Samenvatting Functional Genomics deel 2 - Minor CADSDT & DSDT
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Course
Functional Genomics
Institution
Universiteit Leiden (UL)
Samenvatting van de colleges van Functional Genomics van de BFW minor CADSDT en DSDT. Dit is alleen het tweede deel van de samenvatting, omdat het bestand te groot was. De rest van de samenvatting is te vinden onder 'Samenvatting Functional Genomics deel 1 - Minor CADSDT & DSDT'. De samenvatting is...
College 12:
Proteomics: proteomic profiling – MS | phosphoproteomic profiling – MS after immunoprecipitation with pTyr-specific ABs
Proteomics vs. Genomics:
- Similarities: static picture of dynamic processes | high-throughput analysis | technology-driven | computational
intensive
- Differences: proteomics – “closer” to activity (function: PTM, location, turnover, protein complex, enzyme function) |
protein dynamics & RNA dynamics do not always correlate
Protein purification & visualization: Chromatography (size-exclusion, anion exchange,
reversed phase) | SDS-PAGE | 2D-PAGE
Protein identification:
- Mass spectrometry: = mass of every amino acid
- Edman degradation: zie plaatjes ---------------------->
Amino acid: amino – H & R-group – carboxyl | peptide bond to connect 2 amino acids
Proteomics:
Top-down: protein blijft heel = full mass of protein CON: moeilijk om hele protein zo te verwerken
Bottom-up: digest protein with enzymes into small peptides MS
Trypsin is used as enzyme cuts after Arg (R) & Lys (K) all proteins end with Arg & Lys = tryptic peptides
Proteins purification/extraction – digestion into (tryptic) peptides – peptides are analysed by LC-MS/MS –
identification by database searching
Different approaches of proteomics sample preparation: vb. SDS-PAGE – in-gel digestion | in solution – LC-MS/MS
LC-MS/MS: Liquid Chromatograhy separates peptides MS
TOEPASSING: MS is used in clinic to measure PKU in blood & bacterial species identification
MS = determines the mass-to-charge ratio (m/z) of gas-phase ions by subjecting them to known electric or magnetic fields
& analysing their resultant motion (ionization source = how ions are made)
Sample ionization source Mass analyser | Behaviour in electric/magnetic field m/z ratio
Ionization Source: MALDI | ESI Mass Analyzers: ToF | Iontrap | FT-ICR | Orbitrap
MS/MS for protein identification: MS spectrum recording (= peptide mass) select
particular intact peptide (precursor ion) fragment detect m/z of fragment (mass
analysis of ions eluting from column)
Plaatje: rechtsonder = ion making
MS/MS = mass analysis of fragment ions | From MS to MS/MS: LC MS MS/MS
Wat komt vd kolom (MS) pick one peak MS/MS = waaruit bestond piek
→ MS/MS: Each peptide is reflected in different peaks = different isoforms (vb. 13C, 15N)
MS/MS: fragment of particular peptide | verschillen in pieken: A fragmenteert makkelijker dan B
Peptides enter collision cell collision with gas fragmentation
CID = collision induced dissociation | HCD = high collision dissociation
MS select 10 peptides MS/MS again with other MS 10 peptides … repeat = MS duty cycle
Fragmentation of peptide bonds: breaks at b b & y ions (CID & HCD) | breaks at c c & z ions (ETD)
MS/MS spectrum to peptide identification: breek steeds net anders difference between peaks komt
overeen met 1 aminozuur puzzle
Identification of peptides:
Search algorithms: comparison of experimental MS/MS spectra with in silico MS/MS spectra
Spectral libraries: comparison of experi MS/MS spectra with previously recorded & validated spectra
Build libraries recorded on 1 instrument | useful if analysing same type of cells many times
Bottom-up protein identification: spectra assigned to peptides (database search) | peptide hits assembled to proteins
Protein identification characteristics: confidence of identification | #identified peptides | #unique peptides
#IDed proteins depends on: source of material (tissue, cell) | sample prep | pre-fractionation | length of LC-MS/MS run
Comparative proteomics = study effect of drug, gene KO/overexpression, disease vs control, etc
MS is a priori qualitative technique
Intensity of peptide ion signal depends on instrument response, which is dependent on biophysical property of peptide
Quantitation methods:
- 2D PAGE: no or chemical labelling gel matching OR differential gel electrophoresis (DIGE; green vs red – yellow)
- MS based: chemical/spiking/metabolic/enzymatic labelling
Heavy stable isotope labelling: only chemically identical peptides may be quantitatively
compared in 1 measurement (same physicochemical properties, different mass)
Same elution time in LC & same efficiency of ionization
Heavy stable isotopes are introduced into ≥ 1 samples proteins from control & labelled sample(s) are mixed control &
sample(s) are analysed in same mass spectrum Peak pairs for ‘native’ & labelled peptide species
Calculate peak intensity ratios to determine relative protein abundance
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