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Mass Spectrometry: Recombinant HIV-1IIIBp26
ANALYTICAL BIOCHEMISTRY ARTICLE NO.
239, 25–34 (1996)
0286
Monitoring Cleavage of Fusion Proteins by Matrix-Assisted Laser Desorption Ionization/Mass Spectrometry: Recombinant HIV-1IIIB p26 Carol E. Parker, Damon I. Papac, and Kenneth B. Tomer1 Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, North Carolina 27709
Received November 9, 1995
Matrix-associated laser desorption ionization/mass spectrometry (MALDI/MS) has been used to examine whole bacteria for the presence of a recombinant HIV p26 fusion protein. MALDI/MS, combined with affinitypurification techniques, is also shown to be very useful in monitoring the enzymatic cleavage of both affinitybound fusion protein and fusion protein in solution. The combination of mass resolution, sensitivity, and speed of analysis makes MALDI/MS an attractive alternative to SDS–PAGE. q 1996 Academic Press, Inc.
One of the most common methodologies for producing large amounts of recombinant proteins is by protein expression of genetically altered bacteria or yeast (1). The specific end-use of the recombinant protein can influence the expression and purification strategies employed for protein production. The most stringent end-use in terms of both expression and purification is one in which the natural conformation is retained. This usually requires that the protein be made in a soluble form that does not require a denaturation/renaturation step in its purification because exact refolding of a denatured protein into its native conformation can be difficult to verify. The protein must also be expressed in a system that minimizes proteolytic degradation. In addition, the cloned gene is often introduced into the cell in combination with a gene that codes for a carrier protein, i.e., as a fusion protein. This makes the product more resistant to proteolytic cleavage by the bacterial enzymes, and can also be exploited in protein purification. One example of a carrier protein is glutathione-S1
To whom correspondence should be addressed.
transferase (GST).2 The recombinant protein is expressed as a fusion protein with GST (1, 2). The fusion protein can be purified by affinity chromatography with an immobilized glutathione-agarose column. In general, fusion proteins containing GST are soluble, and can be purified under nondenaturing conditions. After the fusion protein is bound to the glutathione (GSH)agarose column and impurities are removed by washing, the fusion protein can be eluted from the column using reduced glutathione. An additional DNA sequence, coding for additional amino acids which correspond to a recognition sequence for a specific protease, is often incorporated into the plasmid so that this sequence is expressed between the carrier protein and the desired protein. This protease can later be used to cleave the fusion protein into the carrier protein and the desired product after elution from the GSH-agarose column. Alternatively, cleavage reactions can be performed on the fusion protein while it is still affinity-bound to the glutathione-agarose beads. In plasmids designed for the expression of GST fusion proteins, one of the recognition sequences commonly used is that for restriction protease blood coagulation factor Xa (3). Cleavage of the fusion protein is usually monitored by SDS–PAGE gel electrophoresis. Matrix-assisted laser desorption ionization/mass spectrometry (MALDI/MS) is a recently developed mass spectrometric technique which is proving to be of great value in the molecular weight determination of proteins and peptides (4). Errors of mass assignment are usually within 0.1%, with an external mass calibration standard, and detection limits are in the subpicomole range (5). 2 Abbreviations used: GST, glutathione-S-transferase; GSH, glutathione; MALDI/MS, matrix-assisted laser desorption ionization/mass spectrometry; PBS, phosphate-buffered saline.
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0003-2697/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG. 1. (A) MALDI mass spectrum of induced whole bacteria, treated with hexafluoroisopropanol (*, proteins whose expression was induced by IPTG), and (B) SDS–PAGE analysis of an aliquot from the 2-h reaction mixture from the factor Xa cleavage reaction (beads plus solution). Lane A, molecular weight markers; Lanes B and C, bacterial pellets after lysis; Lane D, bacterial supernatant after treatment with GSH-agarose beads; Lane E, affinity-purified p26 released by a 2-h treatment with factor Xa; Lane F, 2-h GSH-agarose beads plus supernatant.
We are currently studying the epitopes on the HIV1IIIB p24 capsid protein. In this project we are using MALDI/MS of affinity-bound analytes (6) combined with proteolytic footprinting (7, 8) for epitope determination (9, 10). To obtain material of sufficient quantity and purity for this study, we expressed recombinant p26, which includes the sequence for the p24 protein plus 18 residues from p17 at the N-terminal end and 18 residues from p12 at the C-terminal end. These recombinant Escherichia coli express the HIV p26 as a fusion protein with GST (11). The preparation of p24 from the p26 fusion protein involves cleavage of the p26 from the GST and/or proteolytic cleavage of the recombinant p26 to p24 with HIV protease. In this paper, we report the use of MALDI/MS as an alternative to SDS–PAGE for monitoring cleavage of a GST-fusion protein. MATERIALS AND METHODS
Bacterial Strain The recombinant E. coli (strain DH5) used in this study were provided by Dr. Ian M. Jones (NERC Institute of Virology, Oxford, UK). These bacteria contain
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the pGEX-3X cloning vector (Pharmacia Biotech, Inc., Piscataway, NJ), which includes the gene for the expression of GST followed by the factor Xa recognition sequence, the tac promoter for induction of fusion protein expression by IPTG (isopropyl-b-D-thiogalactopyranoside), the Laclq gene for repressing transcription from the tac promoter when inducer is not present, and an ampicillin-resistance gene (2). Upon induction with IPTG, these bacteria express the GST–HIVIIIB p26 fusion protein (11).
Chemicals and Reagents Inorganic compounds used for making the buffer solutions were from Baker Chemical Co. (Phillipsburg, NJ), Boehringer Mannheim (Indianapolis, IN), Fisher Scientific, (Pittsburgh, PA), Sigma Chemical Co. (St. Louis, MO), Aldrich Chemical Co. Inc. (Milwaukee, WI), and Mallinckrodt Inc. (St. Louis, MO). All solutions were prepared with 18 MOhm water from a Hydro Services and Supplies, Inc. (Durham, NC) Model RO40 deionized water system. Organic solvents were from Baker Chemical Co.
