Proteomics/Post-translational Modification

Presentation

 

Protein Primary Structure Post-translational Modification Protein - Protein Interactions List of Topics   Protein - Protein Interactions

Introduction

I. Definition

    A. Spontaneous or enzymatic alteration to one or more of a protein's amino acids
    B. Most often manifests as an addition or deletion to a side-chain
    C. Can occur at any point during or following full translation of a protein
    D. Often drastically effects overall structure and function of protein and associated complexes
    E. Are highly conserved among all living organisms

II. Types of modifications

    A. Acetylation
    B. Amidation/Deamidation
    C. Glycosylation
    D. Oxidation
         i. S-Glutathionylation
         ii. S-Nitrosylation
    E. Phosphorylation
         i. Histidine
         ii. Serine
         iii. Theronine
         iv. Tyrosine
    F. Proteolysis
    G. Ubiquitinylation/SUMOylation
    H. Others?

III. Manipulating in-vivo modifications

    A. Modifications can be prevented or induced in organism, tissue, and cell based model systems
    B. May allow for the detection of target proteins or dissection of related processes and pathways
    C. Exogenous introduction of stimuli
         i. endocrine/paracrine signals (aka hormones)
         ii. environmental (temp, UV, heavy-metals, peroxide, etc.)
         iii. antigens (virus, bacteria, allergens, lysates, etc.)
         iv. chemical/medicinal activators and inhibitors
    D. Genetic Approaches
         i. deletion, mutation, or reorganization of genetic elements (enhancers, promoters, genes, etc.)
         ii. gene inactivation or silencing by nucleic acid hybridization
         iii. transgenics (tansformations and transfections)
    E. Advantages and Draw-backs
         i. Qualitative but often hard to quantify
         ii. Often expolaratory in nature (observe and report)
         iii. Often very large scale in terms of available data

IV. In vitro reconstitution strategies

    A. Often provide a more quantitative, in depth analysis of a particular post-translational modification
    B. The protein in question is added to a reaction with the appropriate reagents and/or enzymes
    C. Reactions can be followed in real-time more readily

V. Methods of detection

    A. Most commonly used detection method of known modifications is through immuno/Western blotting
    B. Unexpected or novel modifications can be detected with a variety of analytical techniques, most notably mass spectrometry
    C. The best specific method of detection depends on many factors including the stability, frequency, and scale of the modification(s)
    D. In practice ease and cost dictate which methods are used first while the more exhaustive, cumbersome, or expensive methods follow as needed