| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Baxter Hemoglobin Therapeutics, Boulder, Colorado 80301, USA
2 University of California at Berkeley, California 94720, USA
Reprint requests to: Izydor Apostol, Amgen Inc., Mailstop 25-2-A, Thousand Oaks, CA 91320-1799, USA; e-mail: iapostol{at}amgen; fax: (805) 447-8690.
(RECEIVED January 2, 2001; FINAL REVISION March 8, 2001; ACCEPTED March 8, 2001)
3 Present address: Amgen Inc., Mailstop 25-2-A, Thousand Oaks, CA 91320-1799, USA. 
4 Present address: Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251, USA.
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1101/
 |
Abstract |
|---|
 TOP Abstract IntroductionReferences |
|---|
Keywords: Isotope ratio monitoring mass spectrometry; hemoglobin; origin of protein expression
|
Introduction |
|---|
|
TOPAbstractIntroductionReferences |
|---|
It used to be relatively easy to recognize the origin of expressed proteins based on cotranslational and posttranslational modifications (e.g., misincorporation of norleucine, formylation, and glycosylation), which very often can be related to the method of protein manufacture. Also, many heterologous proteins contain N-terminal methionine because of the initiation codon, which differentiates them from their naturally occurring counterparts. However, recent progress in molecular biology and biotechnology allows for the production of heterologous proteins, which are indistinguishable from the wild type by traditional protein chemistry. Methods used to achieve this include the overexpression of methionyl amino peptidase, which can help to produce a protein with the same N-terminal amino acid as its heterolog (Kendall and Bradshaw 1992; Shen et al. 1997). Overexpression of deformylase can eliminate residual levels of formylation characteristic of proteins produced in bacterial expression systems (Solbiati et al. 1999; Warren et al. 1996). Supplementing the fermentation media with methionine has been shown to minimize the misincorporation of norleucine (Bogosian et al. 1989; Tsai et al. 1988). Good control of fermentation conditions can minimize incorporation of other noncoded amino acids (Apostol et al. 1997; Tsai et al. 1988). These types of technological improvements can result in the production of proteins virtually indistinguishable from their wild-type forms.
Hemoglobin is an example of a protein with a very high potential commercial value as an oxygen-carrying therapeutic (blood substitute). The literature contains numerous reports on the production of hemoglobin in Escherichia coli (Shen et al. 1997; Weickert et al. 1999; Looker et al. 1992), yeast (Hofmann et al. 1994; Yanase et al. 1994), transgenic animals and plants (Rao et al. 1994; Theisen 1999; Dieryck et al. 1997), and by extraction from human blood (Azari et al. 2000; Yu et al. 1997; Talarico et al. 2000; Carmichael et al. 2000). Assuming that all of these approaches can produce hemoglobin with an identical primary structure and similar isoform distribution, we show that it is still possible to elucidate the origin of expression using state-of-the-art analytical techniques. We identified several promising protein chemistry experiments and methods, which could provide a unique analytical signature for hemoglobins originating from different organisms, but ultimately, we concluded that high-precision isotope ratio monitoring mass spectrometry (IRM-MS) had the most potential.
IRM-MS allows for the determination of abundance ratios of stable isotopes of elements such as carbon and nitrogen for individual compounds. Before analysis, a sample must be converted into gas, typically by in-line combustion, and introduced via gas chromatography into a mass spectrometer (GC/MS) (Meier-Augenstein 1999). The chemical elements, which together comprise the majority of biomass (H, C, N, O, and S), occur naturally as a mixture of two or more stable isotopes. These isotopes show differences from the average abundance because of relative enrichment or depletion of a particular isotope by geochemical and biological isotopic fractionation processes (Preston 1992; Handley and Raven 2000; Kennedy and Krouse 1990; O'Leary 1988). Consequently, the stable isotope composition of a biomolecule is a convolution of the source material isotopic composition and isotopic fractionation processes occurring during biosynthetic incorporation. Compounds with identical elemental compositions (e.g., proteins with identical primary structures) can show significantly different stable isotope ratios, which allow discrimination among them. The effectiveness of this approach has been shown for detecting testosterone doping in sport (van de Kerkhof et al. 2000; Aguilera et al. 1996; Becchi et al. 1994). IRM analysis requires small samples, shows a high level of precision (± 0.01
–0.2), and is in routine use around the world for the determination of 15N/14N, 13C/12C, and other stable isotope ratios (Brand 1996). IRM analysis has been used extensively in studies of protein metabolism using precursors enriched in stable isotopes (Pont et al. 1997).
