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    L5 Metaproteomics

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    Núria Val
    Metaproteomics analyzes the proteins present in a microbial community to determine taxonomic composition and metabolic functions. It faces challenges including proteome complexity from multiple species, differing cell extraction methods, reliance on reference databases that may lack sequences for unsequenced species, and unknown metabolic pathways. Metaproteomics employs bottom-up shotgun proteomics to identify peptides and match them to protein sequences from metagenomic or reference databases. Protein stable isotope probing uses isotopically labelled substrates to identify which microbes actively consume specific resources by detecting incorporation of the stable isotope into microbial proteins over time.

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    L5 Metaproteomics

    Uploaded by

    Núria Val
    11 views3 pages
    Metaproteomics analyzes the proteins present in a microbial community to determine taxonomic composition and metabolic functions. It faces challenges including proteome complexity from multiple species, differing cell extraction methods, reliance on reference databases that may lack sequences for unsequenced species, and unknown metabolic pathways. Metaproteomics employs bottom-up shotgun proteomics to identify peptides and match them to protein sequences from metagenomic or reference databases. Protein stable isotope probing uses isotopically labelled substrates to identify which microbes actively consume specific resources by detecting incorporation of the stable isotope into microbial proteins over time.

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    L5_Metaproteomics

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    Metaproteomics analyzes the proteins present in a microbial community to determine taxonomic composition and metabolic functions. It faces challenges including proteome complexity from multiple species, differing cell extraction methods, reliance on reference databases that may lack sequences for unsequenced species, and unknown metabolic pathways. Metaproteomics employs bottom-up shotgun proteomics to identify peptides and match them to protein sequences from metagenomic or reference databases. Protein stable isotope probing uses isotopically labelled substrates to identify which microbes actively consume specific resources by detecting incorporation of the stable isotope into microbial proteins over time.

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    11 views3 pages

    L5 Metaproteomics

    Uploaded by

    Núria Val
    Metaproteomics analyzes the proteins present in a microbial community to determine taxonomic composition and metabolic functions. It faces challenges including proteome complexity from multiple species, differing cell extraction methods, reliance on reference databases that may lack sequences for unsequenced species, and unknown metabolic pathways. Metaproteomics employs bottom-up shotgun proteomics to identify peptides and match them to protein sequences from metagenomic or reference databases. Protein stable isotope probing uses isotopically labelled substrates to identify which microbes actively consume specific resources by detecting incorporation of the stable isotope into microbial proteins over time.

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    Núria Val

    Proteomics (Omics)

    LECTURE 5: INTRODUCTION INTO METAPROTEOMICS


    1. THEORY METAPROTEOMICS
    Metaproteomics = microbial community proteomics

    What would we like to know about microbial communities?

    - What species are there? How many? (taxonomy) →


    Difficult to know since there may be hundreds or
    thousands of species.
    - What are their substrates? What do they eat/consume?
    (Glucose, fructose, CO2…)
    - Which proteins (pathways) are expressed for feeding?
    - What eats each specie?

    CHALLENGES IN METAPROTEOMICS
    1) Proteome complexity. From which species the protein comes? Remember that each yeast
    species has like 6000 proteins, so with 100 species in a community, we would have 600.000
    proteins...
    2) Protein extraction. There are many different cell types, and the same extraction method does
    not work for all of them.
    3) Reference sequence database: many unsequenced species.
    4) Functions: possibly unknown metabolic routes, many new enzymes.

    Metaproteomics usually employs the conventional bottom-up shotgun proteomics approach: the
    peptides obtained in the digestion of the community proteins are compared in databases to find the
    protein sequences.

    Where do we get the protein sequence reference database?

    • Option 1: from metagenomics. We sequence massively the genomes of the microorganisms in


    the sample.
    o Disadvantages:
    ▪ Extract DNA from a certain part of the community.
    ▪ You amplify all the DNA, the one that is in very small amounts and reverse, so
    you don't really know what the most abundant specie in the community is.
    ▪ High costs
    ▪ Time consuming

    • Option 2: use generic reference sequence databases such as NCBI and UniProt.
    o Advantage: no amplification, only the proteins that are sufficiently high abundant will
    be detected. Realistic data from the community.
    o Disadvantage: you will not find new species since you are comparing the peptides
    sequences with known proteins.
    Núria Val
    Proteomics (Omics)

    2. APPLICATIONS
    WHO IS THERE? WHO DOES THAT?

    From an environmental sample, a metaproteomics (and maybe a metagenomics) experiment is


    carried out to identify the proteins and, therefore, the species.

    WHO IS ACTIVE? WHICH MICROBE EATS WHAT?

    Protein stable isotope probing (SIP) allows to measure utilisation of substrates by individual
    microbes. A stable isotope (i.e., C13) labelled substrate is used to feed the microbes, so the ones that
    consume it will assimilate and acquire the label.

    Ex: labelled glucose. It is consumed and the carbon finishes in the aminoacids, so it can be detected
    later.

    Ex: peptides mass spectrum which shows stable isotope incorporation over time. Each coloured peak
    corresponds to a specific time: more time = more assimilation of the stable isotope.
    Núria Val
    Proteomics (Omics)

    Two key parameters in stable isotope probing: RIA and LR


    RIA = Relative isotope abundance LR = Labelling ratio
    How much 12C has been replaced by 13C? What is the fraction of labelled proteins?

    The peak moves to the right each time we have


    another 13C (m/z increases because mass
    increases).
    If you don't really see a big move to the right, it's
    LR let us know how fast the microbe is in
    because the microbe is using another feeding
    terms of making new proteins, because
    source apart from the one you labelled (there is a
    this ratio expresses the assimilation of
    2nd carbon source or multiple carbon sources).
    labelled carbons.
    The more a labelled carbon source is consumed,
    the higher is RIA. But if the microbe uses other
    carbon sources, it will assimilate the labelled carbon
    source more slowly so its RIA will be 'smaller'.

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