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v/v) was prepared fresh each day. A 1-ml aliquot of the sample (as a solution or as an affinity bead slurry) was placed on the MALDI target, followed by a 1-ml aliquot of the matrix solution containing a-cyano-4-hydroxycinnamic acid, and the mixture was allowed to dry at room temperature. Spotting a sample on the target, adding matrix, drying, and obtaining a spectrum can often be carried out in under 5 min, with the drying time often being the most time-consuming step. Expression of Fusion Protein Fig. 1—Continued
Instrumentation The SDS–PAGE separation was done on a PhastSystem gel electrophoresis instrument (Pharmacia Biotech, Inc.) using Homogeneous 20 pHastGels, and a Coomassie blue staining procedure. Proteins were denatured by boiling for 1 min in a buffer containing SDS and dithiothreitol (12), before being applied to the gel. Two MALDI mass spectrometers were used to acquire the mass spectra: preliminary work was conducted on a VG Fisons TofSpec (Fisons, Manchester, UK), and the research was completed on a Voyager RP (PerSeptive Biosystems, Framingham, MA). Both instruments are equipped with nitrogen lasers (l Å 337 nm) to desorb and ionize the samples. The accelerating voltages used were 24 kV for the TofSpec and 30 kV for the Voyager RP. External calibration was used, using two points which bracketed the mass range of interest. Approximately 1 ml of a 10 nM solution of calibration protein was used. Calibration compounds included bovine serum albumin (Mr 66430.0), carbonic anhydrase (Mr 29,027.8), and horse heart cytochrome c (plus heme, Mr 12,360.1), all from Sigma Chemical Co. Both MALDI instruments use stainless-steel targets, on which the samples are deposited. The Voyager RP instrument was equipped with a video camera, which displays a real-time image on a monitor, and allowed aiming the laser at specific features within the area of the target. For the experiments described here, the laser was aimed at or near the affinity beads on the target. MALDI Target Preparation All samples were analyzed using a modification of the matrix reported by DeLlano et al. (13). The a-cyano4-hydroxycinnamic acid (Aldrich Chemical Co., Inc.) was recrystallized from hot methanol and stored in the dark. A saturated solution of a-cyano-4-hydroxycinnamic acid in 45:45:10 ethanol (Pharmco Products, Inc., Bayonne, NJ):water:concentrated formic acid (v/
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A 50-ml flask of ampicillin-containing Luria Broth medium was inoculated with a single colony of the recombinant bacteria, and the culture was grown overnight in a shaking incubator at 377C. The overnight culture was then added to 500 ml ampicillin-containing LB medium and grown for 4 h, after which fusion protein expression was initiated by the addition of IPTG. After a 2-h induction period, the cells from the 500-ml culture were pelleted by centrifugation, and resuspended in 15 ml pH 7.2 phosphate-buffered saline (PBS). The whole cells were stored frozen at 0207C. Fusion Protein in Whole Bacteria For the whole bacteria experiments, aliquots of the centrifuged cells were treated three ways: first, a suspension of the cells was spotted on the target and the standard matrix solution was added; second, the proteins were precipitated with methanol:water (50:50) (14), and the precipitate was dissolved in the matrix solvent; and third, 1 ml of 1,1,1,3,3,3-hexafluoroisopropanol (Sigma Chemical Co.), to dissolve the cell membranes, was spotted onto the cell slurry prior to addition of the matrix solution (15). Purification of Fusion Protein For purification of the fusion protein, a mixture of protease inhibitors (phenylmethylsulfonyl fluoride, leupeptin, and pepstatin) (Boehringer Mannheim), was added to the collected cells, the cells were lysed and centrifuged, and the supernatant was collected. The GSH-agarose beads (Pharmacia Biotech, Inc.) were washed and added to the lysate (Ç0.6 ml bead slurry in 15 ml bacterial lysate), and the mixture was rotated slowly at room temperature for 2 h. The samples were centrifuged gently (õ1000 rpm) to settle the beads, which were transferred to compact reaction columns (United States Biochemical, Cleveland, OH). The columns, containing Ç0.2 ml beads each, were then drained and rinsed three times with four column volumes of pH 7.2 PBS.
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FIG. 2. (A) MALDI mass spectra of 1-ml aliquots of the washed column bed at various times during the cleavage of affinity-bound p26 fusion protein (GST–p26) by restriction factor Xa, and (B) plot of the absolute peak areas of the starting material (GST–p26) and the product (GST) throughout the course of the factor Xa cleavage reaction.
Factor Xa Cleavage Reaction Each minicolumn containing the affinity-bound fusion protein was rinsed three times with four column volumes of factor Xa cleavage buffer, containing 50 mM Tris–HCl, 150 mM NaCl, and 1 mM CaCl2 , pH 8.0. A 0.5-ml volume of cleavage buffer and an aliquot containing 15 mg of Factor Xa were added to each column. Columns were rotated slowly (2 rpm) at 377C. Aliquots of the packing bed (20 ml each) were removed at a series
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of time intervals, and the reaction was stopped by adding 5 ml of 100 mM EDTA (pH 8.0) to each removed aliquot. At 14 h, the remaining beads were washed with 200 ml of deionized water. A 1-ml sample of column bed from each time point was used for MALDI analysis. HIV Protease Cleavage Reactions of p26 The cleavage buffer consisted of 50 mM sodium acetate and 20 mM sodium chloride (Baker Chemical Co.),
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respond to those predicted from the DNA sequences. It is possible that this protein had already been cleaved by bacterial proteases. The presence of other induced proteins is, apparently, often detected in SDS–PAGE analyses of induced E. coli (20), and whole bacteria contain many proteins in the mass range of interest (Fig. 1B, Lanes B and C). The peaks of lower m/z in the MALDI spectrum (Fig. 1A) are due to these native bacterial proteins, and have not been further characterized. The low abundance of the fusion protein signals relative to some of the other proteins can be attributed to the large abundance of low mass ions, which is known to reduce the signal of high mass proteins.
