Analyze the Data 3-3: Protein Purification The purification of a microtubule mot

Question

Analyze the Data 3-3: Protein Purification

The purification of a microtubule motor protein has been undertaken (see Vale et al., 1985, Cell 42:39–50). A partially purified cell fraction containing the motor protein was subjected to an additional purification step using gel filtration chromatography. Aliquots of each fraction eluted from the gel filtration column were subjected to SDS gel electrophoresis. Fractions 21 to 41 are shown on the gel below, which was stained with Coomassie blue.

The band that migrates at about 116 kD in fraction 40 is excised from the gel and is then

      subjected to partial proteolysis with trypsin. Mass spectrometry(not provided) analysis of the peptides

      derived from this proteolysis suggests that this polypeptide is a member of the kinesin motor

      superfamily. Explain how mass spectrometry data could lead to this conclusion.

      This question is assuming that you have mass spectrometry analysis data available, as well as

      access to online sequence databases.

Explanation / Answer

Answer:

Identification of Kinesin motor superfamily polypeptide:

Partial proteolysis of this polypeptide by trypsin will cause hydrolyses on the c-terminal side of lysine and arginine residues resulting into a unique set of smaller peptides. The mass of these peptides is then measured by using a mass spectrometer such as Matrix Assisted Laser Desorption/Ionization-Time of flight analyzer (MALDI-TOF) or Electrospray ionisation time-of-flight (ESI-TOF). This technique is called peptide mass fingerprinting and can be used for identification of proteins with already known sequences.

Protein can be identified based on database searching. The process involves following steps:

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