodium dodecyl sulfate buffer for molecular weight determination. Bovine serum albumin, /3-lactoglobulin A, and soybean trypsin inhibitor have been used as standard proteins for the test of this technique. Using this technique, we have determined the partial N-terminal sequence (26 residues) of an acidic (PI 5.6) glutathione S-transferase isolated from the chicken her. KEY

Q 1988 Academic R-e%%,Inc. WORDS: protein sequencing;

isoelectric focusing; protein blotting; glutathione Strans-

ferase.

Protein sequence analysis is important in the field of molecular biology. Partial amino acid sequences can be used for the prediction and synthesis of unique oligonucleotide probes for gene isolation from cDNA or genomic DNA library ( 1,2) or in confirmation of cloned coding sequences (3). Proteins for sequence determination were usually purified to homogeneity by chromatography (4) or by a combination of SDS’polyacrylamide gel electrophoresis, Coomassie blue dye staining, and electroelution of

the stained protein from the gel slice (5). These processes are time consuming and give variable recoveries of particular proteins. A new process in protein purification for sequencing, which has received considerable attention within the past 2 years, has been reported by Vandekerckhove et al. (6). Proteins were separated on a SDS-polyacrylamide gel and transblotted onto a Polybrene-coated glass-fiber sheet. Each bound protein was then used directly for microsequencing. Aebersold et al. reported a similar process (7), with the exception that the glassfiber sheets were chemically modified with aminopropyl or quarternary ammonium groups. These powerful techniques have one drawback: proteins with molecular weights too close for separation on an SDS gel would give unmeaningful mixed sequences. Proteins with similar molecular weights but different pIs can be resolved in an iso-

’ To whom correspondence should be addressed. ’ Abbreviations used: SDS, sodium dodecyl sulfate; IEF, isoelectric focusing; GST, glutathione Stransferase; acryl, acrylamide; TEMED, N,N, N’,N’-tetramethylethylenediamine; PTH-amino acid, phenylthiohydantoinamino acids; TFA, trifluoroacetic acid, TCA, trichloroacetic acid, Bis, N,N’-methylenebisacrylamide; PCA, perchloric acid; BSA, bovine Serum albumin; CDNB, I-chloro-2,4-dinitrobenzene; NBT, nitroblue tetrazolium; PMS, phenazine methosulfate. 1

0003-2697188

$3.00

Copyright 0 1988 by Academic Press, Inc. All rigbls of reproduction in any form reserved.

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HSIEH,

LIN,

electric-focusing (IEF) gel (8). We report here a general approach for preparing proteins that have been separated on an analytical IEF gel for N-terminal sequencing. Ampholytes in the IEF gel interfere with protein detection and sequencing. In order to solve the problem, we have used perchloric and acetic acids to remove ampholytes from the gels after electrophoresis. The proteins could then be electroblotted onto untreated glassfiber filters (Whatman GF/C or GF/F) and detected by Coomassie blue dye staining. The glass-fiber-bound proteins were excised from the filter and inserted into the reaction chamber of a gas-phase sequenator for the Edman degradation procedure. This technique has been applied on a glutathione S-transferase (GST) isozymes system of chicken liver cells. The isozymes are composed of both anionic and cationic proteins and can be separated into three major groups according to their molecular weights (Tam et al., manuscript in preparation). The anionic proteins could further be resolved into at least 15 bands on a native IEF gel. We show in the following that by the method described above, we were able to determine the first 26 amino acids on the N-terminal of one of these anionic proteins with a minute amount of the proteins. MATERIALS

AND METHODS

Materials. Acrylamide, N,N’-methylenebisacrylamide (Bis), sodium dodecyl sulfate (SDS), ampholytes (Bio-lyte), and ammonium persulfate are all electrophoresis grade chemicals from Bio-Rad Laboratories. Coomassie brilliant blue R-250, perchloric acid (PCA), Tris(hydroxymethyl)aminomethane, ethanol, acetic acid, Omni-Szintisol, and Hz02 are reagent grade chemicals from Merck. Bovine serum albumin (BSA), soybean trypsin inhibitor, fl-lactoglobulin, glutathione, Shexyl glutathione, l-chloro-2,4dinitrobenzene (CDNB), nitroblue tetrazolium (NBT), and phenazine methosulfate (PMS) were from Sigma. Chemicals used in