Fig. 2—Continued
Affinity-Bound GST–p26 pH 4.9. The recommended cleavage buffer for this enzyme also contains dithiothreitol and glycerol (16). These were omitted from the cleavage buffer used for these studies because of possible interference with the MALDI analysis. For the study of the cleavage reactions of p26 in solution and affinity-bound, the enzyme was not physically separated from the protein. Instead the reactions were stopped by the addition of 1 ml of matrix solution to a 1-ml aliquot of the solution or the bead slurry directly on the MALDI target. RESULTS AND DISCUSSION
Detection of Fusion Protein in Whole Bacteria There have been several reports of bacterial protein fingerprinting and characterization using MALDI/MS (14, 17). In addition, fusion protein has been reported to have been detected by MALDI/MS in crude E. coli lysate (18), but no spectra were shown. Because bacterial cell cultures are known to ‘‘drift,’’ resulting in loss of recombinant protein expression (19), we hypothesized that screening by MALDI of a bacterial culture might be a way of verifying recombinant protein expression prior to extensive workup. We, therefore, examined crude E. coli lysates and whole cells in order to see if the recombinant protein could be observed. No peaks corresponding to the fusion protein were detectable by direct analysis of the bacterial lysate solutions, or in the uninduced whole bacteria. Small peaks attributable to the fusion protein could be detected only in the induced whole bacteria, and only by using hexafluoroisopropanol (Fig. 1A). A protein at m/z Ç28,800 was observed in both induced and uninduced bacteria. Another protein, at m/z Ç28,400, was observed in the spectra of the induced bacteria, but was not present in the uninduced bacteria. We attempted to correlate the mass of this other induced protein with proteins encoded by Laclq gene from the pGEX-3X plasmid, but the molecular weights did not cor-
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After the bacterial cell culture was determined to have expressed the desired recombinant protein, the cells were lysed and centrifuged, and the supernatant was incubated with GSH-agarose beads. After washing with PBS to remove non-affinity-bound analytes, an aliquot of the beads was removed for direct analysis by MALDI. A MALDI mass spectrum of the GSH-agarose–fusion protein affinity complex, obtained from a 1-ml aliquot of the column bed, is shown in Fig. 2A. Ions can be observed which correspond to the calculated protonated molecular ion (M / H)/, of the GST–p26 fusion protein (Mr 55,484 Da), while the ions of m/z 27,743, 18,495, and 13,872 correspond to the (M / 2H)2/, (M / 3H)3/, and (M / 4H)4/ ions, respectively. The absence of other protein signals is indicative of the high selectivity of the GSH-agarose affinity column. The potentially interfering proteins at m/z Ç28,400 and 28,800, seen in the whole-cell experiments (Fig. 1A), were not seen in the MALDI mass spectrum of the affinity-bound fusion protein or were of sufficiently low abundance as to be hidden by the (M / 2H)2/ peak (Fig. 2A, 0 min). The signal enhancement obtained using affinity purification can be easily seen by comparing Figs. 1A and 2A, 0 min. The improvement in the signal from the affinity-bound fusion protein may be due partly to concentration of the protein by the GSH-agarose beads. It may also be partially due to removal of suppression effects from other components present in the whole bacteria, which may have interfered with the ionization of the fusion protein in the MALDI analysis. Proteolysis Time Course Studies After a fusion protein has been purified by affinity chromatography, the recombinant protein must be proteolytically cleaved from the carrier protein. This procedure can be performed either after elution of the fusion protein from the affinity column, or it can be performed by treating the affinity-bound fusion protein with the enzyme. When the cleavage occurs in solution, the car-
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FIG. 3. MALDI mass spectrum of HIV-1IIIB p26.
rier protein must be removed in a further purification step. Both procedures are usually monitored by SDS– PAGE. Because of the sensitivity, simplicity, and the applicability of MALDI/MS to direct analysis of affinity-bound analytes, we next explored the utility of this technique for monitoring proteolytic cleavages of proteins both in solution and noncovalently bound to the affinity column. Factor Xa Cleavage of Affinity-Bound Fusion Protein Spectra obtained from PBS-rinsed 1-ml aliquots of the column bed at various time points are shown in Fig. 2A. The ions of m/z 26,271, 13,136, and 8758 correspond to the (M / H)/, (M / 2H)2/, and (M / 3H)3/ of GST. The spectra show a steady increase in GST relative to the abundance of the GST–p26 fusion protein. Because the beads were washed prior to analysis, no ions due to free p26 in solution were observed. Only analytes still affinity-bound to the GSH column will be observed using this procedure. Peak areas are plotted in Fig. 2B. These areas do not correspond directly to the relative amounts of each molecular species present, as the signal intensities are compound-dependent. This
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is especially true when comparing the abundances of ions differing significantly in mass. Changes in relative ion abundances do, however, reflect changes in relative amounts of the analytes. The SDS – PAGE analysis of the 2-h beads is shown in Fig. 1B. Lane D, the supernatant after treatment with GSH-agarose, shows a band at Mr Ç55,000 and a band at Mr Ç29,000. The Mr 55,000 component was tentatively assigned to fusion protein still in solution, while the Mr Ç29,000 component was originally attributed to spontaneously cleaved free p26. From the MALDI data, however, it appears likely that this band contains mainly the unknown Mr 28,400 and 28,800 proteins. Lane E shows that the p26 released from the GSH-agarose beads by the factor Xa is quite pure. As can be seen from Fig. 1B, Lane F, where a sample of beads plus supernatant was analyzed, the cleavage reaction is not yet complete after a 2-h digestion with factor Xa; the fusion protein (GST – p26, Mr 55,484), p26 (Mr 29,232), and GST (Mr 26,271) can all be observed on the gel. From the MALDI data in Fig. 4, it can also be seen that the absolute amount of GST is still increasing after 2 h. At 14 h, only the
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FIG. 4. MALDI mass spectra of the reaction mixture at various times during the cleavage of p26 by HIV protease in solution.
bound GST and its dimer ion (Mr 52,413) are seen in the spectrum. A MALDI spectrum of the eluted p26 (Mr 29,232) is shown in Fig. 3. The low abundance peaks observed at Ç1000-Da intervals below the molecular weight of the fusion protein are due to bacterial proteolysis of p26. This proteolysis proceeds fairly rapidly after the factor Xa cleavage reac-
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tion to give, ultimately, only an Mr Ç24,300 product, even in the presence of protease inhibitors. This provides an example of how MALDI data can help in cases where the expected protein expression product is not produced. Observation of the intact fusion protein affinity-bound to the GSH-agarose indicates that the DNA construct is correct, and that the problem is not one
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FIG. 5. (A) MALDI mass spectra of 1-ml aliquots of the column bed/reaction mixture slurry at various times during the cleavage of p26 by HIV protease, and (B) MALDI mass spectra of a 1-ml aliquot of the column bed/reaction mixture slurry 23 h after start of the cleavage reaction of affinity-bound p26 by HIV protease, showing the mass range 20,000 to 36,000 Da.
