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
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