AND

TAM

sequencing and PTH-amino acids analysis were obtained from Applied Biosystem, Inc. (ABI). r4C-carboxylmethylated BSA was purchased from New England Nuclear. Glass-fiber filter papers GF/F and GF/C were purchased from Whatman. Epoxy-activated Sepharose 6B was obtained from Pharmacia. Isoelectric focusing. Proteins were separated by isoelectric focusing on a native polyacrylamide vertical slab gel (140 X 130 X 0.75 mm). A typical gel solution (20 ml) contained either 7.5 (w/v) or 12% (w/v) acrylamide (acryl:Bis, 30:0.8), 0.4% ampholytes, pH 3-10, 1.6% ampholytes, pH 5-7, and 10% glycerol. Polymerization was initiated by the addition of 70 ~1 10% ammonium persulfate and 28 ~1 TEMED. The anionic buffer was 2.5% phosphoric acid and the cationic buffer was 10 mM NaOH. Gels were prefocused at constant voltages of 200, 300, and 400 V for 15, 15, and 30 min, respectively. Samples were placed in 10% glycerol and 2% ampholytes, loaded onto the cationic side, and focused at 400 V for 14 h. The pH gradient formed in the gel was measured according to Hjelmeland et al. (9). For direct protein visualization, gels were fixed with 10% (w/v) TCA for 30 min before staining with Coomassie blue R-250 in 10% (v/v) acetic acid and 50% (v/v) ethanol. Gels were destained in the same solution without dyes. Washing of ampholytes. Ampholytes were removed from IEF gels by a two-step washing procedure. Gels were soaked in 200 ml (10X gel volume) of 3.5% perchloric acid at room temperature for an hour with gentle shaking, and the perchloric acid was changed every 10 min for five times. The proteins were fixed on the gel with perchloric acid. Then the gels were blotted, as the second step of the washing procedure, in a Southern blotting unit (10) for 3 h with 2.0% acetic acid as carrier buffer. Electroblotting. Electroblotting was carried out in a Bio-Rad Trans-Blot Cell using 0.5% (v/v) NP-40 and 1.O% acetic acid as the buffer. The ampholyte-free slab gel was

PROTEIN

SEQUENCING

FROM ISOELECTROFOCUSING

sandwiched between prewet GF/F or GF/C glass-fiber papers, three layers of Whatman No. 1 papers, and sponges. Electroblotting was performed at 350 mA constant current for 4 h at room temperature. The buffer in the blotting chamber was circulated by stirring. After electroblotting, the glass-fiber filter papers were stained with 0.5% Coomassie blue R-250 in 30% isopropanol and 10% acetic acid for 2 min (7), and destaining was carried out in Milli-Q (Millipore) treated water. The protein bands were visualized against a light blue background and excised from the filter with a sharp razor blade. The filter-bound proteins were dried in a Speed Vat concentrator and stored at -20°C in an Eppendorf tube until sequencing. Radioactivity assay. The efficiency of the electroblotting procedure was quantitated by determining the amount of 14C-labeled BSA remaining in the gel or glass-fiber filter after each step. Gels were sliced into 0.5 X 1.O-cm pieces and each piece was treated with 1 ml of 30% H202 and 60% HC104 at 70°C overnight. The solutions were neutralized with NaOH and counting was performed in a Beckman Model LS 3801 liquid scintillation counter after addition of Omni-Szintisol. Protein sequencing. Automated cycles of Edman degradation were performed with an ABI gas/liquid phase Model 470A sequencer (11) with an on-line Model 120A PTHamino acids analyzer. The protein-bound filter was cut into small pieces, loaded on top of the Zitex support in the reaction cartridge (7), and then covered with a Polybrenetreated filter. The Polybrene-treated filter was used to prevent peptide loss from the sequenator (12) and avoided channeling of reagents and solvents during sequencing. The sequencing program was carried out with a subroutine consisting of a 5-min TFA treatment followed by 180-s butyl chloride and 240-s ethyl acetate washes. The resulting PTH-amino acids were analyzed on an ABI PTH-Cl8 reversed phase cartridge column (2.1 mm i.d. X 22 cm length) and eluted according to the manufacturer’s specification.

