At the end of this assignment, you will be able to:
- Describe the technique of Matrix Assisted Laser Desorption Ionization (MALDI) for creating gas phase ions of peptides and proteins.
- Explain how ions of different m/z ratios separate in a Time of Flight (TOF) mass spectrometer.
- Define resolution and explain how the reflectron design increases resolution in TOF mass spectrometry.
- Explain the process of peptide mass mapping for identifying a protein.
- Understand how variations in search parameters influence the probability score in a peptide mass mapping search.
- Given MALDI-TOF data for a digested protein, identify the protein using the technique of peptide mass mapping in the MASCOT database.
Often the first step in analyzing a complex mixture of proteins is to separate them using two-dimensional (2D) gel electrophoresis. (The Introduction section of this module includes a paper on protein analysis using 2D gel electrophoresis.) If 2D gel analysis indicates that new protein is being expressed or the expression level of a protein has changed, that protein must be identified. Peptide mass mapping is a method used to determine the identity of a protein spot in a gel. The protein is cut from the gel, destained, and extracted. Then the protein is digested (cut into pieces) with a proteolytic enzyme such as trypsin. The digest contains a mixture of peptides whose mass is analyzed using Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) mass spectrometry. In this module you will learn the principles of MALDI-TOF mass spectrometry and the method of peptide mass mapping for identifying a protein.