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All about Protein Sequencing

written by: Rishi Prakash•edited by: Paul Arnold•updated: 7/29/2008

Protein sequencing uses many kinds of analysis tools and methods to help determine the structures of proteins and amino acids.

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    Proteins are found in all living cells and are pivotal to every single biological process that takes place. Protein structures are very complex. In order to determine a protein’s structure, one first has to use protein sequencing. Protein sequencing determines the amino acid sequences and peptides and is sued to determine what formations are adopted and if it is complexed with other molecules that are not peptides. By discovering the various structures and functions of proteins in organisms scientists are able to understand cell processes.

    There are two major methods of conducting protein sequencing:

    1. Mass spectrometry – this is an analytic technique that involves the identification of chemical compositions of compounds and charged particles. It uses chemical fragments of s ample into ions and takes measurements of the two different properties of charge and mass. This method is useful in identifying unknown kinds of compounds and in quantifying the amount of compounds.
    2. Edman degradation reaction – this is a method of sequencing amino acids within a peptide. In this method the residue is labeled from the peptide and does not disrupt the bonds between other amino acids.

    Scientists often want to be able to now the amino acid composition of proteins before they attempt to find an ordered sequence. This knowledge can be useful in discovering errors in sequencing. By knowing the frequency of amino acids it is then possible to choose which protease to be using for digestion purposes of the protein. There is a generalized method of this:

    1. Hydrolese a known quantity of a protein into amino acids
    2. Separating amino acids the same way

    Another method used in protein sequencing is hydrolysis. Hydrolysis is conducted by heating samples of a protein in a hydrochloric acid for 24 hours or even longer. Proteins that have bulky groups sometimes require a longer heating time. In separation, the amino acids can be separated by both ion exchange and hydrophic interactions.

    Once the amino acids have become separated, their quantities are then determined by adding in an agent that forms a colored derivative. If the amounts of amino acids in this mixture are over 10nmol, then it creates a yellow color when it is reacted with praline, and forms a vivid blue with other amino acids. Determining which amino acids form the N terminus of a peptide chain can be useful to scientists for two reasons:

    1. Aids the ordering of individual peptide fragments and sequences into a whole chain.
    2. Often, the first test of Edman degradation can be contaminated.

    The generalized method for N terminal amino acid analysis is as follows:

      1. React the peptide with an agent that will label the terminal amino acid
      2. Hydrolyze the proteins
      3. Determine the amino acids by comparison with standards.

    These are just a few of the methods of analysis that are used in protein sequencing and to determine the structures of proteins and amino acids.