Friday, February 27, 2009

Mass spectrometry for protein identification

There are various types of mass spectrometry depending on the type of ionization source or the ion analyzers. They are used for specialized studies in proteomics such as identification of proteins by generating mass fingerprints or the ionization pattern of the protein molecule, amino acid sequencing of peptides, and thereby the detailed studies of the three-dimensional structure of the protein and modification of the proteins if any can be detected very easily and efficiently. Some of the common types of mass spectrometry are discussed below.

MALDI-TOF Mass Spectrometry :
Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) is another method of ionization that does not require any heating for the production of ions and can be used for heat-sensitive molecules. In this method, the sample to be analyzed is mixed with a matrix compound and crystallized. The compound of this matrix is usually a weak inorganic acid. This mixture (matrix mixed with sample) is then excited with a laser, which results in the evaporation of the matrix compound. The matrix compound also carries the sample molecules into the vapor phase, resulting in indirect vaporization of the sample. Sample ions are formed by the exchange of electrons and protons with the matrix compound.

MALDI is especially useful for protein and peptide identification using masses alone, since the masses of ions can be determined with great accuracy. Time of flight is the technique used for the sorting and separation of ions generated by laser ionization.

Electrospray Ionization (ESI):
This is a newer method of ionization that does not cause excessive fragmentation. ESI generates ions directly from the solution without heating. Therefore, this method can be used for heat-sensitive molecules, which cannot be ionized by heating. The sample is finely sprayed in the presence of an electrical field. Charge accumulates on the sample droplets, which then explode due to mutual repulsion of charges leading to the formation of ions. Both single and multiple-charged ions can be produced in this manner.

Tandem Mass Spectrometry (MS/MS or Two-dimensional MS):
Tandem MS is a procedure in which multiple rounds of mass spectrometry are performed to obtain greater resolution of the ions generated by the unknown sample molecule. Short stretches of polypeptides can be sequenced with tandem MS. In general a polypeptide is cleaned into a number of fragments enzymatically and the products are taken into the mass spectrometer for analysis one by one to generate the primary structure or the amino acid sequence.

The protein solution is cleaned into short stretches of pep tides preferably by an enzyme such as trypsin or chymotrypsin. The mixture of short pep tides thus generated is injected into the tandem MS for analysis. The tandem MS is an instrument with two MS in a series. They are designated as MS1 and MS2. In the first mass analyser (MS1) the peptide mixture (precursor ions) is sorted resulting in the selection of only one peptide from the several types of pep tides produced by cleavage, which comes out at the outlet of the MSl. The selected peptide (precursor ion) is then transmitted to a collision cell where it undergoes collision-induced dissociation (CIO). The peptide is further fragmented by a high-energy impact with a small amount of inert gas such as helium or argon known as the collision gas. The fragment ions, thus produced, are transmitted to the second mass analyzer (MS2), which measures the m/z ratio of all the charged fragments. During the process of fragmentation the peptide molecule breaks at the peptide bonds resulting in the product ions. This fragmentation does not involve the addition of water as in the case of enzymatic cleavage.

The MS2 scans the product ions and generates one or more sets of peaks. A set of peaks consists of all the charged fragments (product ions) generated by breaking the same type of bond (peptide bond) by CIO. Each peak in a set represents one amino acid less than the previous peak. The difference in mass (m/z) between two adjacent peaks indicates the amino acid that was lost during CID, thus revealing the sequence of amino acids of the peptide. This type of mass spectrometry, also known as 2D MS, is usually used for the analysis of complicated mass spectrum, particularly useful in the amino acid sequencing of pep tides and post-translational modifications.

Tags: Bio Technology, Bio Genetics , Protein Purification

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