Protein separation and identification

Jun BH, Noh MS, Kim G, Kang H, Kim JH, Chung WJ, Kim MS, Kim YK, Cho MH, Jeong DH, Lee YS (2009) Protein separation and identification using magnetic beads encoded with surface-enhanced Raman spectroscopy. Anal Biochem 391 24—30... 

Guo, X., Chan-Park, M.B., Yoon, S.F. et al. (2006) UV embossed polymeric chip for protein separation and identification based on capillary isoelectric focusing and MALDl-TOF-MS. Anal Chem, 78 (10), 3249-3256. 

Li, J., Kelly, J.F., Chernushevich, I., Harrison, D.J., and Thibault P., Separation and identification of peptides from gel-isolated membrane proteins using a microfabricated device for combined capillary electrophoresis/nanoelectro-spray mass spectrometry, Anal. Chem. 72, 599, 2000. 

Concerning food peptides, most of the HPLC applications deal with food characterization (based on peptide profile), peptide separation, and identification [172,173] and detection of frauds in milk-based products, meat, and protein hydrolysates [115],... 

Proteome analysis in general involves two stages protein separation and subsequent identification and analysis. Multidimensional separations are required in order to result in an adequate resolution of complex protein or peptide... 

Because of the different interactions involved in partition and adsorption processes, they may be applied to different separation problems. Partition processes are the most effective for the separation of small molecules, especially those in homologous series. Partition chromatography has been widely used for the separation and identification of amino acids, carbohydrates, and fatty acids. Adsorption techniques, represented by ion-exchange chromatography, are most effective when applied to the separation of macromolecules including proteins and nucleic acids. 

However, during the past three decades, an analytical method has been developed that currently rivals and may soon surpass the traditional liquid chromatographic techniques in importance for analytical separations. This technique, high-performance liquid chromatography (HPLC), is ideally suited for the separation and identification of amino acids, carbohydrates, lipids, nucleic acids, proteins, pigments, steroids, pharmaceuticals, and many other biologically active molecules. 

Proteomics in parasitic flatworms can be completed on intracellular fractions (e.g. microsomal or cytosol) or at the host-interface on excretory-secretory (ES) products. ES analysis can be completed during in vitro culture or in vivo by, for example, bile or gut content analysis. In all cases, a rapid and careful preparation is vital to prevent altered pro-teomic profiles due to stress responses (upreg-ulation of heat shock proteins) and action of proteases. Parasitic flatworms are best extracted from fresh host material, washed with a buffered saline solution at approximately the host s body temperature. In F. hepatica, for example, this will allow regurgitation of gut contents to remove digested material from, and removal of host material adherent to the outer surfaces of the parasite (Jefferies et al., 2001), both of which can subsequently complicate separation and identification. 

Isolation, concentration, separation, and Identification of thermally-derived aromas 1s an Important area. These aromas are frequently associated with other non-volatile products Including pigments, fats, carbohydrates, and proteins. Techniques are required to separate the volatiles from the non-volatiles without causing chemical deterioration. 

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. 

All proteomic platforms generally involve separation and identification of proteins. Toxicoproteomics Proteomics applied to toxicology with the aims of identifying critical proteins and pathways affected by chemical and environmental exposures. 

Proteomic platforms combine separation and identification technologies that can be completed as an experiment in a timely fashion. Proteomic platforms strive to accomplish five critical objectives (1) a high level of information about one or more protein attributes (2) a large number of samples per analysis session (sample throughput) (3) quantitative and comparative protein expression among samples (4) timely analysis of raw and processed data and (5) use of a discovery-oriented or open platform system. 

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