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BI407: Tumor Biology > L4 > Flashcards

L4 Flashcards

1
Q

Why are peptides preferred in mass spectometry

A
  • easier than proteins to fractionate by LC
  • fragment more efficiently than proteins
  • resulting spectra are easier to interpret for protein identification
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2
Q

sample prep for in-solution sample proteins

A
  • Denaturation with strong chaotropic agents (urea or thiourea)
  • Disulfide reduction using diothiothretiol (DTT)
  • Alkylation with iodoacetamide
  • Digestion with endoproteinases eg trypsin, chymotrypsin
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3
Q

Alternative to in-solution protein DRAD

A

protein separation by 1D or 2D gel electrophoresis

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4
Q

Sample. buffer for MS contains

A
  • Chaotropes, e.g. Urea and thiourea
  • Ionic, non-ionic or zwitterionic detergent (e.g. SDS)
  • Reducing Agent (e.g. DTT)
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5
Q

How do the neutral chaotropes work

A

they solubilize and unfold most proteins to their fully random conformation

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6
Q

Urea is used at what concentration

A

5-7M. Competes with H-Bonds.Denatures proteins

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7
Q

Thiourea is used at what concentration

A

2M

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8
Q

Function of SDS

A

ensures complete sample solubilisation and prevents aggregation through hydrophobic interactions. disrupts non-covalentinteractions in native proteins

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9
Q

use of reducing agent DTT

A

– to break any disulphide bonds
– maintain proteins in their fully reduced state

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10
Q

alternative to DTT as a reducing agent

A

β-mercaptoethanol

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11
Q

Methods of physical lysis

A
  • dounce pestles
  • sonication
  • homogenisation
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12
Q

why is proteolysis important

A

to break down large proteins into smaller peptides that are compatible with the mass spectrometer’s detection capabilities

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13
Q

Why trypsin?

A
  • its ability to generate peptides of suitable size for MS analysis
  • impart a consistent charge to peptides
  • exhibit high enzymatic activity and specificity
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14
Q

Trypsin

A
  • cleaves at the carboxylic side of lysine and arginine side chains
  • The sizes of the peptide fragments obtained after trypsin digestion, represent the peptide mass fingerprint and are characteristics of each protein.
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15
Q

How Does Mass Spec “Sequence” Proteins?

A
  1. Database eg UNIPROT
  2. Digestion in silico
  3. Measurement of peptide masses and MS/MS spectra
  4. Theoretical MS/MS spectra recorded
  5. Match barcode to the MS/MS spectrum
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16
Q

Challenges With Proteolysis

A
  • Protein folded too tightly –Protease can’t access
  • Protein is insoluble and require additives
17
Q

Solution to problem

A
  • Urea, Guanidine HCl and organic solvents (e.g., acetonitrile) –denaturing agents
  • Detergents such as Triton X-100 and SDS
18
Q

Limitations to solution

A
  • Protease Inhibition
  • Unwanted side effects (i.e., protein modification).
  • Detergents hurt the hardware (LC and Mass Spec)!
19
Q

Why is trypsin not enough for proteolysis

A
  • inadequate length for MS analysis
  • Tryptic cleavage sites might not be accessible due to PTM’s
  • Inefficient Digestion of Certain Proteins by trypsin
    To address these limitations, the use of additional proteases alongside trypsin is warranted. Shown to increase protein identication by 20%
20
Q

Alternative Proteases For Mass Spec

A
  • Lys-C, Glu-C, Asp-N, Chymotrypsin, Arg-C, Pepsin
21
Q

Chymotrypsin

A
  • serine endoprotease derived from bovine pancreas
  • hydrolyzes at the carboxyl side of aromatic aa
  • optimum pH: 7-9
22
Q

Thermolysin

A
  • thermostable methalloproteinase
  • cleaves at N-terminus of hydrophobic residues leucine, phenylalanine, valine
  • optimum temp; 65-85
  • optimum pH: 5-8.5
23
Q

Compound separation modes

A

1.Reversed-phase chromatography
2.Normal-phase and adsorption chromatography
3.Ion exchange chromatography
4.Size exclusion chromatography

24
Q

HPLC

A

consists of a tube (column) packed with tiny absorbent particles eg silica, alumina, and cellulose (stationary phase) with a liquid (mobile phase) forced through the column by high pressure delivered by a pump

25
Q

Liquid chromatogram

A

output measured by detector after components have been separated by interactions with packing material

26
Q
A

BI407: Tumor Biology (3 decks)

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