GELS

3

The PTH-amino acids eluted off the column were quantitated by a Model 740 data module from Waters Association. Preparation of glutathione S-transferases (GST). Glutathione S-transferases from chicken liver were partially purified using an S-hexyl glutathione affinity column as described previously ( 13). The transferases were eluted off the affinity column with a mixture of glutathione and S-hexyl glutathione and dialyzed against 10 mM sodium phosphate, pH 6, to remove the desorbing agents. The samples were concentrated in a Speed Vat concentrator before they were loaded onto an IEF gel or for biological assay. The glutathione S-transferase activity was measured according to Warholm et al. (14), using CDNB as substrate, or detected directly on the gel using the method of Ricci et al. (15). Protein extraction from jilter for SDS electrophoresis. The filter-bound proteins were extracted from the filter with 60 ~1 of SDS buffer (62.5 mM Tris-HCl, pH 6.8, 2.0% SDS, 10% glycerol, and 5% 2-mercaptoethanol) at 90°C for 5 min, and 20 ~1 of each individual protein sample was electrophoresed in a 12% SDS-polyacrylamide gel prepared according to Laemmli for molecular weight determination ( 16). RESULTS AND DISCUSSION

Vandekerckhove et al. (6) and Aebersold et al. (7) have reported previously a method for separating proteins by molecular weight differences on a SDS gel, then transblotting the separated proteins onto glass-fiber sheets for direct sequencing. In this study, proteins were separated according to their isoelectric points, and then the resolved proteins were electroblotted onto glass-fiber sheets for direct sequencing. Ampholytes in the IEF gel interfere with protein detection as well as sequencing and had to be removed after electrophoresis. Reisner et al. (17) reported that proteins on IEF gels can be stained with Coomassie

4

HSIEH, LIN, AND TAM

blue dye in dilute perchloric acid without a prefixing step. Therefore, perchloric acid should be able to remove ampholytes without washing too much of the proteins off the gel. IEF gels were soaked in various concentrations of perchloric acid with gentle shaking at room temperature. The amount of ampholytes washed into the perchloric acid solutions was determined by Bradford assay (18). The absorptions at 595 nm were checked against a calibration curve using known concentrations of ampholytes as standards. The results were expressed as percentage ampholytes remaining in the gel and summarized in Fig. 1. After 60 min of washing, approximately 98-99% of ampholytes were removed from IEF gels irrespective of the concentration of the perchloric acid solutions used. The remaining ampholytes and perchloric acid were replaced with 2% acetic acids in a Southern blotting unit (10) before the electroblotting process. After the gel

20

40 Time

60 (min)

FIG. 1. Washing of ampholytes in IEF gel with perchloric acid. Gel slices were washed with 10X volume of perchloric acid (0, 2.0%; A, 3.5%; n , 5.0%), and the solutions were changed every 10 min. Ampholytes in the washes were determined by Bradford assay(17), and the results were expressed as percentage ampholytes remaining in the gel. Gels without perchloric acid wash contained 2% ampholytes.

1 0

. 60 Time

I

.

120

180

I

(min)

FIG. 2. Washing of ampholytes in IEF gel with Southem blotting method. Gel slices washed with 3.5% perchloric acid were blotted on a Southern blotting unit with 2% acetic acid as carrier buffer, and one gel slice was removed every 30 min. The gel slices were crushed and the ampholytes were extracted with 2% acetic acid overnight. Ampholytes in acetic acid were determined with Bradford assay (17), and the results were expressed as percentage ampholytes remaining in the gel. Ampholytes extracted from gel slices that had been washed with perchloric acid but without Southern blotting were considered as 100%.

slices were washed with acetic acid, they were crushed and the ampholytes were extracted with 2% acetic acid overnight. The amount of ampholytes in the acetic acid was determined with a Bradford assay (18) and the results are summarized in Fig. 2. Approximately 3 of the ampholytes which remained after the perchloric acid wash were removed from the gel in 3 h of Southern blotting. The washing steps remove also some of the proteins from the gel. These losses were measured by focusing several samples of equal amount of “C-labeled BSA ( 1.1 ccg,3.3 x lo4 cpm) on a 7.5% IEF gel and then washed with perchloric acid and acetic acids as mentioned above. The radioisotopes on the gel were determined after various washing steps. It was found that 10 and 36% of the samples were lost after 1.5 and 3 h, respec-

