> Post-Translational Modifications and Processing
- Mass Spectrometry of Proteins and Protein Fragments
- Protein/Peptide Chromatography
- Protein/Peptide Electrophoresis
- Protein/Peptide Isoelectric Focusing
The repertoire of services to assist in identifying and localizing post-translational modifications and to define processing events in vivo or in vitro include, but are not limited to the services for Protein Expression Analysis and Protein Identification. The procedures conducted in any particular project are chosen in discussion with investigators. Frequently, the approach involves several of the options listed below, as well as those described on the accompanying pages indicated above.
Mass Spectrometry of Intact Proteins and Protein Fragments
The mass of intact proteins can be measured with an accuracy of 10,000±2 Da. This ability provides a wealth of chemical information about protein chemical structure, including confirmation of amino acid sequence, assessment of the degree of microheterogeneity, detection and identification of chemical groups added post-translationally, and localization of proteolytic cleavage sites. The latter may be extended to include enzymatic C-terminal sequencing by carboxypeptidase digestion, and the definition of boundaries between domains, recognized by their elevated susceptibility to protease digestion in the native state. Relative signal strengths of unmodified and modified forms of proteins may be used as indicators of the stoichiometry of modification. Such measurements are, of course, independent of in vivo turnover rates, which affect assessment of stoichiometry in metabolic radiolabeling experiments.
In order to localize modifications within the amino acid sequence, proteins are cleaved into pieces of appropriate size, which are then re-analyzed for the presence of the modification of interest. Localization to an individual residue is ultimately performed by Collision-Induced Dissociation in a Tandem Mass Spectrometer system.
Protein/Peptide Chromatography
Chromatography can be employed both to purify and to characterize proteins/peptides. Chromatographic homogeneity is classically used as a criterion for protein purity, and can also be employed to assess the complexity of a protein mixture. Retention time in a chromatographic experiment may be used both to indicate the physical properties of peptides, and to provide a criterion for establishing the identity of particular peptide species of interest. Chromatographic techniques are particularly suitable for the discrimination of peptides and smaller polypeptides.
Protein/Peptide Electrophoresis
Gel electrophoresis may be used in any of the applications listed for chromatography above. However, it often shows greater discriminating power for larger species such as intact proteins. SDS electrophoresis is routinely employed to fractionate protein mixtures for identification of the components (see Protein Identification). In this context the ability of mass spectrometric methods to deconvolute protein mixtures of a high degree of complexity is employed to resolve species not discriminated by the electrophoretic separation. (See also 2-D Electrophoresis)
Protein/Peptide Isoelectric Focusing
Isoelectric focusing is sometimes performed to yield information similar to that provided by gel electrophoresis. Although more expensive and time consuming, it has the advantage that measurement of the pI value is made possible by this technique. (See also 2-D Electrophoresis)
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