of protein expression, but of stability of the expressed product. Since a corresponding series of ions below the molecular weight of the fusion protein is not observed while the fusion protein is still attached to the GSH-
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agarose, the fusion protein itself appears to be stable. Thus, proteolysis occurs only after the p26 is cleaved from the GST. If proteolysis were occurring from the C-terminal end of the molecule, then cleavage products should
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Fig. 5—Continued
be observed in both cases. The data therefore indicate that proteolysis of p26 occurs only when its N-terminal end is exposed. In fact, the resolution of the MALDI technique is sufficient to assign the degradation products to cleavages between specific residues at the N-terminal end of p26 (21). HIV Protease Cleavage Reactions of p26 in Solution HIV protease cleaves p26 at two cleavage sites, generating part of p12 (here designated p12*), p24, and part of p17 (designated p17*). Thus, in addition to the original p26 (Åp17*–p24–p12*) (Mr 29232), possible products of the HIV protease cleavage reaction in solution are p17*–p24 (Mr 27,462), p24–p12* (Mr 27,351), p24 (Mr 25,581), p12* (Mr 1790), and p17* (Mr 1900). As can be seen in Fig. 4, (M / H)/ and (M / 2H)2/ ions of p26, p17*–p24 and/or p24–p12*, and p24 are observed. The resolution and mass accuracy of the MALDI technique were not sufficient to allow separate quantitation of p17*–p24 (Mr 27,462) and p24–p12* (Mr 27,351), which would also have been indistinguishable by SDS–PAGE gel electrophoresis. A low-mass cutoff of 10,000 Da was used for the MALDI analyses to enhance the sensitivities of the higher-molecularweight components; thus p12* and p17* were not analyzed. The enzymatic cleavage of p26 by HIV protease in solution is very rapid. The 1-min time point shows mainly intact p26. Some cleavage of the p12* and/or the p17* bond(s) has already occurred giving p24–p12* and/or p17*–p24. At 25 min, both bonds have been cleaved, giving the peak for p24 (m/z 25581) as the dominant ion in the spectrum, with only a small
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amount of the p26 still remaining. At 1.5 h, the cleavage reaction is almost complete, with cleavage of both ends of the p26 giving p24. HIV Protease Cleavage Reactions of Affinity-Bound GST-p26 When the fusion protein, still attached to the affinity beads, is cleaved with HIV protease, bound and unbound proteins can be detected in the reaction mixture if the slurry is analyzed by MALDI. The GST–p26 fusion protein produced by the Jones’ E. coli consists of GST–p17*–p24–p12* (Mr 55484), with the N-terminal end of the p17* fragment attached to the GST. This means that possible components include some of the products listed above: p24–p12* (Mr 27,351), p24 (Mr 25,581), and p12* (Mr 1790). In addition, there can be proteins still bound to the GST moiety, and thus still attached to the beads, i.e., GST–p26 (Mr 55,484), GST– p17* (Mr 28,152), and GST–p17*–p24 (Mr 53,713). One-microliter aliquots of the reaction mixture (beads and solution) were spotted on the target, and the reaction was quenched with matrix solution. MALDI mass spectra taken of the various time points are shown in Fig. 5A. Although the HIV-1 protease cleavage reaction is very rapid in solution, binding the fusion protein to the affinity column significantly reduces the reaction rate. We previously noted this effect in earlier studies of proteolytic cleavages of affinitybound proteins (10). An expanded spectrum showing the m/z 25,000– 27,000 region of the 23-h time point of the reaction mixture is shown in Fig. 5B. All of the possible reaction products within the mass range displayed can be seen
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in the spectrum. This figure illustrates both the complexity of the reaction mixture, and the resolving power of the MALDI technique. CONCLUSION
We have demonstrated that, although fusion protein can be detected by MALDI/MS of whole bacterial cells after treatment with hexafluoroisopropanol, the presence of normal and induced bacterial proteins limits the utility of MALDI as a screening technique. The advantages of MALDI/MS analysis are more fully realized after affinity purification of the bacterial lysate. With interferences removed, the resolving power of the MALDI technique can be used for directly analyzing fusion proteins and their cleavage products, with or without prior removal of the fusion protein from the affinity beads. In addition, the speed of the MALDI technique allows the monitoring of enzymatic cleavage reactions in nearly ‘‘real time.’’ Although the MALDI/MS method for following the release of recombinant proteins from affinity-bound fusion proteins is not meant to supplant SDS–PAGE analysis, it does offer a rapid and sensitive technique for those laboratories with access to MALDI instrumentation. ACKNOWLEDGMENTS Support for the purchase of the Voyager RP MALDI mass spectrometer by the NIH Office of AIDS Research is gratefully acknowledged. We thank Dr. Ian M. Jones (NERC Institute of Virology, Oxford, UK) for the gift of the E. coli which express HIV p26. We also express our appreciation to Dr. Hirao Kohno (Kansai Medical University, Osaka, Japan) for helpful discussions concerning the plasmid.
REFERENCES 1. Smith, D. B., and Corcoran, L. M. (1991) in Current Protocols in Molecular Biology (Ausbel, F. M., Brent, R., Kingston, R. E.,
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2. 3. 4. 5. 6. 7. 8.
Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K., Eds.), Vol. 2, pp. 16.7.1–16.7.8, Green/Wiley-Interscience, New York. Smith, D. B., and Johnson, K. S. (1988) Gene 67, 31–40. Nagai, K., and Thorgersen, H. C. (1984) Nature 309, 810–812. Karas, M., Bahr, U., and Geissmann, U. (1991) Mass Spectrom. Rev. 10, 335–358. Cerpa-Poljak, A., Jenkins, A., and Duncan, M. W. (1995) Rapid Commun. Mass Spectrom. 9, 233–239. Papac, D. I., Hoyes, J., and Tomer, K. B. (1994) Anal. Chem. 66, 2609–2613. Jemmersen, R., and Paterson, Y. (1986) Science 232, 1001–1004. Sukau, D., Kohl, J., Karwath, G., Schneider, K., Casaretto, M., Bitter-Suermann, D., and Przybylski, M. (1990) Proc. Natl. Acad. Sci. USA 87, 9848–9852.
9. Zhao, Y., and Chait, B. T. (1994) Anal. Chem. 66, 3723–3726. 10. Papac, D. I., Hoyes, J., and Tomer, K. B. (1994) Protein Sci. 3, 1485–1492. 11. Mills, H. R., Berry, N., Burns, N. R., and Jones, I. M. (1992) AIDS 6, 437–439. 12. Pharmacia Biotechnology, PhastSystem Separation Technique File No. 112. 13. DeLlano, J. J. M., Jones, W., Schneider, K., Chait, B. T., Manning, J. M., Rodgers, G., Benjamin, L. J., and Weksler, B. (1993) J. Biol. Chem. 269, 27004–27011. 14. Cain, T. C., Lubman, D. M., and Weber, W. J., Jr. (1994) Rapid Commun. Mass Spectrom. 8, 1026–1030. 15. Schey, K. L., Papac, D. I., Knapp, D. R., and Crouch, R. K. (1992) Biophys. J. 63, 1240–1243. 16. HIV Protease Analytical Data Sheet, Bachem Biosciences Inc., King of Prussia, PA. 17. Liang, X., Cain, T. C., Bai, J., Liu, Y.-H., and Lubman, D. M. (1995) Presented at the 43rd Annual Conference on Mass Spectrometry and Allied Topics, May 21–26, 1995, Atlanta, GA. 18. Chait, B. T., personal communication in (14). 19. Malling, H. V., Environmental Toxicology Program, NIEHS, personal communication. 20. Schaaper, R., Laboratory of Molecular Genetics, NIEHS, personal communication. 21. Parker, C. E., Papac, D. I., Trojak, S. K., and Tomer, K. B. (1996) J. Immunol., in press.
