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Proteomic technologies - Coggle Diagram

- - - - Different techniques:
        
        Label-free
        
        Workflow:
        
        Protein samples from different conditions are collected and digested into peptides
        
        Separation through Liquid Chromatography
        
        Electrospray ionization to make the peptides ionized and volatile
        
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        Straightforward and easy, but less accurate than label-based systems
        
        Label-based: stable isotope labelling
        
        In vivo labelling
        
        SILAC (Stable Isotope Labeling by Amino Acids in Cell Culture) / metabolic labelling
        
        Workflow:
        
        In cell cultures
        
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        In animal models
        
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        Lower reproducibility, as 2 experiments have to run in parallel
        
        In vitro labelling
        
        Principle:
        
        Labelling samples from 2 conditions with a chemical group in 2 isotopic forms, to obtain a relative quantification of the protein levels between the 2 conditions
        
        Depending on the labels, we distinguish among:
        
        Amino acid-based labeling
        
        ICAT (Isotope-Code Affinity Tag)
        
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        N-terminal peptide labeling
        
        iTRAQ
        
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        Make use of internal standards to compensate for variability in MS analysis, given by differences in ionization efficiency + influence of other ions. Such standards are peptides with the same sequence as those to analyze, but labelled with different stable isotopes
        
        Isotope labels are very similar in terms of chemical and physical properties, but the MW is slightly different
    - - 2D gel-based proteomics
        
        Workflow:
        
        Protein samples from 2 different conditions to compare are collected
        
        Protein separation based on isoelectric point (horizontal separation via pI strips) + MW (vertical separation via SDS-PAGE). In this way, proteins are separated on 2 dimensions
        
        Staining with dyes for easier protein recognition
        
        Analysis via image scanner to compare the gels of the 2 conditions and identify differences in the intensity of each protein spot
        
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        Depending on the number of gels used, we can distinguish between:
        
        1! gel for both conditions
        
        2D DIGE (2D fluorescent Difference Gel Electrophoresis)
        
        Involves labeling proteins from different conditions with structurally similar but spectrally distinct fluorophores
        
        Increased reproducibility, but higher cost
        
        2 gels, 1 for each condition
  - - - Sample inlet
        
        Ion source: ionizes the sample molecules by bombarding them with an electron beam
        
        Mass analyzer: ions are separated based on mass when they pass through a magnetic field
        
        Detector
        
        Recorder: receives signal from detector, amplifies and records it to create a mass spectrum
- - - - Studying abundance + localization + PTMs allows to better characterize the disease
    - - Useful to monitor disease progression + response to therapy
- - - - This cannot be used to predict the gene product levels for a specific gene, as it's only an average estimation
      - Some correlation between the abundance RNA/protein, no correlation at all between the half-life RNA/protein
        
        Lazy step function
        
        Describes the probability of observing protein given a certain RNA level. After a certain concentration there is a plateau at probability = 1
  - - - Transcriptional control
        
        RNA processing control
        
        RNA transport and localization
        
        Translational control
        
        mRNA degradation control
        
        Protein activity control
        
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  - - - Polysome profiling
        
        Uses centrifugation to separate mRNA molecules into polysomal fractions. The RNA is then analyzed via RNAseq. mRNAs which are more expressed in the polysomal fractions are assumed to be more actively transcribed
        
        Polysome = aggregate of numerous ribosomes that are actively translating the same mRNA
      - Ribosome footprinting / RiboSeq / Ribosome profiling
        
        Uses RNAse to digest exposed RNAs, leaving the RNA which is bound and protected by ribosomes undigested ("RNA footprints"). RNAseq on the protected RNAs reveals density + locations of ribosomes, which helps to understand which transcripts are preferentially translated
- - - - Studies how protein levels vary in response to different conditions
- - - - Protein arrays
    - - IHC, ELISA, WB
    - - MS proteomics
  - - - ELISA, MS proteomics
    - - Protein arrays
    - - WB, IHC
- - - - Proteins probes can be used to evaluate the binding of other proteins, liposomes, drugs, substrates of certain enzymes
    - - ex. Auto-Ab arrays