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  • Lecture4 CSB450 2016 Three Slides For Notes

    Загружено:

    Zikai Wang
    47 просмотров10 страниц
    This document discusses several topics related to proteomics analysis techniques: - Peptide sequencing by mass spectrometry involves the sequential removal of amino acids from peptides to generate "y- and b- ions" that allow identification of the peptide sequence. - Multidimensional protein identification techniques (MudPIT) combines ion exchange chromatography and reverse phase chromatography to improve coverage over 2D gel electrophoresis alone by separating proteins/peptides in multiple dimensions prior to mass spectrometry. - Quantitative proteomics techniques like isotope-coded affinity tags (ICAT) and isobaric tag for relative and absolute quantitation (iTRAQ) allow quantification of protein abundance and comparative analysis through chemical labeling.

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    Lecture for CSB450 u of t

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    Lecture4 CSB450 2016 Three Slides for Notes

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    This document discusses several topics related to proteomics analysis techniques: - Peptide sequencing by mass spectrometry involves the sequential removal of amino acids from peptides to generate "y- and b- ions" that allow identification of the peptide sequence. - Multidimensional protein identification techniques (MudPIT) combines ion exchange chromatography and reverse phase chromatography to improve coverage over 2D gel electrophoresis alone by separating proteins/peptides in multiple dimensions prior to mass spectrometry. - Quantitative proteomics techniques like isotope-coded affinity tags (ICAT) and isobaric tag for relative and absolute quantitation (iTRAQ) allow quantification of protein abundance and comparative analysis through chemical labeling.

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    © All Rights Reserved
    Скачайте в формате PDF, TXT или читайте онлайн в Scribd
    Отметить как неприемлемый контент
    47 просмотров10 страниц

    Lecture4 CSB450 2016 Three Slides For Notes

    Загружено:

    Zikai Wang
    This document discusses several topics related to proteomics analysis techniques: - Peptide sequencing by mass spectrometry involves the sequential removal of amino acids from peptides to generate "y- and b- ions" that allow identification of the peptide sequence. - Multidimensional protein identification techniques (MudPIT) combines ion exchange chromatography and reverse phase chromatography to improve coverage over 2D gel electrophoresis alone by separating proteins/peptides in multiple dimensions prior to mass spectrometry. - Quantitative proteomics techniques like isotope-coded affinity tags (ICAT) and isobaric tag for relative and absolute quantitation (iTRAQ) allow quantification of protein abundance and comparative analysis through chemical labeling.

    Авторское право:

    © All Rights Reserved
    Скачайте в формате PDF, TXT или читайте онлайн в Scribd
    Отметить как неприемлемый контент
    из 10

    University of Toronto

    CSB450
    Lecture 4
    Professor D. Christendat
    ESC4052

    Peptide sequencing by MS
    Sequential removal of a.a
    either C-term or N-term
    Y- and b- ions
    peptide ladder
    mass differences adjacent peaks

    Hydrolysis of peptide

    A V A G C A G A R

    14

    MudPIT
    Rationale
    Multiple approaches give better
    coverage
    Lack of sensitivity and resolution
    with 2D-PAGE

    Ion Exchange
    Chromatography
    Proteins bind at low
    ionic strength

    Elution with increasing


    ionic strength

    Reverse Phase Chromatography


    Hydrophobic interactions
    Water to organic phase elution
    least to most hydrophobic

    Typical MudPIT
    SCX

    R-Phase
    To MS

    ionic
    strength
    Time

    2D-PAGE vs MudPIT
    separate proteins
    with 2D-PAGE

    Protein complex
    trypsin digest

    Spot excision and


    trypsin digest

    Peptides separated
    by biphasic
    chromatography and
    identification

    (detailed discussion last


    lecture)

    MALDI or LC-MS
    Cap-LC separate
    peptides and
    identify

    Very high
    resolution
    Detection depends
    on MS

    Proteomic survey of metabolic pathways in rice


    Posted on Blackboard

    Protein Identification
    2D-PAGE vs MudPIT
    leaf

    Root

    Total

    Leaf

    Root

    Total

    Peptides

    1,707

    706

    3261

    6182

    7181

    22162

    Unique
    Peptides

    859

    445

    1509

    1626

    2358

    5189

    Proteins

    348

    199

    556

    867

    1292

    2363

    Seed Proteins
    Starch metabolism and catabolism
    Starch synthesized in amyloplast
    4 known allergenic proteins
    a-amylase/trypsin inhibitor gene
    family

    Quantitative
    proteomics
    Why LC-MS? Protein ID not
    quantitative!
    How can we quantify protein
    abundance, comparative analysis?

    Quantitative
    Proteomics
    In vivo (metabolic or natural
    isotopes)
    In vitro - Chemical labelling
    ICAT, Itraq

    Quantitative MS

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