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0286
Monitoring Cleavage of Fusion Proteins by Matrix-Assisted Laser Desorption Ionization/Mass Spectrometry: Recombinant HIV-1IIIB p26 Carol E. Parker, Damon I. Papac, and Kenneth B. Tomer1 Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, North Carolina 27709
Received November 9, 1995
Matrix-associated laser desorption ionization/mass spectrometry (MALDI/MS) has been used to examine whole bacteria for the presence of a recombinant HIV p26 fusion protein. MALDI/MS, combined with affinitypurification techniques, is also shown to be very useful in monitoring the enzymatic cleavage of both affinitybound fusion protein and fusion protein in solution. The combination of mass resolution, sensitivity, and speed of analysis makes MALDI/MS an attractive alternative to SDS–PAGE. q 1996 Academic Press, Inc.
One of the most common methodologies for producing large amounts of recombinant proteins is by protein expression of genetically altered bacteria or yeast (1). The specific end-use of the recombinant protein can influence the expression and purification strategies employed for protein production. The most stringent end-use in terms of both expression and purification is one in which the natural conformation is retained. This usually requires that the protein be made in a soluble form that does not require a denaturation/renaturation step in its purification because exact refolding of a denatured protein into its native conformation can be difficult to verify. The protein must also be expressed in a system that minimizes proteolytic degradation. In addition, the cloned gene is often introduced into the cell in combination with a gene that codes for a carrier protein, i.e., as a fusion protein. This makes the product more resistant to proteolytic cleavage by the bacterial enzymes, and can also be exploited in protein purification. One example of a carrier protein is glutathione-S1
To whom correspondence should be addressed.
transferase (GST).2 The recombinant protein is expressed as a fusion protein with GST (1, 2). The fusion protein can be purified by affinity chromatography with an immobilized glutathione-agarose column. In general, fusion proteins containing GST are soluble, and can be purified under nondenaturing conditions. After the fusion protein is bound to the glutathione (GSH)agarose column and impurities are removed by washing, the fusion protein can be eluted from the column using reduced glutathione. An additional DNA sequence, coding for additional amino acids which correspond to a recognition sequence for a specific protease, is often incorporated into the plasmid so that this sequence is expressed between the carrier protein and the desired protein. This protease can later be used to cleave the fusion protein into the carrier protein and the desired product after elution from the GSH-agarose column. Alternatively, cleavage reactions can be performed on the fusion protein while it is still affinity-bound to the glutathione-agarose beads. In plasmids designed for the expression of GST fusion proteins, one of the recognition sequences commonly used is that for restriction protease blood coagulation factor Xa (3). Cleavage of the fusion protein is usually monitored by SDS–PAGE gel electrophoresis. Matrix-assisted laser desorption ionization/mass spectrometry (MALDI/MS) is a recently developed mass spectrometric technique which is proving to be of great value in the molecular weight determination of proteins and peptides (4). Errors of mass assignment are usually within 0.1%, with an external mass calibration standard, and detection limits are in the subpicomole range (5). 2 Abbreviations used: GST, glutathione-S-transferase; GSH, glutathione; MALDI/MS, matrix-assisted laser desorption ionization/mass spectrometry; PBS, phosphate-buffered saline.
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0003-2697/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG. 1. (A) MALDI mass spectrum of induced whole bacteria, treated with hexafluoroisopropanol (*, proteins whose expression was induced by IPTG), and (B) SDS–PAGE analysis of an aliquot from the 2-h reaction mixture from the factor Xa cleavage reaction (beads plus solution). Lane A, molecular weight markers; Lanes B and C, bacterial pellets after lysis; Lane D, bacterial supernatant after treatment with GSH-agarose beads; Lane E, affinity-purified p26 released by a 2-h treatment with factor Xa; Lane F, 2-h GSH-agarose beads plus supernatant.
We are currently studying the epitopes on the HIV1IIIB p24 capsid protein. In this project we are using MALDI/MS of affinity-bound analytes (6) combined with proteolytic footprinting (7, 8) for epitope determination (9, 10). To obtain material of sufficient quantity and purity for this study, we expressed recombinant p26, which includes the sequence for the p24 protein plus 18 residues from p17 at the N-terminal end and 18 residues from p12 at the C-terminal end. These recombinant Escherichia coli express the HIV p26 as a fusion protein with GST (11). The preparation of p24 from the p26 fusion protein involves cleavage of the p26 from the GST and/or proteolytic cleavage of the recombinant p26 to p24 with HIV protease. In this paper, we report the use of MALDI/MS as an alternative to SDS–PAGE for monitoring cleavage of a GST-fusion protein. MATERIALS AND METHODS
Bacterial Strain The recombinant E. coli (strain DH5) used in this study were provided by Dr. Ian M. Jones (NERC Institute of Virology, Oxford, UK). These bacteria contain
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the pGEX-3X cloning vector (Pharmacia Biotech, Inc., Piscataway, NJ), which includes the gene for the expression of GST followed by the factor Xa recognition sequence, the tac promoter for induction of fusion protein expression by IPTG (isopropyl-b-D-thiogalactopyranoside), the Laclq gene for repressing transcription from the tac promoter when inducer is not present, and an ampicillin-resistance gene (2). Upon induction with IPTG, these bacteria express the GST–HIVIIIB p26 fusion protein (11).
Chemicals and Reagents Inorganic compounds used for making the buffer solutions were from Baker Chemical Co. (Phillipsburg, NJ), Boehringer Mannheim (Indianapolis, IN), Fisher Scientific, (Pittsburgh, PA), Sigma Chemical Co. (St. Louis, MO), Aldrich Chemical Co. Inc. (Milwaukee, WI), and Mallinckrodt Inc. (St. Louis, MO). All solutions were prepared with 18 MOhm water from a Hydro Services and Supplies, Inc. (Durham, NC) Model RO40 deionized water system. Organic solvents were from Baker Chemical Co.
