Protein and Peptide Separations

The development of techniques and methods for protein purification is very important for bioscience and biotechnology. Various chromatographic techniques with different selectivities are powerful methods for the purification of biomolecules, especially the separation of proteins. 

New developments in high-resolution liquid chromatography made this method compatible with mass spectrometry and an indispensable tool in proteomic research. 

In general, a chromatographic separation depends on the differential partition of proteins between the stationary phase (the chromatographic medium or the adsorbent) and the mobile phase (the buffer or organic solvent). Several liquid chromatographic methods are used for the separation of proteins. They differ mainly in the type of stationary phase employed (Table 7.1). 

All these bulk materials are designed to fulfill a general matrix requirement for protein chromatography—to minimize the interaction between the sample and the surface of the support. 

A combination of optimal chemical and physical properties is achieved by the use of hydroxyl or amino groups, and such standard chromatographic media, based on neutral polysaccharides and modified polyamides, are widely used for protein separations [2]. 

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Two-dimensional SECxRP can be also used for protein and peptide separation, and this approach has been used for separation of peptides in tryptic digests of ovalbumin and serum albumin [14],... 

Analytical Properties Similar separations as obtained using C3 bonded silica, with a much larger pH stability range than silica-based phases useful for protein and peptide separations using TFA (trifluoroacetic acid) as a mobile phase modifier less mechanical stability than silica-based phases Reference 19... 

Capillary zone electrophoresis has been used for the separation of a wide variety of analytes, ranging from inorganic ions8-10 to proteins,11 peptides,1213 and nucleotides.14 Protein and peptide separations are by far the most common application areas, and CE is able to provide information complementary to that obtained by HPLC. 

Short-chain ion-pairing reagents, e.g., hexane sulfonic acid, have been used in HPLC for protein and peptide separations. This reagent can also be used in CE for hydrophobic peptides that are difficult to separate. 

There are exceptions to this, so do not rule out the separation of zwitter-ionic biomolecules as cations. Some proteins and peptides separate well at low pH on bare silica or coated capillaries. 

While a wide range of opportunities exist, such as environmental, clinical, and trace analysis, the principal application for labs-on-a-chip is in the analysis of biological samples. The miniaturized dimensions allow extremely small sample volumes to be analyzed, and a microchip format can allow chemical reaction, mixing, sample manipulation, and multiplexing to be performed. Single-cell analysis, immunoassays, protein and peptide separations, DNA analysis and sequencing, and polymerase chain reactions have all been performed on microchip devices [48]. 

Alternative buffer systems include zwitterions and dual-buffering reagents. Zwitterionic buffers such as bicine, tricine, CAPS, MES, and Tris may be useful for protein and peptide separations. An advantage of a zwitterionic buffer is low conductivity when the buffer pH is adjusted to its p7. There is little buffer capacity when the and p7 are separated by more than 2 pH... 

Capillary isoelectric focusing (CIEF) is a high-resolution technique for protein and peptide separation performed at academic sites and in the biotechnology and pharmaceutical industries for the analysis and characterization of, for example, recombinant antibodies and other recombinant proteins, isoforms of glycoproteins, point mutations in hemoglobin, and peptide mapping. Also, hyphenation to mass spectrometry and chip-based CIEF (microfabrication) have shown promise. CIEF kits and specific recipes/application notes are available from vendors of capillary electrophoresis (CE) equipment, as are a vast amount of publications and handbooks of CE published over recent years. 

In addition to reducing the adsorption, the dynamic additives also play important roles in selectivity enhancement. pH is an important option for protein and peptide separation. An increase in the buffer ionic strength can increase the resolution. Any ion that displays a preferential affinity with the peptides has a potential modifying effect on the selectivity. The addition of organic modifiers, e.g., methanol, ethanol, and acetonitrile, in the BGE, can induce different solvation of the peptide chains and modify the migration order and selectivity of peptides. For separation of very hydrophobic proteins, e.g., lipoproteins, surfactants can act as buffer additives to improve solubilization. 

Formic or acetic acid concentrations of 0.1—1% (v/v) are recommended when preparing low pH mobile phases to enhance ionization in electrospray. Trifluoroacetic acid is preferred for protein and peptide separations but should be avoided when negative ion mode is utilized. Ammonium hydroxide, or, in rare cases, triethylamine, are recommended for high pH mobile phases. 

Several publications have demonstrated effective separations of proteins under high salt conditions [19-22]. McCormick first demonstrated that by using phosphate buffers, a phosphosihcate surface would form [19]. This biocompatible surface is produced through a simple preconditioning step of the silica capillary with phosphate buffer [22]. The conditioned caphlary, in conjunction with various neutral salt additives can have an impact on selectivity and resolution for protein and peptide separations (Figure 3). 

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. 

As described in Section 6.2.5, a family of ion exchangers can be obtained from polysuccinimide silica poly(2-sulfoethyl aspartamide) is an aliphatic strong cation exchanger, and polyaspartic acid is a weak cation exchanger. The primary use of this family of products is in the area of protein and peptide separations. These packings exhibit a superior column efiiciency compared to th coimterparts based on organic polymers. 

Pesek, J., et al.. Protein and peptide separations on high surface area capillaries. Electrophoresis, 20, 2343, 1999. 

Multidimensional Microfluidic Systems for Protein and Peptide Separations... 

Affmity separation, advantages for protein and peptide separation from biological broths and blood, 297 Aging, proteins, 247-248 Alkylene oxalate polymers, application, 202 Amine-derivatized poly(ethylene terephthal-ate), preparation, 139,141/... 

Controlling the quality of milk and its derivatives is a very demanding field with a great need for development of more economical, time-saving, and accurate methods of ensuring product quality. This entry presents various reversed-phase (RP) high-performance liquid chromatography, HPLC/UV methods for analysis of miUt proteins, and describes how these techniques are used in the study of bovine, ovine, and human native milk proteins, casein fractions, cheese proteolysis evaluation, detection of product adulteration, enzyme hydrolysis of whey proteins, and peptide separation. 

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