We subjected six samples of highly purified hemoglobin from three sources for simultaneous nitrogen and carbon isotope measurement. The samples included three different batches of recombinant hemoglobin expressed in E. coli produced over several years, one sample of recombinant hemoglobin expressed in yeast, and two samples of human hemoglobin purified from pooled blood collection in Europe and the United States. All samples were prepared in triplicate except the yeast recombinant hemoglobin (a gift from Dr. J. Manning), which was prepared in duplicate. Information about the type of hemoglobins, their origin, and year of production is included in Table 1
. To remove all buffer and formulation components, which could interfere with analysis, samples were repurified using a Sephadex G25 PD10 column to bring them into pure water. The identity and concentration of proteins was confirmed by visible region spectrophotometry. About 0.7 mg of each hemoglobin was lyophilized into 9 x 5 mm tin capsules, which were rolled into small balls and submitted for IRM analysis. The tin capsules were analyzed with a carbon-nitrogen analyzer interfaced to a PDZ Europa Scientific 2020 isotope ratio mass spectrometer (Barrie et al. 1989; Preston 1992). In brief, the tin capsules are dropped automatically into a 1000°C furnace with a burst of oxygen. The oxygen ignites the tin, which burns at 1700°C, and all of the hemoglobin in the sample is burned to a mixture of CO2, NOx, and H2O. These gases are swept by helium carrier gas through a copper column that reduces NOx to N2 and then through a water trap to remove water. N2 and CO2 are then separated on a GC column. The mass spectrometer is tuned to m/z of 28, 29, and 30 when the N2 peak comes out, and 44, 45, and 46 when the CO2 peak comes out. The relative abundance of isotopes was then determined for nitrogen and carbon, respectively. Because of the relatively high amount of protein sample burned, the precision is usually better than ± 0.2 for N2, and ± 0.1 for carbon.
|
) using the delta notation. This is defined as (Preston 1992):
(minor isotope) = [(minor/major)sample/(minor/major)standard) - 1] x 1000 (Brand 1996; Preston 1992). Negative and positive values indicate depletion and enrichment of the minor isotope, respectively. The 13C/12C and 15N/14N ratios were measured for each sample relative to Pee Dee Belemnite (PDB, CaCO3), and air standards, respectively. The results are shown in Figure 1. Significant differences were observed between the 13C and 15N values for hemoglobins expressed in human, yeast, and E. coli. The 13C (-10.19 ± 0.73) and 15N (5.31 ± 1.20) values for recombinant hemoglobin samples from E. coli are significantly different from those of hemoglobins from the other sources. The 13C (-18.73 ± 0.04 and -24.43 ± 0.25) and 15N values (7.7 ± 0.14 and 8.96 ± 0.22) for hemoglobin from North Americans and Europeans, respectively, are dramatically different from rHb1.1 made in E. coli. Hemoglobin synthesized in yeast had a 13C value (-23.50 ± 0.32) closest to the human-derived protein, but 15N (2.19 ± 0.26) was significantly different from E. coli or human hemoglobin.
|
15N for ammonium hydroxide should be constant and close to zero. This has been observed for several samples of ammonium nitrate and ammonium sulfate fertilizers in which 15N (ammonium ion) was in the range -4 to +0.2 (Heaton 1986). The 15N for rHb1.1 (-6.58 to -3.83) appears to principally reflect 15N of the ammonium hydroxide used in the controlled fermentation, although a small degree of nitrogen isotope fractionation by E. coli may be present. This is indicated by the small changes in 15N observed for hemoglobin produced over several years during which fermentation conditions were altered. The 15N for human-derived hemoglobin undoubtedly reflects the more diverse sources of nitrogen in the human diet, and the enrichment of 15N over dietary 14N (typically, 
15N = 3.4 ± 1.1) observed in many animals (Minigawa and Wada 1984; DeNiro and Epstein 1981). Consequently, 15N values for the human-derived hemoglobins (SFHB and DCLHB) are significantly different from 15N values for the recombinant human hemoglobins.
Analogous arguments can account for the range of 13C values observed for the different hemoglobin samples. The primary carbon source in controlled microbial fermentations is corn dextrose syrup, whose 13C value is relatively invariant and specific to corn, which fixes carbon by the C4 pathway. Widely different and nonoverlapping 13C values are observed for C3 (13C 
-27) and C4 plants (13C -13) because of the different contributions of diffusion and kinetic isotope effects in discriminating against 13C in these biosynthetic pathways (O'Leary 1988). The 13C value of hemoglobin expressed in E. coli is thus consistent with its source of carbon (Kennedy and Krouse 1990). As with dietary nitrogen, humans obtain carbon from diverse sources, including C3 and C4 plants and animal products. This could explain the significant differences in 13C between hemoglobin expressed in humans and E. coli. Our observation of greater 13C depletion in hemoglobin samples derived from Europeans (13C -22.43 ± 0.25) compared to North Americans (13C -18.73 ± 0.04) could be significant. These values are comparable to data reported previously for human blood (13C = -18.22) (Lyon and Baxter 1978) and are consistent with the suggestion that extensive use of C4 plant maize in the North American diet leads to less depletion of 13C (Brand 1996). Similarly to human-derived hemoglobin, hemoglobin from yeast grown in YPD media presumably shows greater depletion of 13C than hemoglobin from E. coli because of the mixture of C3 (soy hydrolysate) and C4 (dextrose) plant-derived carbon used in this media (Kennedy and Krouse 1990). Consequently, 13C for hemoglobin expressed in yeast is significantly different from E. coli hemoglobin and closer to human hemoglobin.
