Anal. Chem. 1998, 70, 2188-2190
Technical Notes
Use of Mass Spectrometry To Ensure Purity of Recombinant Proteins: A Cautionary Note Bosong Xiang, James Ferretti, and Henry M. Fales*
Laboratory of Biophysical Chemistry, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892-1676
Electrospray mass spectrometry was used to confirm the identity and purity of the unlabeled and isotopically labeled vnd/NK2-A35T homeodomain protein prepared for NMR experiments. A series of events involving adduction by urea, carbamoylation of amino acid groups, and the presence of nickel ion resulted in protein masses shifted from their expected values, thus emphasizing that observed discrepancies in mass may have sources other than an error in amino acid sequence. Procedures used to detect and remedy these problems are described. The application of mass spectrometry to confirm the identity and purity of proteins prepared by recombinant methods for X-ray and NMR structural analysis, including those isotopically labeled, is now fairly routine,1,2 and anyone undertaking these timeconsuming investigations is well advised to use this procedure. When differences between experimental and calculated molecular masses are encountered, one might initially suspect a protein sequence error. However, other, sometimes less serious conditions can occur as a result of the complex experimental conditions used in protein purification. The purpose of this note is to describe, as an example, one series of such events encountered in the course of a protein preparation using Ni2+ affinity column chromatography. EXPERIMENTAL SECTION Luria-Bertani broth (LB) was purchased from Advanced Biotechnologies Inc., Columbia, MD; Martek 9-N (15N > 98%) medium from Martek Biosciences Corp., Columbia, MD; plasmid vector pET-15b, expression host BL21(DE3)plyS, 8× binding buffer (40 mM imidazole, 4 M NaCl, and 160 mM Tris-HCl, pH 7.9), and 4× elute buffer (4 M imidazole, 2 M NaCl, and 80 mM Tris-HCl, pH 7.9) from Novagen, Inc., Madison, WI; isopropyl β-Dthiogalactopyranoside (IPTG) and calcium chloride (CaCl2) from Sigma Chemical Co., St. Louis, MO; urea (ultrapure) and guanidine (ultrapure) from Life Technologies, Grand Island, NY; sodium chloride (NaCl) from J.T. Baker, Phillipsburg, NJ; and * To whom correspondence should be addressed. E-mail: hmfales@ helix.nih.gov. Fax: 301-402-3404. (1) Beavis, R. C.; Chait, B. T.; Creel, H. S.; Fournier, M. J.; Mason, T. L.; Tirrell, D. A. J. Am. Chem. Soc. 1992, 114, 7584. (2) For a recent review of electrospray mass spectrometry, see: Gaskell, S. J. J. Mass Spectrom. 1997, 32, 677-688.
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thrombin from Calbiochem-Novabiochem Intl., La Jolla, CA. Ni2+ affinity columns were prepared by using His‚Bind resin and charge buffer (Novagen) following the procedure described in ref 3. Mutant vnd/NK2-A35T homeodomain protein analyzed in this study was prepared for NMR structural determination.4 The sitedirected A35T mutation was constructed by PCR from the wildtype vnd/NK2 homeobox DNA. The gene was inserted between Nde1 and BamH1 restriction sites of pET-15b plasmid vector that was then transformed into expression host BL21(DE3)plysS. The unlabeled and 15N-labeled vnd/NK2-A35T were expressed in LB and Martek 9-N media. The expressed protein contained a HisTag at its N-terminal side to facilitate purification by Ni2+ affinity chromatography.3 The protein was purified under denaturing conditions. Initially, urea was used as the denaturant. Cells harvested after the protein expression induced by IPTG were suspended in the binding buffer (8-fold dilution of the 8× binding buffer) containing 6 M urea. A French press was used to break cells, and cell debris was removed by centrifugation. The supernatant containing vnd/NK2-A35T protein was loaded onto a Ni2+ affinity column that was prebalanced with the binding buffer. After extensive washing with the binding buffer, vnd/NK2-A35T was eluted with the elute buffer (4-fold dilution of the 4× elute buffer) containing 6 M urea. The buffer of the protein solution was then changed into a cleavage buffer containing 2 M urea, 2.5 mM CaCl2, 150 mM NaCl, and 20 mM Tris-HCl at pH 7.5 by dialysis. Thrombin was added to the solution to cut off the His-Tag and removed by centrifugation after completion of the reaction. The reaction mixture was concentrated, dialyzed against the binding buffer for buffer exchange, and loaded onto another Ni2+ column. The solution passing through the second column contained vnd/NK2-A35T without the His-Tag and was collected and dialyzed against water extensively to refold the protein and remove low-molecular-weight impurities. In subsequent purifications, where guanidine was used as the denaturant, the protocol was similar to that described above, except that 6 M guanidine was used in the binding and elute buffers and about 0.1 M guanidine in the cleavage buffer instead of urea. The SDS gel of the protein showed a single band at the expected molecular weight of the pure protein. The molecular (3) pET System Manual, 6th ed.; Novagen Inc.: Madison, WI, 1995. (4) Gruschus, J. G.; Tsao, D. H. H.; Wang, L.; Nirenberg, M.; Ferretti, J. A. Biochemistry 1997, 36, 5372-5380. S0003-2700(97)01258-4 Not subject to U.S. Copyright. Publ. 1998 Am. Chem. Soc.
