Lecture 4 Flashcards
(22 cards)
What are proteomics
Study of entire set of proteins produced by an organism
Problem of proteomics
- Genome of each cell in an organism is identical
- Proteome differs
- Protein abundance varies >6 orders of magnitude
- Co/post-translational modifications widespread
How does MS solve protein
- Identifies protein in complex mixture
- Identify/localize protein modifications
- Relative/absolute quantitation
- Detect attomolar levels of proteins
- Limitation - dynamic range (detects 1 in 1000)
Overview of MS
- Several interconnected chambers
- Under high vacuum
- Sample ionized/accelerated
- Mass selection - ionized sample defected by EM field (depends on m/z ratio)
- Ions detected - mass/charge ratio plotted against signal intensity
theory surrounding MS
- Charged particles experience force proportionate to charge
- Ions travelling non-linear path requires given acceleration
Experimental variations:
- Ionisation methods
- Different geometries
- Fragmentation chambers
Tandem mass spectrometry
- Standard MS spectrum acquired
- m/z of interest selected
- Ion fragmentation induced
- Second mass selection event gives fragmentation induced spectrum
- Provides identity of ion
- MS instruments allow sequential fragmentation
MALDI-TOF-MS
Matrix-assisted laser desorption ionization-time of flight
- Sample dried on inset solid in chemical matrix
- Laser beam cause ionisation with single +ve charge
- Mass calculated by time taken to travel to detector
Advantages: Ionise peptides, intact proteins, carbs
Disadvantages: Sample prep limits throughtput
Liquid chromatography-mass spectrometry
- Analysis of eluted peptides
- Electrospray ionisation
Overview of proteomics
Whole protein -> Complex peptide mixture -> MS/MS -> Protein list
Proteomics sample prep
- Isolate proteins from biological sample e.g. tissues/cells
- Denature and reductively alkylate - break disulphide bonds
‘ - Digest into peptides - use trypsin
- Fractionation used to reduce sample complexity
- Enrichment or depletion improves sensitivity
Analysis of proteomics
- Nano-flow C18 column with acetlonitrile gradient
- Positive electrospray ionization
- Data dependent acquisition: 1 MS scan and 10 MS/MS per duty cycle
- Dynamically exclude peaks for set time
Identifying peptides using proteomics
- peptides fragment in predicable manner from each end of peptide
- Peptide sequence read from MS/MS spectrum
protein modifications
Phosphorylation of Ser/Tyr/Thr
N-glycans on Asn
O-glycans on Ser/Thr
Ubiquitination of Lys
- Modifications occur at low abundance
- Introduced in vitro as deliberate strategy or accidently during sample processing
- Modified peptides identified by m/z
- Increasing modifications increases sequence space/CPU time
phosphorylation
- Enrichment of phosphopeptides essential
- Sites localised and occupancy calculated
Tryptic digest -> Phosphopeptide enrichment -> IMAC/Anti-pY IgG/TiO2/SCX -> LC-MS/MS
N-glycosylation
Complex branched structures
Mapped by ezymatic cleavage e.g. EndoH cleaves N-glycan and leaves GlcNAc residue
Identified by GC-MS
Inact glycopeptides identified by MS^n
Ubiquitinylation
- Ubiquitin attached to lysine side chain
- Contains multiple lysines allows polyubiquitinylation
Mapped by tryptic digest which digests protein and Ubq modification
SILAC quantification
- Stable isotope labelling by amino acids in cell culture
- Metabolic labelling technique
Use 2H, 13C, 15N labelled amino acids
- Labelled L-Arginine and L-lysine used
Light - C12-Arg label
Medium - 13C6-Arg or 13C4-Lys used
Heavy - 13C6/15N4-Arg or 13C4/15N4-Lys used
Proteomics data processing
- Peptides and theoretical fragments calculated from sequence/allowed modifications
- Theoretical fragments matched to peaks
- proteins assembled from peptide data
- False discovery rate estimated using decoy sequences
iTRAQ quantification
- Isobaric tagging for relative/absolute quantification
Chemically reactive multiplexed tag:
- All tags have same mass in MS
- Each tag produces distinct mass in MS/MS
- Up to 10 conditions can be simultaneously labelled
Affinity purification MS
Isolates biologically relevant complexes
Distinguishing contaminant from real components is major problem
AP-MS
qMS can identify non-specific contaminants
Stoichiometry can be obtained
Complex of interest is bound by a GFP affinity tag
Anti-GFP antibodies bind GFP where they have a non-specific contaminant
GFP tagging produces greater intensity than untagged complexes
Unspecific background binders same across untagged and tagged complexes
Cross-linking MS
- Cross linking agents gain distance information
- CHemically reactive cross-linkers form covalent bonds
- Reveal weak interactions
Process:
Protein complex crosslinked
Proteolysis forms peptide mixture
Peptide mixture undergoes LC-MS/MS
Map of cross-links and set of distance restraints formed
Selection of structural models
