Process of Characterization of Proteins

The purified protein needs to be identified and characterized. There are various methods to accomplish this. The purity of the protein can be determined by the SDS-PAGE or by isoelectric focusing (IEF) or by two-dimensional gel electrophoresis.

Identification or characterization of protein can be carried out with the help of anyone of the following techniques or by a combination of these:

1) Two-dimensional gel electrophoresis,
2) Peptide fingerprinting,
3) Protein sequencing, and
4) Mass fingerprinting by mass spectrometry. Here we are discussing the technique of Mass Spectrometry.

Mass Spectrometry: It is a chemical-analysis technique that is used to measure the mass of unknown molecules by ionizing, separating, and detecting ions according to their mass-to-charge ratios. Mass spectrometry is also used to determine the structure of molecules.Mass spectrometry helps us in the following areas:

1) To determine the sequence of proteins and peptides.
2) To identify the structure of other biomolecules such as lipids, carbohydrates, oligonucleotides, etc.
3) To detect the presence of banned substances in athletes.
4) To determine the composition of rocks and thus determine their age and origin.
5) To identify isotopes of various elements.
6) To perform forensic analysis to detect the presence of certain substances.

A mass spectrometer creates charged particles (ions) from molecules. It then analyzes those ions to provide information about the molecular weight of the compound and its chemical structure. There are different types of mass spectrometers and sample introduction techniques, which allow a wide range of analyses. This discussion will focus on the principle of mass spectrometry and its use in the study of protein chemistry. The techniques can be used to sequence peptides and proteins and to study their interactions. It is useful in the identification of protein molecules, their accurate molecular weight, the post-translational modifications, and structural functional relationship of proteins.

It is now possible through mass spectrometer to 'see' proteins of molecular weight as high as 100,000 daltons. A big advantage is that a very small sample, as small as picomoles, is required. Thus, it is possible to do two-dimensional gel electrophoresis of proteins of a cell (to separate the few thousand different proteins) and identify them using mass spectrometer. This approach of protein analysis and identification is one of the important techniques of what is called proteomics-the study of the complete protein complement of a cell.

Let us understand as to what is a Mass Spectrum:
A mass spectrum is a plot that shows the relative abundance of ions of various mass-to-charge ratios. The X-axis represents the mass-to-charge ratio of the ions and the Y-axis represents the relative abundance of each ion.

Mass spectrometry can be in association with or linked to one or more separating techniques. For example, in organic chemistry mass spectrometry is always linked to Gas chromatography and therefore, is called GC-MS. Gas chromatography will convert the samples into gaseous phase, which is introduced to the mass spectrometer. In addition to this, GC can be used to separate the components in a mixture and the separated pure compounds can be directly identified and analyzed by a mass spectrometry spectrum. This is the advantage of linking a separating technique to mass spectrometry. Similarly, in the case of protein studies mass spectrometry can be linked to various types of liquid chromatographic systems such as GPC, ion exchange, or affinity chromatography individually or in tandem, or to capillary electrophoretic systems, which can also be operated automatically, like HPLC.

Tags: Bio Technology, Bio Genetics , Protein Characterization

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