PROTEIN

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tively, on the Southern blotting unit. After electroblotting, 48-54% of the 14C labels were located on the filter; 16% of the radioisotopes still remained on the gel. The efficiency of the IEF/electroblotting method depends on the physical characteristics of the protein as well as the pore size of the gel. The gel pores should be small enough to retain the protein during the washing steps, but without hindering the protein from leaving the gel during the electroblotting step. With the protein standards that we were using, fllactoglobulin (molecular weight 18,400 Da) and soybean trypsin inhibitor (molecular weight 2 1,000 Da) focused on a 7.5% gel were mostly lost during the perchloric acid wash step. Therefore, we used 12% gel for these two proteins. In previous reports (6,7), the investigators used modified glass-fiber filters for protein immobilization during the electroelution step. Vandekerckhove et al. (6) used Polybrene-coated filters, while Aebersold et al. (7) used TFA-treated or quarterary-ammonium-salt-treated glass-fiber filters. The Polybrene-coated or quarternary-ammonium-salt-treated filters have slightly higher binding capacity for proteins than untreated filters, but still do not prevent proteins from migrating through the filters. The procedures in preparing these filters are tedious and time consuming, and special fluorescent dyes are needed to detect the proteins. In this study, unmodified GF/C or GF/F filters were used and sufficient amounts of proteins were immobilized for sequencing experiments. Based on the 14C-labeled BSA data, the proteins immobilized on the filter alter the washing and electroblotting steps were about half of the amounts applied to the gel. Since relatively crude samples could be applied to IEF gel, the amount lost during washing and electroblotting should be tolerable. The partial N-terminal sequences of BSA, .&lactoglobulin A, and trypsin inhibitor were determined according the present procedure, and the results are summarized in Table 1. Commercially available &lactoglobulin is a

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mixture of A and B forms with different isoelectric points. BSA and trypsin inhibitor also showed multiple bands on an IEF gel stained with Coomassie blue dye. It is thus difficult to estimate the amounts of filterbound proteins that were loaded into the sequenator. We therefore loaded known amounts of BSA, trypsin inhibitor, and fllactoglobulin (20 pg each, 303, 714, and 1090 pmol, respectively) directly into the sequenator, and the initial yields of the sequencing reaction for each sample were determined. We obtained an initial yield of 3 10,264, and 676 pmol for BSA, trypsin inhibitor, and ,&lactoglobulin, respectively. The low initial yield was due mainly to Nterminal blockages on some of the proteins and to a lesser extent the efficiency of the sequenator, which could be considered as a machine constant. The ratio of initial yield/. amount of sample applied would give a fair estimate of the percentage of “sequenceable” proteins for each sample. The BSA sample is 100% sequenceable, while 37 and 62% of trypsin inhibitor and /3-lactoglobulin are sequenceable, respectively. Assuming the steps of electrophoresis, washing, and electroblotting did not cause any further protein N-terminal blockages, and the machine constant is not a variable factor, the amount of filterbound proteins loaded into the sequenator can be calculated from the initial yield of the filter-bound sample and the percentage sequenceable protein data. We thus estimated that 8.0 pg (121 pmol) of BSA, 3.0 pg (150 pmol) of soybean trypsin inhibitor, and 1.9 pg (106 pmol) of /3-lactoglobulin A were loaded into the sequenator. This IEF/electroblotting method is best suited for partial N-terminal sequence determination of isozymes. GSTs from chicken liver can be separated into three classes according to their mobilities on SDS gel (Fig. 3a), and their molecular weights range from 25,000 to 26,500 Da. Most of these enzymes are basic proteins and can be absorbed onto a cation exchanger (Mono S, Pharmacia) at neutral pH (data not shown). However, it has

HSIEH,

LIN,

AND

TABLE EDMAN Chicken

Cycle 1 2 3 4 5 6 7 8 9 10 11 12 13 14 i5 16 17 18 19 20 21 22 23 24 25 26

Identified residue Val Val Thr LeU GlY Tyr Trp Asp Be A% ‘3~ LtXl Ala His Ala Be Arg LeU LeU

Leu Glu Tyr Thr Glu Thr Pro

DEGWDAT~ON

OF PROTEINS

liver GST Yield bmol) 42.9 40.3 23.2 26.1 17.5 19.2 8.1 14.0 20.9 16.8 5.6 11.7 8.5 3.5 6.2 8.0 10.5 8.1 12.0 9.7 4.8 3.7 2.9 4.3 1.9 5.8

Asp Thr His LYS Ser Glu Ile Ala His A% Phe LYS Asp Lell

GUY Glu Glu

1

PERFORMED

ON GAS-PHASE Trypsin

BSA Identified residue

TAM

Yield (pmol) 123.6 79.0 57.2 53.8 45.7 51.9 32.5 46.4 11.4 35.4 24.9 35.4 38.7 34.6 18.3 35.7 42.2

Identified residue Asp Phe Val LeU

Asp Asn Glu GUY Asn Pro LeU Glu Asn GUY GUY NW Tv W Be Lell

SEQUENCER

inhibitor Yield (Pm4 55.2 48.9 22.1 18.3 15.0 13.8 9.4 11.7 8.6 4.7 5.7 5.4 4.7 5.8 5.8 2.8 3.1 6.3 4.4

