C4 Flashcards
Types of protein identification methods
- Edman Degradation: Chemical
- Western Blotting: Gel based
- PMF & Tandem MS: MS
Explain Edman degradation
- Long peptide were cut to smaller pieces using protease or cynagen bromide
- Require peptide amount of 1 picomole or above
- Each peptide sequenced by derivatizing N terminal residue using PITC
- Derivatized residue cleaved off & identify
- New molecules of PITC undergo same process
What is PITC
Fluorescent label
Pros & cons of Edman digestion
Pros
- No need sample pre treatment
- Identify unknown protein that are not in database
- Guarantee N terminal sequence of protein
Cons
- Not work if N terminal chemically modified
- Sequencing stop if non alpha amino acid found
- Not useful to determine position of disulfide bond
Explain western blotting
- Protein transfer from gel to membrane
- Probed with antibody against protein of interest or lectins
- Based on specificity of antigen-antibody interactions
Primary & secondary Ab specific to
- Primary Ab: protein of interest
- Secondary Ab: Primary Ab & carry signalling molecules
Procedure of western blotting
- Protein transfer
- Blocking skimmed milk
- Wash PBST/TBST
- Primary Ab or lectin
- Wash
- Secondary Ab
- Wash
- Develop signal
Name of primary & secondary antibody as well as protein
Primary
- Host anti protein
- Rabbit anti GAPDH
Secondary
- Anti primary Ab
- Anti rabbit
Protein
- GAPDH
Types of protein transfer for western blotting
- Wet system
- Semi dry system
- Dry system
Types of signal detection in western blotting
- Visualisation based on chemiluminiscene (densitometry)
- Visualisation based on fluorescence
- Visualisation based on colour
Steps in initial protein fragmentation in PMF & Tandem MS
- Protein spot/band excision from gels
- Protein digestion & extraction
- Endoglycosidase digestion
Explain protein spot/band excision from gel
- Automated/ manual removal of spot containing protein of interest
- Spot transfer directly to tube / 96 well for protease digestion
- Careful to avoid contamination
Steps in protein digestion & extraction
- Destaining: remove dye from gel spot
- Reduction/ alkylation: break disulphide bond
- Proteolytic cleavage: digestion by trypsin or cleave on C side of Arg & Lys
- Extraction of digested peptide by incubation with extraction buffer
- Supernatant collected for MS
Explain endoglycosidase digestion
- If protein known to be N-glycosylated & need to be analysed the N-glycan, it must be digested with trypsin
- Removal using endoglycosidase
- Either cleave glycan completely from Asn or leave 2 GlcNAcs
Procedure of PMF
- Sample preparation: Extraction & Digestion
- Mass spectrometry: Ionisation & Mass analysis
- Data analysis: PMF generation & database search
- Protein identification: Matching. & Validation
Principle of Tandem MS
- Use 2 mass analyser in series (tandem)
- First mass analyser acts as ion selectors by allowing only ion in given m/z to pass
- Second mass analyser use as mass analyser for fragment
Explain tandem MS/MS for peptide sequencing
- Tandem MS/MS generate daughter ion leading to peptide sequence & correct identification of peptide
Explain tandem MS/MS with post source decay (PSD)
- Ion decay after leaving the ion source but before reaching collector, this term corresponds to metastable dissociation
- 2 mass analyser with collision cell in between
- First analyser select peptide with specific m/z ratio
- Send into collision cell where peptide collides with gas
- Fragmentation always at the peptide bond
Procedure of ESI-TRIPLE QUADRUPOLE
- Sample ionisation in ion source
- Quadrupole 1: Ion selection in quadrupole mass filter
- Quadrupole 2: ion fragmentation in quadrupole collision chamber
- Quadrupole 3: fragment ion selection in quadrupole mass filter
- Ion detection
