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BMS661: PROTEOMICS > C4 > Flashcards

C4 Flashcards

1
Q

Types of protein identification methods

A
  • Edman Degradation: Chemical
  • Western Blotting: Gel based
  • PMF & Tandem MS: MS
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2
Q

Explain Edman degradation

A
  • 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
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3
Q

What is PITC

A

Fluorescent label

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4
Q

Pros & cons of Edman digestion

A

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

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5
Q

Explain western blotting

A
  • Protein transfer from gel to membrane
  • Probed with antibody against protein of interest or lectins
  • Based on specificity of antigen-antibody interactions
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6
Q

Primary & secondary Ab specific to

A
  • Primary Ab: protein of interest
  • Secondary Ab: Primary Ab & carry signalling molecules
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7
Q

Procedure of western blotting

A
  • Protein transfer
  • Blocking skimmed milk
  • Wash PBST/TBST
  • Primary Ab or lectin
  • Wash
  • Secondary Ab
  • Wash
  • Develop signal
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8
Q

Name of primary & secondary antibody as well as protein

A

Primary
- Host anti protein
- Rabbit anti GAPDH

Secondary
- Anti primary Ab
- Anti rabbit

Protein
- GAPDH

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9
Q

Types of protein transfer for western blotting

A
  • Wet system
  • Semi dry system
  • Dry system
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10
Q

Types of signal detection in western blotting

A
  • Visualisation based on chemiluminiscene (densitometry)
  • Visualisation based on fluorescence
  • Visualisation based on colour
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11
Q

Steps in initial protein fragmentation in PMF & Tandem MS

A
  • Protein spot/band excision from gels
  • Protein digestion & extraction
  • Endoglycosidase digestion
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12
Q

Explain protein spot/band excision from gel

A
  • Automated/ manual removal of spot containing protein of interest
  • Spot transfer directly to tube / 96 well for protease digestion
  • Careful to avoid contamination
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13
Q

Steps in protein digestion & extraction

A
  • 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
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14
Q

Explain endoglycosidase digestion

A
  • 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
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15
Q

Procedure of PMF

A
  • Sample preparation: Extraction & Digestion
  • Mass spectrometry: Ionisation & Mass analysis
  • Data analysis: PMF generation & database search
  • Protein identification: Matching. & Validation
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16
Q

Principle of Tandem MS

A
  • 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
17
Q

Explain tandem MS/MS for peptide sequencing

A
  • Tandem MS/MS generate daughter ion leading to peptide sequence & correct identification of peptide
18
Q

Explain tandem MS/MS with post source decay (PSD)

A
  • 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
19
Q

Procedure of ESI-TRIPLE QUADRUPOLE

A
  • 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

BMS661: PROTEOMICS (6 decks)

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