Electrophoresis isoforms

Caldini, A., Moneti, G., Fanelli, A., et al. (2003) Epoetin alpha, epoetin beta and darbepoetin alfa Two-dimensional gel electrophoresis isoforms characterization and mass spectrometry analysis. Proteomics, 3,937-941. 

The principal molecular constituent of thin filaments is actin. Actin has been highly conserved during the course of evolution and is present in all eukaryotes, including single-celled organisms such as yeasts. Actin was first extracted and purified from skeletal muscle, where it forms the thin filaments of sarcomeres. It also is the main contractile protein of smooth muscle. Refined techniques for the detection of small amounts of actin (e.g., immunofluorescence microscopy, gel electrophoresis, and EM cytochemistry) subsequently confirmed the presence of actin in a great variety of nonmuscle cells. Muscle and nonmuscle actins are encoded by different genes and are isoforms. 

Middle panel Cell wall proteins were isolated, 10 pgm of each resolved by non-denaturing polyacrylamide gel electrophoresis and PGl and PG2 isoforms detected by activity staining. 

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. 

An interesting twist to the story is provided by studies on N. brasiliensis, which secretes three distinct isoforms of AChE, designated A, B and C (Ogilvie et al, 1973). These enzymes can be easily separated by nondenaturing electrophoresis due to their distinct pis, and this is illustrated in Fig. 11.1, which also shows the distinct electrophoretic properties of the amphiphilic enzyme (arrowed) found only in somatic extracts and therefore presumably associated with neuromuscular function. The overall amount of AChE produced by this parasite increases dramatically following establishment in the jejunum, and a switch in isoform expression occurs,... 

Molecular masses of the same enzymes of different species are different. Molecular mass of the laccase of Pleorotus ostreatus was found to be 66.8 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) [48]. Purified enzyme of T. versicolor having a single band with a molecular mass of 68 kDa was in the same range with the molecular weights of laccase isoforms isolated from 2,5-xylidine-induced cultures of T. versicolor [49]. 

Finally, work that may facilitate understanding the role of oq-acid glycoprotein variants in inter-individual variations in plasma protein binding, pharmacokinetic behavior, and drug action has been described. A capillary zone electrophoresis method that allows for the determination of 11 intact forms (i.e., isoforms, glycoforms) of oq-acid glycoprotein has been described [84],... 

Fig. 5. Thirty-four out of 35 potential apo(a) isoforms, separated by SDS-agarose gel electrophoresis. The photograph represents a composite of two separate gels with the reference mixture (St) and 17 different samples applied to each gel. Samples were selected to represent each of the observed isoforms. Twenty-nine samples had single-handed patterns and five samples had double-banded patterns. The double banded types and every fifth single-handed phenotype are indicated at the bottom of each gel lane. [With permission of Marcovina et al. (M12).]...

Electroimmunoassay (rocket electrophoresis) and radial immunodiffusion (A5) lack sensitivity at low Lp(a) concentrations, and the response is influenced by the size of the apo(a) isoforms (A5, K28). Differences in migration velocity in the agarose gel lead to an underestimation of the samples with large apo(a) isoforms and to an overestimation of samples with small apo(a) isoforms. Moreover, the detection limit lies around 0.07-0.08 g/liter Lp(a), so that this method is better suited for screening and detection of individuals with elevated Lp(a) levels than for the exact measurement of the plasma Lp(a) concentration. 

F5. Farrer, M., Game, F. L., Adams, P. C., Laker, M. F., and Alberti, K. G. M. M., A simple sensitive technique for classification of apolipoprotein(a) isoforms by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Clin. Chim. Acta 207, 215-225 (1992). 

L5. Lackner, C., Boerwinkle, E., Leffert, C. C., Rahming, T., and Hobbs, H. H., Molecular basis of apolipoprotein(a) isoform heterogeneity as revealed by pulse-field electrophoresis. J. Clin. Invest. 87, 2153-2161 (1991). 

Bristow, A., and Charton, E. (1999). Assessment of the suitability of a capillary zone electrophoresis method for determining isoform distribution of erythropoietin. Pharmeuropa 11(2), 290—300. 

