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FAQ-Post Translational Modification

  1. What are the concerns about mapping a phosphorylation site in my protein?

    You will have two major concerns:
    Coverage: Can the mass spectrometry technique(s) deliver high sequence coverage? With higher coverage, there is a greater statistical chance to detect and identify the peptide containing a modification group.
    Occupancy of the modification site: What percent of the protein preparation is modified? If it is low, then the chance of detecting the modified peptide decreases. If the occupancy is high (>30%), then this works in the favor of identifying a modification site.

  2. How can coverage be increased for mapping phosphorylation or methylation sites?

    First, note that, due to its unique sequences, every protein has a different potential to be mapped at high sequence coverage.
    Coverage for all proteins increases by:
    a)    Starting with a higher amount of protein (>5 μg of protein is desirable) for MS analysis
    b)    Making sure that the protein is pure
    c)    Using multiple MS instruments/techniques for analysis will increase coverage
    d)    Enriching the phosphopeptides using a TiO2 column

  3. Will glycosylation sites inhibit the identification of sample protein?

    If purity and concentration of the protein is high, then in most cases MS-based protein identification is still favorable.
    Attached carbohydrate (CHO) chains, however, can impair MS-based identification of specific peptides through the following mechanism:
    a)    CHO chains can block trypsin cleavage
    b)    The variable mass of attached CHO chains can reduce glycopeptide detection.
    c)    Options: Cleave N-linked CHO chains with N-glycanase (PNGaseF) prior to gel loading and MS analysis
    4)    I want a preliminary analysis for potential phosphorylation sites in my protein. What is your lowest cost service? What are the limitations? What are your suggestions?
    A lowest-cost preliminary analysis is a method which uses MALDI-TOF/TOF on a pure protein from a gel or a highly pure preparation. Limitations: The coverage of the protein will be low, if the protein is very large, or if it is impure, or if it is in low yield (<500 ng).
    Suggestions: For a preliminary, low cost analysis, provide only a high quality sample (this would be a single band on a gel that is in the 1 μg range).


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