Protein modification analysis

C. Wa, R. Cemy, and D. S. Hage, Obtaining high sequence coverage in matrix-assisted laser desorption time-of-flight mass spectrometry for studies of protein modification Analysis of human serum albumin as a model, Anal. Biochem., 349 (2006) 229-241. 

The systematic characterization of secondary protein modifications is different from sequencing a protein. For a systematic analysis of protein modifications, the complete protein sequence should be covered by the investigation. This is difficult to achieve since the enzymatic digestion of a protein is not homogeneous. The sequence coverage depends on the nature of the protein, its quantity, and the enzyme used. Typical... 

The easiest way to detect a protein modification seems to be the mass measurement of all peptides generated by enzymatic digestion. The comparison with the predicted peptide masses from the sequence of the protein identifies unmodified peptides and unexplained masses would give indications to modified peptides. Unfortunately, this is not a suitable approach in practice. In many peptide mapping experiments done with the MALDI mass mapping technique, up to 30% of the measured masses remain unexplained. This is probably due to protein contaminations from human keratins, chemical modifications introduced by gel electrophoresis and the digestion procedure, and other proteins present at low levels in the piece excised from the sodium dodecyl sulfate gel. The detection of a protein modification requires a more specific analysis. 

The CFR 21 part 11 compliant software newly released by Convergent Bioscience makes imaged cIEF a viable technique for use in QC environments for release testing of therapeutic proteins or antibody molecules. i-cIEF has also been used heavily for characterization of protein modifications such as deamidation and isomerization, as well as oligosaccharide structure analysis. ... 

The use of MALDI-MS analysis alone to study in vivo protein modifications has several limitations, especially when it comes to identifying the specific amino acid... 

Characterization of Purified Proteins Assessing purity, 182, 555 determining size, molecular weight, and presence of subunits, 182, 566 amino acid analysis, 182, 587 limited N-terminal sequence analysis, 182, 602 peptide mapping, 182, 613 analysis for protein modifications and nonprotein cofactors, 182, 626 protein crystallization, 182, 646. 

Proteomics includes a variety of technologies that include differential protein display on gels, protein chips, quantitation of protein amoimts, analysis of post-translational modifications, characterization of protein complexes and networks and bioinformatics. All this information in combination with genome and phenotype studies will ultimately yield a comprehensive picture of a cellular or tissue proteome (Wasinger and Corthals 2002). 

It may be assumed that a protein mixture consists of protein molecules with different molecular weights, solubilities, and modifications. Analysis of the mixtures begins with separation of the proteins from each other and then, cleavage of these proteins to peptides by digestion as mass spectrometry cannot be performed using intact proteins. The resultant peptides are analyzed employing one of the following techniques... 

The diverse nature of proteins leads to the problem of applying a unique, single method in preparing samples for 2-DE analysis. The most important point to be taken into consideration should be minimizing protein modifications while preparing samples. The method with minimum modification should be chosen, otherwise artifactual spots may form on the gel and mislead the operator. 

McGregor, A., Murray, J.B., Adams, C.J., Stoddey, P.G. and Connolly, B. A. (1999) Secondary structure mapping of an RNA ligand that has high affinity for tire MeLT repressor protein and interference modification analysis of tlie protein-RNA complex, J. Biol. Chem. 274, 2255-2562. 

Steinberg RA, Coffino P. Two-dimensional gel analysis of cyclic AMP effects in cultured S49 mouse lymphoma cells protein modifications, inductions and repressions. Cell 1979 18 719-733. 

---