/Xactoglobulin Identified residue Lell

Be Val Thr Gln Thr Met LYS GUY LI2l.l

Asp Be Gln LYS Val Ala GUY Thr ND“ Vr

A Yield (pmol) 62.2 68.6 40.5 25.7 25.4 17.1 18.4 21.7 26.6 19.0 15.8 16.0 11.8 5.9 8.8 13.1 11.3 6.4 6.5

’ Not determined.

been reported that basic GSTs from rat liver have blocked N-termini (19). Therefore, we chose to isolate acidic GSTs for sequencing. The GSTs were partially purified by an 9 hexyl glutathione affinity column (13). The uv-absorbing fractions from the column were first dialyzed against a phosphate buffer and then assayed for GST activity (14). The active fraction was resolved further on an IEF gel without urea. The isoelectric focusing system was optimized for resolution between pH 3 and 7, and the acidic GSTs were separated into IO- 15 dye-binding bands on the gel (Fig. 3b). The enzymes remained active in a dimeric form on the gel and locations were further confirmed by directly detecting

the GST activity in the IEF gel (15). The protein bands visualized by Coomassie blue dye were all active GST enzymes (data not shown). An active GST with ~15.6 was chosen for a sequencing experiment, and the first 26 amino acid residues from the N-terminus were determined (Table 1). The sequence show a high degree of homology with rat liver and human liver GSTs. Out of 26 amino acids, 21 are identical to either an acidic protein from the rat liver Yb family (8 1.5% homology) (20) or a protein from human liver Hb family (Ming F. Tam, unpublished result). The molecular weight of the sequenced protein was determined by SDS gel electro-

PROTEIN

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GELS

FIG. 3. Electrophoretic patterns of glutathione Stransferases. (a) Rat liver GSTs, chicken liver GSTs, and a chicken liver GST with ~15.6 extracted from glass-fiber filter were separated on a 12% SDS gel (lanes 1, 2, and 3, respectively). Molecular weights for rat liver GSTs are Y,, 25,600 Da; Yb, 27,000 Da; Y,, 28,000 Da (22). Molecular weights for chicken liver GSTs are class I, 26,300 Da; class II, 25,600 Da; class III, 25,200 Da. (b) IEF pattern of a GST sample after affinity column. The sequenced GST (PI 5.6) was indicated.

phoresis. The two-dimensional gel system of O’Farrell (2 1) is normally used to correlate the p1 and molecular weight of a protein. On the slab gel (second dimension), the protein appears as a spot instead of a sharp narrow band. In the chicken liver GST system, three classes of proteins were separated by less than 1500 Da, with classes II and III differing by less than 500 Da. The G’Farrell gel system cannot resolve class II from class III for a clear identification. Therefore, we resort to extracting the protein from the IEF gel and then running it again on a SDS gel with Laemmli’s system (16). After extraction, the electrophoretic mobility of the sequenced protein was compared with that of a chicken liver GST preparation from the affinity column on an SDS-polyacrylamide gel (Fig. 3a), and the molecular weight was estimated to be 26,300 +: 400 Da. Proteins embedded in polyacrylamide gels are difficult to extract. The published electroelution technique required a fair amount of sample and time (5). With the electroblotting method, the filter-bound proteins were extracted with SDS buffer as an alternative to electroelution. Samples of 14C-labeled BSA

(3.4 pg, 1.O X IO5 cpm) were focused on a 7.5% IEF gel and electroblotted onto a glassfiber filter after the ampholytes were washed away. The protein bands were located by Coomassie blue dye staining, excised from the filter and extracted with SDS buffer. Radioisotopes in the extract were determined by scintillation counting and recovery was 22 f 4% of the original sample applied on the gel. In summary, a technique of preparing proteins for N-terminal sequencing by IEF gel purification and electroblotting is presented. A simplified method to extract proteins from an IEF gel for accurate molecular weight determination on a SDS gel is also reported. The partial N-terminal sequence and molecular weight of an anionic glutathione Stransferase (p1 5.6) from chicken liver was determined with these methods. ACKNOWLEDGMENTS The authors thank Drs. C.-F. Hui and James C. Wang for their critical reading of the manuscript and Ms. LiHsueh Chang for preparing the GST samples. This project has been supported in part by Grant NSC 76-020 l-BOO l-32 from the National Science Council, Republic of China.

HSIEH, LIN, AND TAM

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