Protein/peptide analysis affinity chromatography

Collins, M.O. and Yu, L. et al. (2005b) Robust enrichment of phosphorylated species in complex mixtures by sequential protein and peptide metal-affinity chromatography and analysis by tandem mass spectrometry. Sci. STKE 2005,16. 

Other methods that are related to affinity chromatography include hydrophobic interaction chromatography and thiophilic adsorption. The former is based on the interactions of proteins, peptides, and nucleic acids with short nonpolar chains on a support. This was first described in 1972 [113,114] following work that examined the role of spacer arms on the nonspecific adsorption of affinity columns [114]. Thiophilic adsorption, also known as covalent or chemisorption chromatography, makes use of immobilized thiol groups for solute retention [115]. Applications of this method include the analysis of sulfhydryl-containing peptides or proteins and mercurated polynucleotides [116]. 

A comprehensive proteomics approach has been developed to identify the components of phosphoprotein complexes and includes the following experimental steps isolation of native phosphoproteins and associated proteins by affinity chromatography, ID PAGE separation of the affinity-isolated proteins, proteolysis of the protein spots, LC-ESI-MS/MS analysis of the cleaved peptides, and database searching for indentification of the proteins involved in the complex formation.178... 

Proteins and Amino Acids Total protein in food and feed samples is commonly determined by Kjeldahl (acid digestion/titration) or Dumas (pyrolysis) or elemental analysis.14 FIPLC can separate major proteins and furnish protein profiles and speciation information. HPLC can be used to further characterize specific proteins via peptide mapping and amino acid sequence analysis. HPLC modes used for protein include IEC, SEC, RPC, and affinity chromatography with typical UV detection at 215 nm or MS analysis. Details on protein separations are discussed in the life sciences section. 

Bernhard, O.K., Burgess, J.A., Hochgrebe, T., Shell, M.M., and Cunningham, A.L., 2003, Mass spectrometry analysis of CD4-associating proteins using affinity chromatography and affinity tag-mediated purification of tryptic peptides. Proteomics. 3 139-146. 

Detection of phosphorylated proteins and peptides can be enhanced by selectively isolating these species. Online immobilized metal affinity chromatography (IMAC)-CE-ESI-MS is such a powerful analytical tool. The IMAC resin retains and preconcentrates phosphorylated proteins and peptides, CE separates the phosphorylated species and MS/MS identifies the components and their phosphorylation sites. Cao and Stults applied this method to the analysis of phosphorylated angiotensin II and tryptic digests of a- and /3-casein (CE conditions buffer, 0.1% acetic acid/10% methanol uncoated capillary). Beta-casein is a well-characterized protein with five phosphorylation sites and is widely used as a standard for protein phosphorylation studies. 

Chemical proteomics consists of the classical drug-affinity chromatography and modern high-resolution MS analysis for protein identification [3, 11]. The procedure typically involves immobilization of the compound of interest to a solid support through a spacer arm, and the affinity matrix is then used to purify specific interacting proteins from cellular lysate. The complex proteomic mixture is then proteolytically digested, and the resulting peptides are sequenced... 

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