Published on Web 04/08/1998
a
b
Figure 1. (a) Electrospray mass spectrum of vnd/NK2-A35T protein M dissolved in 10% acetic acid with tube lens (skimmer) voltage at 96 V. (b) Mass spectrum of the same sample with tube lens voltage raised to 196 V.
weights calculated for the 80-residue unlabeled and 100% 15Nlabeled vnd/NK2-A35T protein are 9723.7 and 9862.7 Da, respectively. Mass spectra were collected on a Finnigan TSQ700 mass spectrometer (Finnigan Instrument Co., Sunnyvale, CA) equipped with an electrospray source without curtain gas using a heated capillary at 200 °C. Protein samples were dissolved at approximately 20 µM in 10% acetic acid (Mallinckrodt Baker, Inc., Paris, KY) unless otherwise noted and were run in the Finnigan electrospray source, admitting the solution with a Harvard apparatus syringe pump (South Natick, MA) without sheath or auxiliary gas at 0.5-1 µL/min using 5.5 kV on the needle. Spectra were acquired for 1-5 min using 4-s scans from 10 to 2000 Da in the profile mode of data acquisition and deconvoluted with the Finnigan ICIS data system.
RESULTS AND DISCUSSIONS At first, the unlabeled vnd/NK2-A35T protein purified as described above and dissolved in 10% acetic acid displayed its largest peak, M1 (Figure 1a), at a mass 60.3 Da more than calculated (9723.7 Da), along with small amounts of the corresponding sodium salts and an impurity 98 Da still higher in mass. Ions such as the last are common in electrospray mass spectra of proteins, and they are usually thought to be due to traces of phosphate or sulfate, although none were known to be present in this sample. On the other hand, the 60-Da excess was unusual. It was thought not to be due to the presence of acetic acid, since we have used this solvent for hundreds of protein analyses without
Figure 2. Electrospray mass spectrum of 15N-labeled vnd/NK2A35T purified with aged binding buffer containing 6 M urea and dissolved in 10% acetic acid.
detecting adducts at this concentration of the acid. A small peak with the correct mass is visible in Figure 1a, and, upon increasing the tube lens (skimmer) voltage, it became the dominant mass (Figure 1b). This behavior is typical of weakly bound adduct ions, and, after further consideration of the protein purification procedure, formation of the higher mass peak was finally traced to the presence of 6 M urea in its buffer. While annoying, easy loss of such adducts under gentle collision conditions usually provides assurance that the protein has the correct mass. A second, not quite so obvious, problem is also related to the urea buffer. Here it involved the detection of two peaks 43 and 86 Da higher than the largest peak at 9858 Da (Figure 2). The latter was expected since it corresponded to an isotopomer of vnd/ NK2-A35T 15N-labeled to 96.4% (the media was 15N-labeled > 98%). Unlike the first case, these peaks did not diminish upon lowering the tube lens voltage. Their presence was finally traced to the use of an aged binding buffer with 6 M urea that had been standing for several weeks and, therefore, contained HNCO. The presence of this impurity resulted in partial carbamoylation of the lysines or terminal amino groups,5 and the sample was clearly of little further use in NMR. Substitution of urea with guanidine as the denaturant overcame this problem. All of the above analyses were undertaken by dissolving the protein in a small amount of 10% acetic acid, added to reduce the surface tension of the water to aid in the electrospray process. It was some surprise that a solution of the carbamoyl-free, 15Nlabeled vnd/NK2-A35T, purified with guanidine as the denaturant and made up in a solution substituting 25% hexafluoro-2-propanol for acetic acid, now exhibited a mass of 9916 Da, i.e., 58 Da higher than expected (Figure 3a). This result suggested the presence of a nickel (58.7 Da) complex since this ion had been used in the protein preparation (see Experimental Section). Although addition of ethylenediaminetetraacetic acid (EDTA), raising the pH of the solution to pH 8.0, had no effect on the spectrum, acidifying it to pH 3 by addition of acetic acid produced the spectrum shown in Figure 3b. Here the complex has been nearly completely destroyed due to its instability at low pH, and the main peak is at 9858 Da, as expected. We then recalled that the protein had remained at pH > 6.0 during its whole purification process, (5) Lundblad, R. L. Chemical Reagents for Protein Modification; CRC Press: Boca Raton, FL, 1991, pp 137-140.
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a
b
Figure 3. (a) Electrospray mass spectrum of 15N-labeled vnd/NK2A35T purified with buffers containing guanidine as the denaturant and dissolved in 25% hexafluoro-2-propanol (pH 6.0). (b) Electrospray mass spectrum of the same sample after adding acetic acid to pH 3.
including the mass spectrometric analysis. Clearly, nickel ion from the affinity column used in the purification had not been
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removed by the dialysis due to its high affinity for the protein; its presence was subsequently confirmed by atomic absorption analysis. Nickel was later removed from the purified vnd/NK2A35T protein solution by dialyzing against water at pH 3.0 for the NMR study. We note that nickel was not responsible for the 60Da excessive mass in Figure 1a, since that sample had been run in 10% acetic acid, thus prohibiting the formation of such complexes. Whether any of the above modifications of a protein would perturb a given X-ray or NMR analysis is, of course, unknown. The presence of small amounts of urea or phosphate would, hopefully, minimally affect attempts at crystallization for X-ray, while the mere presence of urea should cause minimal problems in NMR. On the other hand, covalent carbamoylation from the aged urea would be expected to have serious consequences for both techniques. The effect of the presence of nickel as an impurity is less clear. In our experiment, it merely shifted slightly some upfield 1H NMR peaks. Nevertheless, one can imagine scenarios where the effect would be much greater. Our conclusion is that, at the very minimum, electrospray mass spectra should be measured under several instrumental conditions and at high and low pH before concluding that there is a sequence error. Additionally, atomic absorption is a simple method that requires little sample and provides assurance that no extraneous metal ions are present. We note that a useful database of excess mass values is presented on the World Wide Web by K. Mitchelhill at http://www.meddstv.unimelb.edu.au/WWW.DOCS/ SVIMRDocs/MassSpec/deltamassV2.html. Received for review November 17, 1997. Accepted March 5, 1998. AC971258J