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v/v) was prepared fresh each day. A 1-ml aliquot of the sample (as a solution or as an affinity bead slurry) was placed on the MALDI target, followed by a 1-ml aliquot of the matrix solution containing a-cyano-4-hydroxycinnamic acid, and the mixture was allowed to dry at room temperature. Spotting a sample on the target, adding matrix, drying, and obtaining a spectrum can often be carried out in under 5 min, with the drying time often being the most time-consuming step. Expression of Fusion Protein Fig. 1—Continued
Instrumentation The SDS–PAGE separation was done on a PhastSystem gel electrophoresis instrument (Pharmacia Biotech, Inc.) using Homogeneous 20 pHastGels, and a Coomassie blue staining procedure. Proteins were denatured by boiling for 1 min in a buffer containing SDS and dithiothreitol (12), before being applied to the gel. Two MALDI mass spectrometers were used to acquire the mass spectra: preliminary work was conducted on a VG Fisons TofSpec (Fisons, Manchester, UK), and the research was completed on a Voyager RP (PerSeptive Biosystems, Framingham, MA). Both instruments are equipped with nitrogen lasers (l Å 337 nm) to desorb and ionize the samples. The accelerating voltages used were 24 kV for the TofSpec and 30 kV for the Voyager RP. External calibration was used, using two points which bracketed the mass range of interest. Approximately 1 ml of a 10 nM solution of calibration protein was used. Calibration compounds included bovine serum albumin (Mr 66430.0), carbonic anhydrase (Mr 29,027.8), and horse heart cytochrome c (plus heme, Mr 12,360.1), all from Sigma Chemical Co. Both MALDI instruments use stainless-steel targets, on which the samples are deposited. The Voyager RP instrument was equipped with a video camera, which displays a real-time image on a monitor, and allowed aiming the laser at specific features within the area of the target. For the experiments described here, the laser was aimed at or near the affinity beads on the target. MALDI Target Preparation All samples were analyzed using a modification of the matrix reported by DeLlano et al. (13). The a-cyano4-hydroxycinnamic acid (Aldrich Chemical Co., Inc.) was recrystallized from hot methanol and stored in the dark. A saturated solution of a-cyano-4-hydroxycinnamic acid in 45:45:10 ethanol (Pharmco Products, Inc., Bayonne, NJ):water:concentrated formic acid (v/
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A 50-ml flask of ampicillin-containing Luria Broth medium was inoculated with a single colony of the recombinant bacteria, and the culture was grown overnight in a shaking incubator at 377C. The overnight culture was then added to 500 ml ampicillin-containing LB medium and grown for 4 h, after which fusion protein expression was initiated by the addition of IPTG. After a 2-h induction period, the cells from the 500-ml culture were pelleted by centrifugation, and resuspended in 15 ml pH 7.2 phosphate-buffered saline (PBS). The whole cells were stored frozen at 0207C. Fusion Protein in Whole Bacteria For the whole bacteria experiments, aliquots of the centrifuged cells were treated three ways: first, a suspension of the cells was spotted on the target and the standard matrix solution was added; second, the proteins were precipitated with methanol:water (50:50) (14), and the precipitate was dissolved in the matrix solvent; and third, 1 ml of 1,1,1,3,3,3-hexafluoroisopropanol (Sigma Chemical Co.), to dissolve the cell membranes, was spotted onto the cell slurry prior to addition of the matrix solution (15). Purification of Fusion Protein For purification of the fusion protein, a mixture of protease inhibitors (phenylmethylsulfonyl fluoride, leupeptin, and pepstatin) (Boehringer Mannheim), was added to the collected cells, the cells were lysed and centrifuged, and the supernatant was collected. The GSH-agarose beads (Pharmacia Biotech, Inc.) were washed and added to the lysate (Ç0.6 ml bead slurry in 15 ml bacterial lysate), and the mixture was rotated slowly at room temperature for 2 h. The samples were centrifuged gently (õ1000 rpm) to settle the beads, which were transferred to compact reaction columns (United States Biochemical, Cleveland, OH). The columns, containing Ç0.2 ml beads each, were then drained and rinsed three times with four column volumes of pH 7.2 PBS.
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FIG. 2. (A) MALDI mass spectra of 1-ml aliquots of the washed column bed at various times during the cleavage of affinity-bound p26 fusion protein (GST–p26) by restriction factor Xa, and (B) plot of the absolute peak areas of the starting material (GST–p26) and the product (GST) throughout the course of the factor Xa cleavage reaction.
Factor Xa Cleavage Reaction Each minicolumn containing the affinity-bound fusion protein was rinsed three times with four column volumes of factor Xa cleavage buffer, containing 50 mM Tris–HCl, 150 mM NaCl, and 1 mM CaCl2 , pH 8.0. A 0.5-ml volume of cleavage buffer and an aliquot containing 15 mg of Factor Xa were added to each column. Columns were rotated slowly (2 rpm) at 377C. Aliquots of the packing bed (20 ml each) were removed at a series
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of time intervals, and the reaction was stopped by adding 5 ml of 100 mM EDTA (pH 8.0) to each removed aliquot. At 14 h, the remaining beads were washed with 200 ml of deionized water. A 1-ml sample of column bed from each time point was used for MALDI analysis. HIV Protease Cleavage Reactions of p26 The cleavage buffer consisted of 50 mM sodium acetate and 20 mM sodium chloride (Baker Chemical Co.),
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respond to those predicted from the DNA sequences. It is possible that this protein had already been cleaved by bacterial proteases. The presence of other induced proteins is, apparently, often detected in SDS–PAGE analyses of induced E. coli (20), and whole bacteria contain many proteins in the mass range of interest (Fig. 1B, Lanes B and C). The peaks of lower m/z in the MALDI spectrum (Fig. 1A) are due to these native bacterial proteins, and have not been further characterized. The low abundance of the fusion protein signals relative to some of the other proteins can be attributed to the large abundance of low mass ions, which is known to reduce the signal of high mass proteins.