Yeung, B., Porter, T. J., and Vath, J. E. (1997). Direct isoform analysis of high-mannose containing glycoproteins by on-line capillary electrophoresis electrospray mass spectrometry. Anal. Chem. 69, 2510-2516. 

Westbrook, J.A., Yan, J.X., Wait, R., Welson, S.Y., Dunn, M.J. (2001). Zooming-in on the proteome very narrow-range immobilised pH gradients reveal more protein species and isoforms. Electrophoresis, 22(14), 2865-2871. 

In vitro. The activity or absolute level of enzymes such as cytochrome P-450 and glucuronosyl transferase can be measured in cells, tissue fractions, or subcellular fractions (e.g., microsomes) and compared with those from control animals. The activity is measured by using a particular substrate for each of the isoforms of the enzyme (e.g., cytochrome P-450 or UDPGT) of interest. The total level of cytochrome P-450 could be determined by spectrophotometry using standard methods (e.g., carbon monoxide binding and difference spectra). Alternatively, the level of protein can be determined by gel electrophoresis and Western blotting, and this would allow the separation of different isoforms. 

J. H. Beattie, R. Self and M. P. Richards, The use of solid phase concentrators for online pre-concentration of metallothionein prior to isoform separation by capillary zone electrophoresis , Electrophoresis 16 322-328 (1995). 

It appears reasonable that because several types of CYP are associated with the endoplasmic reticulum, various inducers may induce one or more of them. Because each of these types has a relatively broad substrate specificity, differences may be caused by variations in the extent of induction of different cytochromes. Now that methods are available for gel electrophoresis of microsomes and identification of specific isoforms by immunoblotting and isoforms-specific antibodies, the complex array of inductive phenomena is being more logically explained in terms of specific isozymes. 

Zanusso, G., Righetti, P. G., Ferrari, S., Terrin, L., Farinazzo, A., Cardone, F., et al. (2002) Two-dimensional mapping of three phenotype-associated isoforms of the prion protein in sporadic Creutzfeldt-Jakob disease. Electrophoresis 23, 347-355. 

Li, M. X. Wu, J. T. Parus, S. Lubman, D. M. 1998. Development of a three-dimensional topographic map display for capillary electrophoresis/mass spectrometry with an ion trap/reflectron time-of-flight mass spectrometer detector applications to tryptic digests of isoforms of myelin basic protein. ./. Am. Soc. Mass Spectrom., 9,701-709. 

E Contiero, R Ferrari, GM Vaselli, M Folin. Apolipoprotein A1 isoforms in serum determined by isoelectric focusing and immunoblotting. Electrophoresis 18 122-126, 1997. 

Conformational isomers of OBPs that are detectable by gel electrophoresis have also been found in three species of scarab beetles (Deyu and Leal, 2002 Wojtasek et al., 1999). We have demonstrated by protein sequencing and mass spectrometry that these electrophoretically distinct bands are from proteins with the same primary sequences (Deyu and Leal, 2002 Wojtasek et al., 1999). Although the term isoform means different forms of a protein it has been... 

The quantification of specific sugars and total carbohydrates should be performed in each lot. In some cases, IEF and capillary electrophoresis can be alternative methods for determining the percentage of each isoform in a product sample lot. It is often important to determine the isoform profile for each lot prior to release. It would be ideal, but not always possible, to relate the isoform profile to the specific activity of the product. 

Methods to determine the potential biological activity of products obtained through recombinant DNA techniques are of fundamental importance. Despite the existence of numerous physicochemical techniques to characterize the protein product structure and the presence of contaminants, they provide little, if any, information about its biological potency. A bioassay is defined as a functional test, and no physicochemical test can measure the function. However, for some peptide hormones, which are less complex in structure than most cytokines, well defined physicochemical tests may be used to estimate biological activity for instance, the capillary electrophoresis analysis of a protein s isoform content if the specific activity of each one is known. 

Capillary electrophoresis of purified rhEPO (recombinant human erythropoietin) protein. From left to right isoform nos 2, 3, 4, 5, 6, 7, and 8 (according to European Pharmacopea). 

Petersen, A., K. Grobe, B. Lindner, M. Schlaak, and W. M. Becker. 1997. Comparison of natural and recombinant isoforms of grass pollen allergens. Electrophoresis 18 (5) 819—825. 

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