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8.11: Protein Cleavage

[ "article:topic", "authorname:ahern2", "MALDI-TOF", "license:ccbyncsa" ]
  • Page ID
    10449
  • Because of their large size, intact proteins can be difficult to study using analytical techniques, such as mass spectrometry. Consequently, it is often desirable to break a large polypeptide down into smaller pieces. Proteases are enzymes that typically break peptide bonds by binding to specific amino acid sequences in a protein and catalyzing their hydrolysis.

    Chemical reagents, such as cyanogen bromide, which cleaves peptide bonds on the C-terminal side of a methionine residue can also be used to cut larger proteins into smaller peptides. Common proteins performing this activity are found in the digestive system and are shown below.

    • Subtilisin - C-terminal side of large uncharged side chains
    • Chymotrypsin - C terminal side of aromatics (Phe, Tyr, Trp)
    • Trypsin - C-terminal side of lysine and arginines (not next to proline)
    • Carboxypeptidase - N-terminal side of C-terminal amino acid
    • Elastase - Hydrolyzes C-side of small AAs (Gly, Ala)
    • Cyanogen Bromide (chemical) - Hydrolyzes C-side of Met

     

    Figure 8.45 - Protease cleavage sites on a polypeptide

    Determining mass and protein sequence

    Mass spectrometry, as its name suggests, is a method that can be used to determine the masses of molecules. Once limited to analyzing small molecules, it has since been adapted and improved to allow the analysis of biologically important molecules like proteins and nucleic acids. Mass spectrometers use an electrical field to accelerate an ionized molecule toward a detector. The time taken by an ionized molecule to move from its point of ionization to the detector will depend on both its mass and its charge and is termed its time of flight (TOF).

    MALDI-TOF

    Figure 8.46 - A desktop MALDI-TOF system

    MALDI-TOF (Matrix-assisted Laser Desorption Ionization - Time of Flight) is an analytical technique allowing one to determine the molecular masses of biologically relevant molecules with great precision. It is commonly used in proteomics and determination of masses of large biomolecules, including nucleic acids. The development of MALDI, which permits the production of ionic forms of relatively large molecules, was crucial to the successful use of mass spectrometry of biomolecules. Figure 8.46 shows a compact MALDI-TOF system.

    The MALDI-TOF process involves three basic steps. First, the material to be analyzed is embedded in solid support material (matrix) that can be volatilized in a vacuum chamber by a laser beam. In the second part of the process, a laser focused on the matrix volatilizes the sample, causing the molecules within it to vaporize and, in the process, to form ions by either gaining or losing protons. Third, the ions thus created in the sample are accelerated by an electric field towards a detector. Their rate of movement towards the detector is a function of the ratio of their mass to charge (m/z). An ion with a mass of 100 and a charge of +1 will move twice as fast as an ion with a mass of 200 and a charge of +1 and at the same rate as an ion with a mass of 200 and a charge of +2. Thus, by precisely determining the time it takes for an ion to go from ionization (time zero of the laser treatment) to being detected, the mass to charge ratio for all of the molecules in a sample can be readily determined.

    Ionization may result in destabilization of larger molecules, which fragment into smaller ones in the MALDI-TOF detection chamber. The size of each of the sub-fragments of a larger molecule allows one to determine its identity if this is not previously known. This fragmentation can be intentionally enhanced by having the accelerated ions collide with an inert gas, like argon.

    Fragmentation of a molecule may also be carried out prior to analysis, as for example, by cleaving a protein into smaller peptides by the use of enzymes or chemical agents. The amino acid sequence of a protein may be determined by using MALDI-TOF by analyzing the precise molecular masses of the many short peptide fragments obtained from a protein. When one amino acid, for example, fragments from a larger peptide, this can be detected as the difference in mass between the fragment with and without the amino acid, since each amino acid will have a characteristic molecular mass. By peptide mass fingerprinting and analysis of smaller fragments of individual peptides, the entire sequence of a polypeptide can, thus, be determined.