In summary, our data show that hemoglobins from different biosynthetic sources have unique and characteristic nitrogen and carbon isotope ratios. Excellent discrimination among hemoglobins derived from controlled E. coli fermentation, humans, and yeast is possible on the basis of 15N values alone. However, the degree of discrimination is improved further by parallel measurements of 13C. These isotopic ratios can thus be used to distinguish otherwise chemically identical proteins and indicates that parallel measurement of several different stable isotope ratios is the best way to determine unambiguously the biosynthetic origin of a protein.
|
Acknowledgments |
|---|
The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
|
References |
|---|
|
TOPAbstractIntroductionReferences |
|---|
Apostol, I., Levine, J., Lippincott, J., Leach, J., Hess, E., Glascock, C.B., Weickert, M.J., and Blackmore, R. 1997. Incorporation of norvaline at leucine positions in recombinant human hemoglobin expressed in Escherichia coli. J. Biol. Chem. 272: 28980–28988.
Azari, M., Boose, J.A., Burhop, K.E., Camacho, T., Catarello, J., Darling, A., Ebeling, A.A., Estep, T.N., Pearson, L., Guzder, S., et al. 2000. Evaluation and validation of virus removal by ultrafiltration during the production of diaspirin crosslinked haemoglobin (DCLHb). Biologicals 28: 81–94.[Medline]
Barrie A., Davies, J.E., Park, A.J., and Workman, C.T. 1989. Continuous-flow stable isotope analaysis for biologists. Spectroscopy 4: 42–52.
Becchi, M., Aguilera, R., Farizon, Y., Flament, M.M., Casabianca, H., and James, P. 1994. Gas chromatography/combustion/isotope-ratio mass spectrometry analysis of urinary steroids to detect misuse of testosterone in sport. Rapid Commun. Mass Spectrom. 8: 304–308.[CrossRef][Medline]
Bogosian, G., Violand, B.N., Dorward-King, E.J., Workman, W.E., Jung, P.E., and Kane, J.F. 1989. Biosynthesis and incorporation into protein of norleucine by Escherichia coli. J. Biol. Chem. 264: 531–539.
Brand, W.A. 1996. High precision isotope ratio monitoring technique in mass spectromemtry. J. Mass Spectrom. 31: 225–235.[CrossRef][Medline]
Carmichael, F.J., Ali, A.C., Campbell, J.A., Langlois, S.F., Biro, G.P., Willan, A.R., Pierce, C.H., and Greenburg, A.G. 2000. A phase I study of oxidized raffinose cross-linked human hemoglobin. Crit. Care Med. 28: 2283–2292.[CrossRef][Medline]
DeNiro, M.J. and Epstein, S. 1981. Influence of diet on the distribution of nitrogen isotopes in animals. Geochim. Cosmochim. Acta 45: 341–351.
Dieryck, W., Pagnier, J., Poyart, C., Marden, M.C., Gruber, V., Bournat, P., Baudino, S., and Merot, B. 1997. Human haemoglobin from transgenic tobacco. Nature 386: 29–30.[Medline]
Handley, L.L. and Raven, J.A. 2000. The use of natural abundance of nitrogen isotopes in plant physiology and ecology. Plant Cell Environ. 15: 965–985.
Heaton, T.H.E. 1986. Isotopic studies of nitrogen pollution in the hydrosphere and atmosphere: A review. Chem. Geol. 59: 87–102.
Hofmann, O.M., Mould, R.M., and Brittain, T. 1994. The incorporation of sulphaem into recombinant adult human haemoglobin produced in a yeast expression system. Protein Eng. 7: 281–283.
Kendall, R.L. and Bradshaw, R.A. 1992. Isolation and characterization of the methionine aminopeptidase from porcine liver responsible for the co-translational processing of proteins. J. Biol. Chem. 267: 20667–20673.
Kennedy, B.V. and Krouse, H.R. 1990. Isotope fractionation by plants and animals: Implications for nutrition research. Can. J. Physiol. Pharmacol. 68: 960–972.[Medline]
Looker, D., Abbott-Brown, D., Cozart, P., Durfee, S., Hoffman, S., Mathews, A.J., Miller-Roehrich, J., Shoemaker, S., Trimble, S., and Fermi, G. et al. 1992. A human recombinant haemoglobin designed for use as a blood substitute. Nature 356: 258–260.[CrossRef][Medline]
Looker, D., Mathews, A.J., Neway, J.O., and Stetler, G.L. 1994. Expression of recombinant human hemoglobin in Escherichia coli. Methods Enzymol. 231: 364–374.
