Mass spectrometry recombinant proteins

Parker, C. E. Papac, D. L Tomer, K. B. Monitoring cleavage of fusion protein by matrix-assisted laser desorption ionization/mass spectrometry Recombinant HIV-1IIIB p26. Anal. Biochem. 1996, 239, 25-34. 

Figure 5.6 Positive-ion electrospray spectrum obtained from the major component in the LC-MS analysis of a purified recombinant 62 kDa protein using a Cig microbore 50 X 1 mm column and a flow rate of 50 p.lmin . The starting buffer (buffer A ) was 0.1% TEA in water, while the gradient buffer (buffer B ) consisted of 0.1% TEA in acetonitrile-water (9 1 vol/vol). The running conditions consisted of 0% B for 5 min, followed by a linear gradient of 100% B for 55 min. Reprinted from J. Chromatogr., B, 685, McAtee, C. P., Zhang, Y., Yarbough, P. O., Fuerst, T. R., Stone, K. L., Samander, S. and Williams, K. R., Purification and characterization of a recombinant hepatitis E protein vaccine candidate by liquid chromatography-mass spectrometry , 91-104, Copyright (1996), with permission from Elsevier Science.

In E. Coli bacterial lysates, the proteome (i.e., the full array of proteins produced) was analyzed by isoelectric focusing and mass spectrometry.97 A comparison of capillary electrophoretic separation and slab gel separation of a recombinant monoclonal antibody demonstrated that the precision, robustness, speed, and ease-of-use of CE were superior.98 Seventy-five proteins from the yeast ribosome were analyzed and identified by capillary electrophoresis coupled with MS/MS tandem mass spectrometry.99 Heavy-chain C-terminal variants of the anti-tumor necrosis factor antibody DE7 have been separated on capillary isoelectric focusing.100 Isoforms differing by about 0.1 pi units represented antibodies with 0,1 or 2 C-terminal lysines. 

Goodacre, R. Karim, A. Kaderbhai, M. A. Kell, D. B. Rapid and quantitative analysis of recombinant protein expression using pyrolysis mass spectrometry and artificial neural networks Application to mammalian cytochrome b5 in Escherichia coli. J. Biotechnol. 1994,34,185-193. 

Jebanathirajah, J. A. Andersen, S. Blagoev, B. Roepstorff, P. A rapid screening method to monitor expression of recombinant proteins from various prokaryotic and eukaryotic expression systems using matrix-assisted laser desorption ioniza-tion-time-of-flight mass spectrometry. Anal. Biochem. 2002, 305, 242-250. 

Winkler, M. A. Hickman, R. K. Golden, A. Aboleneen, H. Analysis of recombinant protein expression by MALDI-TOF mass spectrometry of bacterial colonies. BioTechniques 2000, 28,890,892,894-895. 

A final problem for bioinformatics and bioanalytical scientists is the characterization of engineered microorganisms. Whole-cell analysis by mass spectrometry has been used to confirm the introduction of therapeutic genes into adenovirus vectors,100 to confirm the expression of recombinant proteins in bacteria,101,102 and also in vaccinology.103 In the broader case, identification of... 

Tang, Q., Harrata, A. K., Lee, C. S. (1997). Two-dimensional analysis of recombinant E. coli proteins using capillary isoelectric focusing electrospray ionization mass spectrometry. Anal. Chem. 69(16), 3177-3182. 

K.L.Johnson, T.D. Veenstra,J.M. Londowski, A.J.Tomlinson, R. Kumar, S. Naylor, On-line sample clean-up and chromatography coupled with electrospray ionization mass spectrometry to characterize the primary sequence and disulfide bond content of recombinant calcium binding proteins, Biomedical Chromatography 13 (1999)37-45. 

Weaver, R., Graham, K.S., Beattie, I.G. and Riley, R.J. (2003) Cytochrome P450 inhibition using recombinant proteins and mass spectrometry/multiple reaction monitoring technology in a... 

Monegier, B., Clerc, F.F., Van Dorsselaer, A., Vuihorgne, M., Green, B., Cartwright, T. (1991). Using mass spectrometry to characterize recombinant proteins. Part II. Pharm. Tech-nol., 15(4), 28 10. 

A. Tsarbopoulos, M. Karas, K. Strupat, B. N. Pramanlk, T. L. Nagabhushan, and F. Hillen-kamp, Comparative mapping of recombinant proteins and glycoproteins by plasma desorption and matrix-assisted laser desorption/ionization mass-spectrometry, Anal. Chem., 66 (1994) 2062-2070. 

Fenn JB, Mann M, Meng CK Electrospray ionization for mass spectrometry of large biomolecules. Science (1989) 246 64-71. Patrick JS, Lagu AL Review applications of capillary electrophoresis to the analysis of biotechnology-derived therapeutic proteins. Electrophoresis (2001) 22 4179-4196. Sowell J, Salon J, Strekowski L, et al Covalent and noncovalent labeling schemes for near-infrared dyes in capillary electrophoresis protein applications. Methods Mol. Biol. (2004) 276 39-75. Moini M Capillary electrophoresis mass spectrometry and its application to the analysis ofbiological mixtures. Anal. Bio-anal. Chem. (2002) 373 466 180. Nemunaitis J, Holmlund JT, Kraynak M, et al. Phase I evaluation of ISIS 3521, an antisense oligodeoxynucleotide to protein kinase C-a, in patients with advanced cancer./. Clin. Oncol. (1999) 17 3586-3595. De Frutos M, Cifuentes A, Diez-Masa JC Differences in capillary electrophoresis profiles of urinary and recombinant erythropoietin. Electrophoresis (2003) 24 678-680. 

Coupling CE with electrospray ionization mass spectrometry (MS) can potentially be a very powerful tool for detecting and identifying product-related impurities in recombinant pharmaceuticals. Proteins can be detected in the femtomole range with this mode. Conceivably, the bioanalyst could perform a peptide map with CZE-MS and detect, identify, and sequence aberrant peptides derived from degradates. 

When working with purified enzymes, it can be useful to perform a close examination of their phosphorylation states and molecular masses. Mass spectrometry is often useful for this purpose. Post-translational modifications or sequence truncations can potentially alter the compound binding sites available and can also change the structure of potential inhibitory sites. For example, with protein kinases, phosphorylations distal from the ATP binding site can inactivate the kinase whereas phosphorylations near the ATP binding site can activate the catalytic activity. Often, practice does not permit control of such situations because the purified systems are often mixtures and cannot be controlled in the commonly used recombinant expression technologies. 

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