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Protein Identification Techniques

Proteins can be seperated by 2-D Gel Electrophoresis and Identified with Mass Spectrometry

Protein content of a cell is estimated to consist of thousands of different types of proteins
with a dynamic range of expression. The technology that is currently being used involves the retrieval of the protein components, fractionation of this very complex mixture,
enzymatic proteolysis of each separated and isolated species, extensive mass spectrometric analysis and finally matching the structural data generated against a database of known proteins or anticipated genome expression products.
This procedure involves an initial step using 1- or 2-dimensional electrophoresis (DE). Using 1-DE, proteins are separated on the basis of molecular mass; the technique is reproducible and can be used for proteins in the mass range of 10–300 kDa, but does have limited resolution.
For separation of more complex protein mixtures, 2-DE is the preferred method. This involves separating the proteins first according to their net charge by an isoelectric focusing step and then, in the second dimension, according to their molecular mass employing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) which gives a high separation effciency.
 Mass spectrometry (MS) is the method of choice for the identification
of the separated proteins, and 2-DE and mass spectrometry
now represent a technology by which several thousand proteins can be separated, detected and identified in an automated fashion.
Proteomics methodology is initially done by protein separation and location by 2-DE followed by excision of the proteins of interest which then undergo proteolytic digestion to yield a mixture of peptide fragments. The peptides are analysed by mass spectrometry, initially using MALDI-MS for molecular mass determination and generation of a peptide mass fingerprint. If database searching at this stage yields ambiguous results, a second mass spectrometric analysis, ESI-MS/MS, is used to generate sequence information which is used for more stringent database searching.
From the mass spectrum obtained on analysis of the protein digest mixture, the peptide mass map or peptide mass fingerprint i.e. the molecular masses of the peptide fragments, can be ascertained. Often, if the amino acid sequence is suffciently unique and the mass
accuracy suffciently high, the mass map can be used to search databases 12–15 to identify the protein successfully without the need for further analyses. If, however, database searching leads to ambiguous results, then further MS analyses, involving the use of tandem mass spectrometry (MS/MS), are undertaken sequentially on each peptide in the mixture to generate a sequence, or partial sequence, known as a sequence tag, for these peptides. This is frequently achieved by the use of ESI-MS/MS19 and usually an additional purification step must be performed to separate the peptides from the salts and detergents that may be present which cause attenuation of the peptide signal.

Further database searching with both the molecular mass of the peptide
and the sequence tag information should lead to unambiguous protein identification.

 

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