Lyon, T.D. and Baxter, M.S. 1978. Stable carbon isotopes in human tissues. Nature 273: 750–751.[CrossRef][Medline]
Meier-Augenstein, W. 1999. Use of gas chromatography-combustion-isotope ratio mass spectrometry in nutrition and metabolic research. Curr. Opin. Clin. Nutr. Metab. Care 2: 465–470.[CrossRef][Medline]
Minigawa, M. and Wada, E. 1984. Stepwise enrichment of 15N along food chains: Further evidence and the relation between 15N and animal age. Geochim. Cosmochim. Acta 48: 1135–1140.
O'Leary, M.H. 1988. Carbon isotopes in photosynthesis. Bioscience 38: 328–336.[CrossRef]
Pont, F., Duvillard, L., Maugeais, C., Athias, A., Persegol, L., Gambert, P., and Verges, B. 1997. Isotope ratio mass spectrometry, compared with conventional mass spectrometry in kinetic studies at low and high enrichment levels: Application to lipoprotein kinetics. Anal. Biochem. 248: 277–287.[CrossRef][Medline]
Preston, T. 1992. The measurement of stable isotope natural abundance variation. Plant Cell Environ. 15: 1091–1097.
Rao, M.J., Schneider, K., Chait, B.T., Chao, T.L., Keller, H., Anderson, S., Manjula, B.N., Kumar, R., and Acharya, A.S. 1994. Recombinant hemoglobin A produced in transgenic swine: Structural equivalence with human hemoglobin A. Artif. Cells Blood Substit. Immobil. Biotechnol. 22: 695–700.[Medline]
Shen, T.J., Ho, N.T., Zou, M., Sun, D.P., Cottam, P.F., Simplaceanu, V., Tam, M.F., Bell, D.A.J., and Ho, C. 1997. Production of human normal adult and fetal hemoglobins in Escherichia coli. Protein Eng. 10: 1085–1097.
Solbiati, J., Chapman-Smith, A., Miller, J.L., Miller, C.G., and Cronan, J.E.J. 1999. Processing of the N termini of nascent polypeptide chains requires deformylation before methionine removal. J. Mol. Biol. 290: 607–614.[CrossRef][Medline]
Talarico, T.L., Guise, K.J., and Stacey, C.J. 2000. Chemical characterization of pyridoxalated hemoglobin polyoxyethylene conjugate. Biochim. Biophys. Acta 1476: 53–65.[Medline]
Theisen, M. 1999. Production of recombinant blood factors in transgenic plants. Adv. Exp. Med. Biol. 464: 211–220.[Medline]
Tsai, L.B., Lu, H.S., Kenney, W.C., Curless, C.C., Klein, M.L., Lai, P.H., Fenton, D.M., Altrock, B.W., and Mann, M.B. 1988. Control of misincorporation of de novo synthesized norleucine into recombinant interleukin-2 in E. coli. Biochem. Biophys. Res. Commun. 156: 733–739.
van de Kerkhof, D.H., de Boer, D., Thijssen, J.H., and Maes, R.A. 2000. Evaluation of testosterone/epitestosterone ratio influential factors as determined in doping analysis. J. Anal. Toxicol. 24: 102–115.[Medline]
Warren, W.C., Bentle, K.A., Schlittler, M.R., Schwane, A.C., O'Neil, J.P., and Bogosian, G. 1996. Increased production of peptide deformylase eliminates retention of formylmethionine in bovine somatotropin overproduced in Escherichia coli. Gene 174: 235–238.
Weickert, M.J., Pagratis, M., Glascock, C.B., and Blackmore, R. 1999. A mutation that improves soluble recombinant hemoglobin accumulation in Escherichia coli in heme excess. Appl. Environ. Microbiol. 65: 640–647.
Yanase, H., Cahill, S., Martin de Llano J.J., Manning, L.R., Schneider, K., Chait, B.T., Vandegriff, K.D., Winslow, R.M., and Manning, J.M. 1994. Properties of a recombinant human hemoglobin with aspartic acid 99(beta), an important intersubunit contact site, substituted by lysine. Protein Sci. 3: 1213–1223.[Abstract]
Yu, Z., Friso, G., Miranda, J.J., Patel, M.J., Lo-Tseng, T., Moore, E.G., and Burlingame, A.L. 1997. Structural characterization of human hemoglobin crosslinked by bis(3,5-dibromosalicyl) fumarate using mass spectrometric techniques. Protein Sci. 6: 2568–2577.[Abstract]
CiteULike 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