Fig. 2—Continued
Affinity-Bound GST–p26 pH 4.9. The recommended cleavage buffer for this enzyme also contains dithiothreitol and glycerol (16). These were omitted from the cleavage buffer used for these studies because of possible interference with the MALDI analysis. For the study of the cleavage reactions of p26 in solution and affinity-bound, the enzyme was not physically separated from the protein. Instead the reactions were stopped by the addition of 1 ml of matrix solution to a 1-ml aliquot of the solution or the bead slurry directly on the MALDI target. RESULTS AND DISCUSSION
Detection of Fusion Protein in Whole Bacteria There have been several reports of bacterial protein fingerprinting and characterization using MALDI/MS (14, 17). In addition, fusion protein has been reported to have been detected by MALDI/MS in crude E. coli lysate (18), but no spectra were shown. Because bacterial cell cultures are known to ‘‘drift,’’ resulting in loss of recombinant protein expression (19), we hypothesized that screening by MALDI of a bacterial culture might be a way of verifying recombinant protein expression prior to extensive workup. We, therefore, examined crude E. coli lysates and whole cells in order to see if the recombinant protein could be observed. No peaks corresponding to the fusion protein were detectable by direct analysis of the bacterial lysate solutions, or in the uninduced whole bacteria. Small peaks attributable to the fusion protein could be detected only in the induced whole bacteria, and only by using hexafluoroisopropanol (Fig. 1A). A protein at m/z Ç28,800 was observed in both induced and uninduced bacteria. Another protein, at m/z Ç28,400, was observed in the spectra of the induced bacteria, but was not present in the uninduced bacteria. We attempted to correlate the mass of this other induced protein with proteins encoded by Laclq gene from the pGEX-3X plasmid, but the molecular weights did not cor-
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After the bacterial cell culture was determined to have expressed the desired recombinant protein, the cells were lysed and centrifuged, and the supernatant was incubated with GSH-agarose beads. After washing with PBS to remove non-affinity-bound analytes, an aliquot of the beads was removed for direct analysis by MALDI. A MALDI mass spectrum of the GSH-agarose–fusion protein affinity complex, obtained from a 1-ml aliquot of the column bed, is shown in Fig. 2A. Ions can be observed which correspond to the calculated protonated molecular ion (M / H)/, of the GST–p26 fusion protein (Mr 55,484 Da), while the ions of m/z 27,743, 18,495, and 13,872 correspond to the (M / 2H)2/, (M / 3H)3/, and (M / 4H)4/ ions, respectively. The absence of other protein signals is indicative of the high selectivity of the GSH-agarose affinity column. The potentially interfering proteins at m/z Ç28,400 and 28,800, seen in the whole-cell experiments (Fig. 1A), were not seen in the MALDI mass spectrum of the affinity-bound fusion protein or were of sufficiently low abundance as to be hidden by the (M / 2H)2/ peak (Fig. 2A, 0 min). The signal enhancement obtained using affinity purification can be easily seen by comparing Figs. 1A and 2A, 0 min. The improvement in the signal from the affinity-bound fusion protein may be due partly to concentration of the protein by the GSH-agarose beads. It may also be partially due to removal of suppression effects from other components present in the whole bacteria, which may have interfered with the ionization of the fusion protein in the MALDI analysis. Proteolysis Time Course Studies After a fusion protein has been purified by affinity chromatography, the recombinant protein must be proteolytically cleaved from the carrier protein. This procedure can be performed either after elution of the fusion protein from the affinity column, or it can be performed by treating the affinity-bound fusion protein with the enzyme. When the cleavage occurs in solution, the car-
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FIG. 3. MALDI mass spectrum of HIV-1IIIB p26.
rier protein must be removed in a further purification step. Both procedures are usually monitored by SDS– PAGE. Because of the sensitivity, simplicity, and the applicability of MALDI/MS to direct analysis of affinity-bound analytes, we next explored the utility of this technique for monitoring proteolytic cleavages of proteins both in solution and noncovalently bound to the affinity column. Factor Xa Cleavage of Affinity-Bound Fusion Protein Spectra obtained from PBS-rinsed 1-ml aliquots of the column bed at various time points are shown in Fig. 2A. The ions of m/z 26,271, 13,136, and 8758 correspond to the (M / H)/, (M / 2H)2/, and (M / 3H)3/ of GST. The spectra show a steady increase in GST relative to the abundance of the GST–p26 fusion protein. Because the beads were washed prior to analysis, no ions due to free p26 in solution were observed. Only analytes still affinity-bound to the GSH column will be observed using this procedure. Peak areas are plotted in Fig. 2B. These areas do not correspond directly to the relative amounts of each molecular species present, as the signal intensities are compound-dependent. This
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is especially true when comparing the abundances of ions differing significantly in mass. Changes in relative ion abundances do, however, reflect changes in relative amounts of the analytes. The SDS – PAGE analysis of the 2-h beads is shown in Fig. 1B. Lane D, the supernatant after treatment with GSH-agarose, shows a band at Mr Ç55,000 and a band at Mr Ç29,000. The Mr 55,000 component was tentatively assigned to fusion protein still in solution, while the Mr Ç29,000 component was originally attributed to spontaneously cleaved free p26. From the MALDI data, however, it appears likely that this band contains mainly the unknown Mr 28,400 and 28,800 proteins. Lane E shows that the p26 released from the GSH-agarose beads by the factor Xa is quite pure. As can be seen from Fig. 1B, Lane F, where a sample of beads plus supernatant was analyzed, the cleavage reaction is not yet complete after a 2-h digestion with factor Xa; the fusion protein (GST – p26, Mr 55,484), p26 (Mr 29,232), and GST (Mr 26,271) can all be observed on the gel. From the MALDI data in Fig. 4, it can also be seen that the absolute amount of GST is still increasing after 2 h. At 14 h, only the
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FIG. 4. MALDI mass spectra of the reaction mixture at various times during the cleavage of p26 by HIV protease in solution.
bound GST and its dimer ion (Mr 52,413) are seen in the spectrum. A MALDI spectrum of the eluted p26 (Mr 29,232) is shown in Fig. 3. The low abundance peaks observed at Ç1000-Da intervals below the molecular weight of the fusion protein are due to bacterial proteolysis of p26. This proteolysis proceeds fairly rapidly after the factor Xa cleavage reac-
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tion to give, ultimately, only an Mr Ç24,300 product, even in the presence of protease inhibitors. This provides an example of how MALDI data can help in cases where the expected protein expression product is not produced. Observation of the intact fusion protein affinity-bound to the GSH-agarose indicates that the DNA construct is correct, and that the problem is not one
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FIG. 5. (A) MALDI mass spectra of 1-ml aliquots of the column bed/reaction mixture slurry at various times during the cleavage of p26 by HIV protease, and (B) MALDI mass spectra of a 1-ml aliquot of the column bed/reaction mixture slurry 23 h after start of the cleavage reaction of affinity-bound p26 by HIV protease, showing the mass range 20,000 to 36,000 Da.
