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Section 3: Peptide Mass Mapping

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    79424

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    Learning Objectives

    At the end of this assignment, you will be able to:

    1. Describe the technique of Matrix Assisted Laser Desorption Ionization (MALDI) for creating gas phase ions of peptides and proteins.
    2. Explain how ions of different m/z ratios separate in a Time of Flight (TOF) mass spectrometer.
    3. Define resolution and explain how the reflectron design increases resolution in TOF mass spectrometry.
    4. Explain the process of peptide mass mapping for identifying a protein.
    5. Understand how variations in search parameters influence the probability score in a peptide mass mapping search.
    6. Given MALDI-TOF data for a digested protein, identify the protein using the technique of peptide mass mapping in the MASCOT database.

    Overview

    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.

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