Proteomics noncovalent interactions

Field Proteomics, noncovalent interaction, structural determination, PTM. Technique Electrospray for sequencing, post-translational modification location, and noncovalent interactions. 

Reference E. Damoc, C. S. Eraser, M. Zhou, H. Videler, G. L. Mayeur, J. W. B. Hershey, J. A. Doudna, C. V. Robinson, and J. A. Leary, Structural characterization of the human eukaryotic initiation factor complex by mass spectrometry, Mol. Cell. Proteomics 6 1135-1146 (2007). 

Background The assanbly of the ribosomal complex depends on the presence of proteins called enkaryotic initiation factors (elEs). In mammals the largest of these is eIF3, an -800 kDa noncovalent complex comprising of 13 stoichiometric nonidentical subunits ranging in mass from 25 to 167 kDa. This paper describes the isolation and characterization of human eIE3 obtained from HeLa cells (an inunortal cell line derived from cervical cancer cells from Henrietta Lacks in 1951). 

MS in linear trap neutral losses of 98/z show presence of phosphates 

FIGURE 4.9 Analysis of eIF3. Component proteins (left) and intact noncovalent complex (right). 

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New ionization methods and mass analyzers have extended the applicability and overall sensitivity of to mass spectrometry to macromolecular targets. Mass spectrometry can be used to directly study the covalent and noncovalent interactions of drug molecules and biomolecule targets. Inhibitors that bind irreversibly or covalently to a target are easily studied by use of MS because the covalent complex survives intact in the gas phase. MS is combined with other proteomic technologies as well for use in drug discovery e.g., matrix-assisted laser desorption ionization MS (MALDI-MS) and electrospray ionization (ESI). 

Protein expression encompasses an enormous dynamic range. Since rare proteins cannot be amplified by any type of PCR method, sensitive detection is critical to proteome projects. Fluorescence methods deliver streamlined detection protocols, superior detection sensitivity, broad linear dynamic range and excellent compatibility with modem microchemical identification methods such as mass spectrometry. Two general approaches to fluorescence detection of proteins are the covalent derivatization of proteins with fluorophores or noncovalent interaction of fluorophores through direct electrostatic interaction with proteins. One approach for quantifying fluorescence is to use a photomultiplier tube detector combined with a laser... 

Soft ionization methods in mass spectrometry (MS)—in particular, electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI)—have had an enormous impact in the field of proteomics and simultaneously spurred the industrial development of excellent instrumentation that is robust and designed to be operated by nonspecialists. ESI and MALDI also allow, under appropriate conditions, the observation of noncovalently bound complexes in the gas phase. Recent research suggests that bio-macromolecules taking part in such noncovalent interactions are still in their folded conformation in the gas phase, close to their native conformation in solution. This surprising hypothesis has not yet... 

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