Peptide separations methods

It should be obvious from the above that membrane proteomics strictly follows Murphy s law. This is due to the fact that there is a mutual exclusion, for physicochemical reasons, between on the one hand the conditions that must be used to solubilize in water all membrane proteins, including the most hydrophobic ones, and thus give a fair representation of the protein population in the sample, and on the other hand the conditions prevailing in the high resolution peptide separation methods. On top of this problem, there is a second problem linked to the membrane versus aqueous phase volume in many subcellular preparations, which makes transmembrane proteins rare compared to water-soluble proteins. 

Tang HY, Ali-Khan N, Echan LA et al. A novel four-dimensional strategy combining protein and peptide separation methods enables detection of low-abundance proteins in human plasma and serum proteomes. Proteomics 2005 5(13) 3329-42. 

Liquid chromatography is a separation method that is often applied to nonvolatile, thermally labile materials such as peptides, and, if their mass spectra are required after the separation step, then a mild method of ionization is needed. Since FAB/LSIMS is mild and works with a liquid matrix, it is not surprising that attempts were made to utilize this ionization source as both an inlet... 

Polypeptide Synthesis and Analysis. Sihca or controUed-pore glass supports treated with (chloromethyl)phenylethyltrimethoxysilane [68128-25-6] or its derivatives are replacing chloromethylated styrene—divinylbenzene (Merrifield resin) as supports in polypeptide synthesis. The sdylated support reacts with the triethyl ammonium salt of a protected amino acid. Once the initial amino acid residue has been coupled to the support, a variety of peptide synthesis methods can be used (34). At the completion of synthesis, the anchored peptide is separated from the support with hydrogen bromide in acetic acid (see Protein engineering Proteins). 

Separation methods based on size include size exclusion chromatography, ultra-filtration, and ultracentrifugation (see Chapter Appendix). The ionic properties of peptides and proteins are determined principally by their complement of amino acid side chains. Furthermore, the ionization of these groups is pH-dependent. 

Capillary zone electrophoresis, an up-to-date high resolution separation method useful for proteins and peptides, has been shown to be a useful method for determining electrophoretic mobilities and diffusion coefficients of proteins [3], Diffusion coefficients can be measured from peak widths of analyte bands. The validity of the method was demonstrated by measuring the diffusion coefficients for dansylated amino acids and myoglobin. 

TWO-DIMENSIONAL HPLC-CE METHODS FOR PROTEIN/PEPTIDE SEPARATION... 

For reproducible expression analysis and protein quantification MS methods based on isotopic labeling are available. They were designed in conjunction with two or more dimensional chromatographic peptide separation coupled online to MS and require advanced bioinformatics input to analyze the complex data sets in a reasonable time frame. This is also true for the alternative fluorescence-based technology of differential gel electrophoresis (DIGE Fig. 10.6) with tailor-made software which allows statistical validation of multiple data sets. 

The development of chiral separation methods for dipeptides is of relevance for purity controls, for checking racemization processes in peptide syntheses, and for the investigation of peptide and protein hydrolysates. Since their introduction as chiral... 

As already mentioned the EP wants to replace old TEC tests with separation methods of higher efficiency for example, the purity of amino acids is currently evaluated by a TEC test for ninhydrin-positive substances that is only able to find and limit amino acids to 0.5%. However, this test is only valid in the case the amino acids are produced by the cleavage of peptides/proteins and purification. The ninhydrin method is also used in the amino acid analysis of peptides, utilizing a cation-exchange chromatography with a post-column derivatization and a subsequent UVA is detection. This method is often used in industries for purity evaluation of amino acids. 

Several different analytical and ultra-micropreparative CEC approaches have been described for such peptide separations. For example, open tubular (OT-CEC) methods have been used 290-294 with etched fused silicas to increase the surface area with diols or octadecyl chains then bonded to the surface.1 With such OT-CEC systems, the peptide-ligand interactions of, for example, angiotensin I-III increased with increasing hydrophobicity of the bonded phase on the capillary wall. Porous layer open tubular (PLOT) capillaries coated with anionic polymers 295 or poly(aspartic acid) 296 have also been employed 297 to separate basic peptides on the inner wall of fused silica capillaries of 20 pm i.d. When the same eluent conditions were employed, superior performance was observed for these PLOT capillaries compared to the corresponding capillary zone electrophoresis (HP-CZE) separation. Peptide mixtures can be analyzed 298-300 with OT-CEC systems based on octyl-bonded fused silica capillaries that have been coated with (3-aminopropyl)trimethoxysilane (APS), as well as with pressurized CEC (pCEC) packed with particles of similar surface chemistry, to decrease the electrostatic interactions between the solute and the surface, coupled to a mass spectrometer (MS). In the pressurized flow version of electrochromatography, a pLC pump is also employed (Figure 26) to facilitate liquid flow, reduce bubble formation, and to fine-tune the selectivity of the separation of the peptide mixture. 

The chemistry of this reaction has been extensively studied by Use and Edman. Although the reaction velocity is different for various amino acids, the reaction is complete in 1 N HCl at 80 °C within 10 min, with virtually all amino acids. This treatment, however, would cause partial hydrolysis of the remaining peptide. The method was made amenable to automation when Edman suggested to separate the thiazolinone derivative from the remaining peptide by extraction of the liberated thiazolinone derivative into butyl chloride and to perform the conversion into the PTH-amino acid outside the reaction vessel. 

The acid-base properties, and hence ionic character, of peptides and proteins also can be used to achieve separations. Ion-exchange chromatography, similar to that described for amino acids (Section 25-4C), is an important separation method. Another method based on acid-base character and molecular size depends on differential rates of migration of the ionized forms of a protein in an electric field (electrophoresis). Proteins, like amino acids, have isoelectric points, which are the pH values at which the molecules have no net charge. At all other pH values there will be some degree of net ionic charge. Because different proteins have different ionic properties, they frequently can be separated by electrophoresis in buffered solutions. Another method, which is used for the separation and purification of enzymes, is affinity chromatography, which was described briefly in Section 9-2B. 

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