Chromatographic separation proteins

The preliminary precipitation of proteins from milk is realized through the addition of solutions of acetic acid (1,7 mol/1) and sodium acetate (lmol/1) at t = 40-45°C before chromatographic isolation of OxTC. The precipitated proteins are separated by filtration. OxTC is detenuined in filtrate after its isolation on chromatographic column. Contents of OxTC was determined on calibration curve which is linear within concentration range 0,01-1,0 p.g/ml. 

In theory, SEC of proteins depends only on their molecular size. Sometimes the size of a protein varies with the ionic strength of the buffer (5,6). The concentration of salt not only affects the conformation of the protein, but can also influence the chromatographic separation itself. Additional retention... 

Fig. 30. Chromatographic separation of 1) acid and 2) neutral proteases (Bac. Subtilis) on Biocarb-T biosorbent. A. proteolytic activity units/ml —o— B. protein concentration (E28o) — —...

Kaufmann, M., Unstable proteins how to subject them to chromatographic separations for purification procedures. J. Chromatogr. B 699, 347 (1997). 

The effect of the buffer on the efficiency of electrospray ionization was mentioned earlier in Section 4.7.1. This is a good example of the dramatic effect that this may have - good chromatographic separation and ionization efficiency with formic, acetic and propionic acids, and good separation, although with complete suppression of ionization, with trifluoroacetic acid (TFA), the additive used for the protein application described previously. Post-column addition of propionic acid to the mobile phase containing TFA has been shown to reduce, or even... 

The molecular mass of the protein was redetermined by infusing a 5-10 pmolp.l solution of the protein in 50% aqueous acetonitrile containing 0.2% formic acid at a flow rate of 6 p,lmin into an electrospray source. The scan rate employed on the mass spectrometer was from m/z 60 to m/z 1800 in 12 s. This is a relatively slow scan speed which will lead to a more precise molecular weight determination. Scan speeds of this order may be, and indeed should be, utilized for infusion experiments if sufficient sample is available but it is unlikely to be feasible when chromatographic separations, particularly those involving capillary columns, are employed because of the restriction imposed by the chromatographic peak width (see Section 3.5.2.1 above). 

MALDI-ToF is a technique that allows the molecular weights of proteins and peptides to be determined. It is less susceptible to suppression effects than electrospray ionization and thus is able to be used for the direct analysis of mixtures. In the case of a crude tryptic digest, MALDI-ToF will provide a molecular weight profile of the polypeptides present without the analysis time being extended by the need to use some form of chromatographic separation. 

Column chromatographic separations depend on the relative affinity of different proteins for a given stationary phase and for the mobile phase. Association between each protein and the matrix is weak and transient. Proteins that interact more strongly with the stationary phase are retained longer. The length of time that a protein is associated with the stationary phase is a function of the composition of both the stationary and mobile phases. Optimal separation of the protein of interest from other proteins thus can be achieved by careful manipulation of the composition of the two phases. 

Chromatographic Development The basic concepts of chromatographic separations are described elsewhere in this handbook. Proteins differ from small solutes in that the large number of charged and/or hydrophobic residues on the protein surface provide multiple... 

Xu, W. and Regnier, EE., /. Chromatogr. A, Protein-protein interactions on weak-cation-exchange sorbent surfaces during chromatographic separations, /. Chromatogr. A, 828, 357, 1998. 

Cohen, K. A., Schellenberg, K., Benedek, K., Karger, B. L., Grego, B., and Hearn, M. T. W., Mobile-phase and temperature effects in the reversed phase chromatographic separation of proteins, Anal. Biochem., 140, 223, 1984. 

As noted above, whole-cell MALDI-TOF MS was intended for rapid taxonomic identification of bacteria. Neither the analysis of specific targeted bacterial proteins, nor the discovery of new proteins, was envisioned as a routine application for which whole cells would be used. An unknown or target protein might not have the abundance or proton affinity to facilitate its detection from such a complex mixture containing literally thousands of other proteins. Thus, for many applications, the analysis of proteins from chromatographically separated fractions remains a more productive approach. From a historical perspective, whole-cell MALDI is a logical extension of MALDI analysis of isolated cellular proteins. After all, purified proteins can be obtained from bacteria after different levels of purification. Differences in method often reflect how much purification is done prior to analysis. With whole-cell MALDI the answer is literally none. Some methods attempt to combine the benefits of the rapid whole cell approach with a minimal level of sample preparation, often based on the analysis of crude fractions rather... 

Methanol, isopropanol, and 50 mM hexafluoroisopropanol were also evaluated for use as organic modifiers in the mobile phase. Little improvement to the solubility of whole proteins or their chromatographic separation was observed, however. 

Chloupek, R.C., Hancock, W.S., Marchylo, B.A., Kirkland, J.J., Boyes, B.E., Snyder, L.R. (1994). Temperature as a variable in reversed-phase high-performance liquid chromatographic separations of peptide and protein samples, n. Selectivity effects observed in the separation of several peptide and protein mixtures. J. Chromatogr. A 686, 45-59. 

Remove excess reagent and reaction by-products by dialysis or gel filtration using 0.1M sodium phosphate, 0.15M NaCl, lOmM EDTA, pH 7.5. For chromatographic separation, use a desalting gel filtration support such as the Zeba desalting spin columns (Thermo Fisher) or the equivalent. The SAMSA-modified protein may be stored at -20°C until needed. 

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