of protein expression, but of stability of the expressed product. Since a corresponding series of ions below the molecular weight of the fusion protein is not observed while the fusion protein is still attached to the GSH-
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agarose, the fusion protein itself appears to be stable. Thus, proteolysis occurs only after the p26 is cleaved from the GST. If proteolysis were occurring from the C-terminal end of the molecule, then cleavage products should
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Fig. 5—Continued
be observed in both cases. The data therefore indicate that proteolysis of p26 occurs only when its N-terminal end is exposed. In fact, the resolution of the MALDI technique is sufficient to assign the degradation products to cleavages between specific residues at the N-terminal end of p26 (21). HIV Protease Cleavage Reactions of p26 in Solution HIV protease cleaves p26 at two cleavage sites, generating part of p12 (here designated p12*), p24, and part of p17 (designated p17*). Thus, in addition to the original p26 (Åp17*–p24–p12*) (Mr 29232), possible products of the HIV protease cleavage reaction in solution are p17*–p24 (Mr 27,462), p24–p12* (Mr 27,351), p24 (Mr 25,581), p12* (Mr 1790), and p17* (Mr 1900). As can be seen in Fig. 4, (M / H)/ and (M / 2H)2/ ions of p26, p17*–p24 and/or p24–p12*, and p24 are observed. The resolution and mass accuracy of the MALDI technique were not sufficient to allow separate quantitation of p17*–p24 (Mr 27,462) and p24–p12* (Mr 27,351), which would also have been indistinguishable by SDS–PAGE gel electrophoresis. A low-mass cutoff of 10,000 Da was used for the MALDI analyses to enhance the sensitivities of the higher-molecularweight components; thus p12* and p17* were not analyzed. The enzymatic cleavage of p26 by HIV protease in solution is very rapid. The 1-min time point shows mainly intact p26. Some cleavage of the p12* and/or the p17* bond(s) has already occurred giving p24–p12* and/or p17*–p24. At 25 min, both bonds have been cleaved, giving the peak for p24 (m/z 25581) as the dominant ion in the spectrum, with only a small
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amount of the p26 still remaining. At 1.5 h, the cleavage reaction is almost complete, with cleavage of both ends of the p26 giving p24. HIV Protease Cleavage Reactions of Affinity-Bound GST-p26 When the fusion protein, still attached to the affinity beads, is cleaved with HIV protease, bound and unbound proteins can be detected in the reaction mixture if the slurry is analyzed by MALDI. The GST–p26 fusion protein produced by the Jones’ E. coli consists of GST–p17*–p24–p12* (Mr 55484), with the N-terminal end of the p17* fragment attached to the GST. This means that possible components include some of the products listed above: p24–p12* (Mr 27,351), p24 (Mr 25,581), and p12* (Mr 1790). In addition, there can be proteins still bound to the GST moiety, and thus still attached to the beads, i.e., GST–p26 (Mr 55,484), GST– p17* (Mr 28,152), and GST–p17*–p24 (Mr 53,713). One-microliter aliquots of the reaction mixture (beads and solution) were spotted on the target, and the reaction was quenched with matrix solution. MALDI mass spectra taken of the various time points are shown in Fig. 5A. Although the HIV-1 protease cleavage reaction is very rapid in solution, binding the fusion protein to the affinity column significantly reduces the reaction rate. We previously noted this effect in earlier studies of proteolytic cleavages of affinitybound proteins (10). An expanded spectrum showing the m/z 25,000– 27,000 region of the 23-h time point of the reaction mixture is shown in Fig. 5B. All of the possible reaction products within the mass range displayed can be seen
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in the spectrum. This figure illustrates both the complexity of the reaction mixture, and the resolving power of the MALDI technique. CONCLUSION
We have demonstrated that, although fusion protein can be detected by MALDI/MS of whole bacterial cells after treatment with hexafluoroisopropanol, the presence of normal and induced bacterial proteins limits the utility of MALDI as a screening technique. The advantages of MALDI/MS analysis are more fully realized after affinity purification of the bacterial lysate. With interferences removed, the resolving power of the MALDI technique can be used for directly analyzing fusion proteins and their cleavage products, with or without prior removal of the fusion protein from the affinity beads. In addition, the speed of the MALDI technique allows the monitoring of enzymatic cleavage reactions in nearly ‘‘real time.’’ Although the MALDI/MS method for following the release of recombinant proteins from affinity-bound fusion proteins is not meant to supplant SDS–PAGE analysis, it does offer a rapid and sensitive technique for those laboratories with access to MALDI instrumentation. ACKNOWLEDGMENTS Support for the purchase of the Voyager RP MALDI mass spectrometer by the NIH Office of AIDS Research is gratefully acknowledged. We thank Dr. Ian M. Jones (NERC Institute of Virology, Oxford, UK) for the gift of the E. coli which express HIV p26. We also express our appreciation to Dr. Hirao Kohno (Kansai Medical University, Osaka, Japan) for helpful discussions concerning the plasmid.
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2. 3. 4. 5. 6. 7. 8.
Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K., Eds.), Vol. 2, pp. 16.7.1–16.7.8, Green/Wiley-Interscience, New York. Smith, D. B., and Johnson, K. S. (1988) Gene 67, 31–40. Nagai, K., and Thorgersen, H. C. (1984) Nature 309, 810–812. Karas, M., Bahr, U., and Geissmann, U. (1991) Mass Spectrom. Rev. 10, 335–358. Cerpa-Poljak, A., Jenkins, A., and Duncan, M. W. (1995) Rapid Commun. Mass Spectrom. 9, 233–239. Papac, D. I., Hoyes, J., and Tomer, K. B. (1994) Anal. Chem. 66, 2609–2613. Jemmersen, R., and Paterson, Y. (1986) Science 232, 1001–1004. Sukau, D., Kohl, J., Karwath, G., Schneider, K., Casaretto, M., Bitter-Suermann, D., and Przybylski, M. (1990) Proc. Natl. Acad. Sci. USA 87, 9848–9852.
9. Zhao, Y., and Chait, B. T. (1994) Anal. Chem. 66, 3723–3726. 10. Papac, D. I., Hoyes, J., and Tomer, K. B. (1994) Protein Sci. 3, 1485–1492. 11. Mills, H. R., Berry, N., Burns, N. R., and Jones, I. M. (1992) AIDS 6, 437–439. 12. Pharmacia Biotechnology, PhastSystem Separation Technique File No. 112. 13. DeLlano, J. J. M., Jones, W., Schneider, K., Chait, B. T., Manning, J. M., Rodgers, G., Benjamin, L. J., and Weksler, B. (1993) J. Biol. Chem. 269, 27004–27011. 14. Cain, T. C., Lubman, D. M., and Weber, W. J., Jr. (1994) Rapid Commun. Mass Spectrom. 8, 1026–1030. 15. Schey, K. L., Papac, D. I., Knapp, D. R., and Crouch, R. K. (1992) Biophys. J. 63, 1240–1243. 16. HIV Protease Analytical Data Sheet, Bachem Biosciences Inc., King of Prussia, PA. 17. Liang, X., Cain, T. C., Bai, J., Liu, Y.-H., and Lubman, D. M. (1995) Presented at the 43rd Annual Conference on Mass Spectrometry and Allied Topics, May 21–26, 1995, Atlanta, GA. 18. Chait, B. T., personal communication in (14). 19. Malling, H. V., Environmental Toxicology Program, NIEHS, personal communication. 20. Schaaper, R., Laboratory of Molecular Genetics, NIEHS, personal communication. 21. Parker, C. E., Papac, D. I., Trojak, S. K., and Tomer, K. B. (1996) J. Immunol., in press.
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