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BMS661: PROTEOMICS > C5 > Flashcards

C5 Flashcards

1
Q

Define quantitative proteomics

A
  • Simultaneously quantify level differences between many protein in different samples
  • Use statistical analysis & large population of control & sample
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2
Q

Potential error arise from quantitative proteomics

A
  • Difference in sample preparation
  • Method of sample analysis
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3
Q

Types of quantitative protein

A
  • Gel based
  • MS based
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4
Q

Explain gel based quantification

A
  • Protein bands/spot directly quantify from gels
  • Difference in protein level measure the band/spot intensity by densitometry
  • Use fluorescent dye as signal intensity that represents number of protein copies in spot/band
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5
Q

Accuracy of gel based quantification depends on

A
  • Choice of staining method
  • Evaluation software
  • Shape of spot
  • Background level
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6
Q

Example of 2DE Imaging system

A
  • Bio Rad: PDQuest
  • Gene Data: Impressionist
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7
Q

Explain MS based quantification

A
  • Use internal standard that have similar properties to analyte
  • Use stable heavy isotope for sample & light isotopes for control
  • Added to sample at variable stage which is in vivo or in vitro
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8
Q

Types of MS based quantification

A

In vivo
- Incorporated of light/heavy isotopes into protein in living cell before extraction, separation & analysis
- 15N

In vitro
- Based on estrification of carboxyl group or acetylation of amino group

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9
Q

What is SILAC

A

Stable isotopes labelling amino acid in cell cultures

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10
Q

Pros of MS quantification

A
  • Can be use to all sample
  • High rate of labelling or tagging
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11
Q

Explain SILAC

A

Protein abundance depicted by intensity of labels

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12
Q

Procedure of SILAC

A
  • Cell grown in light & heavy isotopes containing media
  • Harvest & lyse cell
  • Quantify extracted protein
  • SDS PAGE
  • Excise bands & trypsin digestion
  • LC MS MS
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13
Q

Types of labelling of in vitro

A
  • Proteolytic
  • ICAT
  • iTRAQ
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14
Q

Explain proteolytic labelling

A
  • Incorporation of oxygen isotopes in peptide during proteolytic digestion
  • Oxygen isotopes label at C terminal of release peptide
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15
Q

Chemical reaction of protein with reagents in ICAT involve

A
  • Iodoacetamide residue bind SH of cysteine
  • Linker (hydrogen or deuterium atom)
  • Biotin: permit isolation of molecules by affinity chromatography
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16
Q

Pros of ICAT

A
  • High specificity
  • High sensitivity
  • Applicable to most sample
17
Q

Explain iTRAQ

A
  • Simultaneous analysis up to 8 samples
  • Labelling of peptide with isobaric tags & produce different ions during fragments
18
Q

3 components of iTRAQ

A
  • Moiety reactive towards amino acids group
  • Balance group
  • Reporter group
19
Q

Procedure of iTRAQ

A
  • Sample
  • Protein extraction followed by enzymatic digestion
  • iTRAQ label
  • Combine label digest
  • Analysis by LC-MS/MS

BMS661: PROTEOMICS (6 decks)

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