Examples of Protein Separations

Recoveries of Protein Samples after Separation by Reversed-Phase HPLC 

Protein sample Re- covery (%) Sepa- ration condi- tions Protein sample Recovery (%) Sepa- ration condi- tions 

This material was developed by the National Research and Demonstration Center, National Heart, Lung and Blood Institute, National Institutes of Health, Grant. No. HL-17269. Support is also acknowledged from The National Heart Foundation and Medical Research Council of New Zealand (WSH). JTS is an Established Investigator of The American Heart Association. 

Gratzer, in Poly-a-amino acids. Biological Macromolecules Series (G. D. Fasman, ed.), pp. 177-238. Dekker, New York, 1967. 

Hancock and J. T. Sparrow, in A Laboratory Manual for the Separation of Biological Materials by HPLC. Dekker, New York (in preparation). 

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One of the most intriguing recent examples of disordered structure is in tomato bushy stunt virus (Harrison et ah, 1978), where at least 33 N-terminal residues from subunit types A and B, and probably an additional 50 or 60 N-terminal residues from all three subunit types (as judged from the molecular weight), project into the central cavity of the virus particle and are completely invisible in the electron density map, as is the RNA inside. Neutron scattering (Chauvin et ah, 1978) shows an inner shell of protein separated from the main coat by a 30-A shell containing mainly RNA. The most likely presumption is that the N-terminal arms interact with the RNA, probably in a quite definite local conformation, but that they are flexibly hinged and can take up many different orientations relative to the 180 subunits forming the outer shell of the virus particle. The disorder of the arms is a necessary condition for their specific interaction with the RNA, which cannot pack with the icosahedral symmetry of the protein coat subunits. 

Fig. 2.1.11 HPLC example of lust separation of LCM proteins. (From Ref. 50.)...

Figure 15.6 Schematic depiction of one example of protein identification by mass spectrometry. Genes of interest are tagged, then transfected into mammalian cells, and proteins associated with the cognate tagged protein are purified by affinity methods. Separation of the complex is carried out by 2D SDS-PAGE. Identification of the proteins is by MALDI and ESI (Blackwell, 1999).

The floating injection (with no push-back voltage) was successful without sample leakage in some examples of the separations of DNA [554,925] and proteins [555], possibly because of slow molecular diffusion in the viscous gel separation media. 

Each cycle creates additional chemical noise. In TLC or GC, this noise build-up creates a substantial problem. In LC, however, the very polar, noise-causing compounds elute before the PTH-amino acids and, therefore, do not interfere with the analysis. The sequencing of a protein or peptide can be done manually or on a commercial device called a sequencer. The HPLC is the analysis tool which aids in the identification of the amino acid by matching the retention time of the unknown to a known, specific amino acid. An example of the separation and identification of the output of a sequencer is shown in Figure 2-11. 

For many years salting-out by high concentrations of ammoniiun sulfate has been one of the classical methods of protein separation. There is very little literature on the theoretical basis of the method, particularly as applied to the isolation of enzymes, where it has mainly been used quite empirically. The underlying assumption in most cases seems to have been that the different proteins are precipitated at different fixed ammonium sulfate concentrations, provided the pH and temperature are fixed. For example one may commonly read in instructions for the piuification of an enzyme that the enzyme is precipitated at 65% saturation with ammonium sulfate or that the fraction precipitating between 0.62 and 0.68 saturation should be taken. It is, however, a fairly common experience that when one repeats a published method the enzyme fails to precipitate within the limits given. Furthermore, where the purification of a protein involves more than one salt-fractionation stage, the limits are usually found to be different for the different stages. 

Chiral columns are packed with stereo-specific sorbents for the separation of stereoisomers in the sample.27 Many chiral columns operate in hydrophilic interaction mode (e.g., Pirkle-type) whereas others are used in reversed-phase mode (proteins, macrocyclic antibiotics, polysaccharides, etc.). The use of these columns is critical in the development of chiral drugs. Most chiral columns are quite expensive and many older chiral columns have low efficiencies and limited lifetimes. Examples of chiral separations are shown in Chapter 6. Alternately, convention columns can also be used for chiral separations using a chiral selector in the mobile phase.27... 

This section details characteristic examples of protein analysis by CZE, CSE, CIEF, and MEKC. The selected examples were chosen to demonstrate some of the more important theoretical aspects of protein separation by each of these techniques while also highlighting the application of CE for characterization of protein properties, isoform identification, use in mobility shift assays, and multidimensional protein analysis. Table 2.1 lists recent references for specific proteins analyzed by different CE methods and show how CE analysis of proteins can be used in a variety of biological applications. 

Several pure proteins have been purified and checked for their purity, microheterogeneity, or diagnostic significance by CE. In this case, coated capillaries are preferred for this separation. Many of the proteins can be analyzed by CE however, sensitive detectors such as fluorescence are necessary [68-70]. Examples of proteins studied by CE separately or in a profile are transferrin isoforms, which are important as markers of alcoholism [71], a-1 antitrypsin [72], recombinant human erythropoietin glycoforms that stimulates erythopiosis [73], plasminogen tissue activator [74], prions [75], urothelial carcinoma proteins in urine [76,77], and numerous urinary proteins [78]. 

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