Isoelectric focusing variants

Having found genetic variation for AB structure in sibling species, we screened natural populations of IN melanogaster to identify isoelectric focusing variants within the species. So far we have identified four genotypes with variants of AB structure (Fig. 

Manning JM et aJ Normal and abnormal protein subunit interactions in hemoglobins.] Biol Chem 1998 273 19359-Mario N, Baudin B, Giboudeau J Qualitative and quantitative analysis of hemoglobin variants by capillary isoelectric focusing. J Chromatogr B Biomed Sci Appl 1998 706 123-Reed W, Vichinsky EP New considerations in the treatment of sickle cell disease. Annu Rev Med 1998 49 46l. 

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. 

Zone electrophoresis is influenced by adsorption and capillarity, as well as by electroosmosis. Therefore evaluation of mobility (and f) from this type of measurement is considerably more complex than from either microelectrophoresis or moving-boundary electrophoresis. Nevertheless, zone electrophoresis is an important technique that is widely used in biochemistry and clinical chemistry. One particularly important area of application is the field of immunoelectrophoresis, which is described briefly in Section 12.11. Additional information on zone electrophoresis may be obtained from Probstein (1994) and Hunter (1981) and the references given there. Variants of zone electrophoresis also exist see, for example, Gordon et al. (1988) for information on a variant known as capillary zone electrophoresis and Righetti (1983) for information on what is known as isoelectric focusing. 

Capillary electrophoresis (CE) coupled to MS has the advantage of high resolution and soft ionization for biomolecules, which may be used to differentiate post-translational modifications and variants of intact proteins and oligonucleotides. Different modes of CE (capillary zone electrophoresis, capillary isoelectric focusing, capillary electrochromatography, micellar electrokinetic chromatography, nonaqueous capillary electrophoresis) to MS as well as online preconcentration techniques (transient capillary isotachophoresis, solid-phase extraction, membrane preconcentration) are used to compensate for the restricted detection sensitivity of the CE methodology [77, 78]. 

STEADY-STATE VARIANTS OF Sc METHODS ISOELECTRIC FOCUSING AND ISOPYCNIC SEDIMENTATION... 

When transversely oriented focusing forces of the type used in isoelectric focusing or isopycnic sedimentation are used to confine different components to different thin bands or laminae in an FFF channel, we have a variant of FFF called hyperlayer FFF. Suppose component A is focused at y = 15.2 /xm and B at y = 127 /xm (0.005 in). You may assume that the thin bands form a 6-function distribution in coordinate y and that channel thickness w = 254 /xm. What is the R value for each of the two components ... 

Connors, L. H., Ericsson, T., Skare, J., Jones, L. A., Lewis, W. D., and Skinner, M. (1998). A simple screening test for variant transthyretins associated with Familial transthyretin amyloidosis using isoelectric focusing. Biochim. Biophys. Acta 1407, 185-192. 

Capillary isoelectric focusing coupled to mass spectrometry has gained popularity in recent years (by analogy to two-dimensional gel electrophoresis). Additional information obtained from mass spectrometry includes not only precise molecular-weight determination but also the possiblity for peptide sequencing. Analysis of hemoglobin variants, recombinant proteins, and monoclonal antibodies have been demonstrated [1,7,8]. 

Hempe JM, Vargas A, Graver RD. Clinical analysis of structural hemoglobin variants and Hb Ale by capillary isoelectric focusing. In Petersen JR, Mohammad AA, eds. Clinical and forensic applications of capillary electrophoresis. Totowa, NJ Humana Press, 2001 145-63. 

Figure 20-3 Diagrammatic representation of the common variants of tt -antitrypsin, as demonstrated by isoelectric focusing.The five major bands are shown for Mi, but only the 2 and 4 bands for the other variants (with dots to the left of each).Variants F,S,V,and Z are shown in combination with Ml for clarity s sake. If cysteine is added to the samples before focusing, the F-2 and F-4 bands shift to the locations of the faint bands.

CEA is a glycoprotein with a molecular mass of 150 to 300 kDa it contains 45% to 55% carbohydrate. It is a single polypeptide cham consisting of 641 amino acids, with lysine in the N-terminal position. The heterogeneity of CEA can be demonstrated by using isoelectric focusing to separate the variants. 

Figure 31-11 A diagrarn of isoelectric focusing patterns for a variety of Hb variants.The conditions shown represent heterozygotes (traits) unless otherwise indicated.The width of the bars approximates the relative density of the bands observed.The acid anodic (pH 6) side is to the right, and the alkaline cathodic (pH 8) side is to the left.The same pattern Is observed in homozygous patients with Hb S disease who have received Hb A by transfusion.

Q. Tang, A.K. Harrata and C.S. Lee, High-resolution capillary isoelectric focusing-electrospray ionization mass spectrometry for hemoglobin variants analysis. Analytical Chemistry, 68, 2482-2487 (1996). 

At the present time, it seems that the two-dimensional technique of Harris et al. (H6) has been used to identify more electrophoretic variants than has any other method, and is capable of greater resolution than are the other methods that have been employed. Using this method on all the new electrophoretic variants that have been described may make it possible to determine whether they are in fact all different from one another. Probably the best resolution currently available is isoelectric focusing in a gel medium in the first direction, followed by electrophoresis in a polyacrylamide gradient gel in the second direction (A15, W36). There... 

---