Quantification
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Quantification. Setup. ?. ?. Myc-Protein. Flag-Protein. ?. ?. ‘Heavy’. ‘Light’. Arg (6)Lys(4). Immunoprecipitation with FLAG antibody. Specific “Bait” . Specific “Interactor” . Non-specific. 000431_B06_P003075_B07_A00_R1. #. 4734. RT:. 38.00. AV:. 1. NL:. 1.99E6. T:.

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Quantification

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Quantification

Quantification


Setup

Setup

?

?

Myc-Protein

Flag-Protein

?

?

‘Heavy’

‘Light’

Arg(6)Lys(4)

Immunoprecipitation with FLAG antibody

Specific

“Bait”

Specific

“Interactor”

Non-specific


Example quantification of the bait protein

000431_B06_P003075_B07_A00_R1

#

4734

RT:

38.00

AV:

1

NL:

1.99E6

T:

FTMS + p NSI Full ms [300.00-2000.00]

483.22

100

95

90

85

80

75

70

65

60

55

Relative Abundance

50

483.72

45

40

35

30

25

20

484.22

15

10

484.28

486.23

483.08

5

484.72

482.72

483.28

485.23

486.73

487.86

484.07

0

482.5

483.0

483.5

484.0

484.5

485.0

485.5

486.0

486.5

487.0

487.5

488.0

m/z

Example: Quantification of the Bait protein

Bait protein peptide: Scan number 4602, zoom in at m/z 483

Sequence is :VDVCTTDR

As the peptide is doubly charged and contains one heavy R (6)

The heavy labeled peptide should be vissible at +3 m/z

Light peptide


Ratio

Ratio

The ratio can be calculated from:

Comparing intensities in the MS1 scan i.e. summing up all isotope intensities for both heavy and light peptide and dividing H/L

483.22

100

95

90

85

80

75

70

65

60

55

Relative Abundance

50

483.72

45

40

35

30

25

20

484.22

15

10

484.28

486.23

483.08

5

484.72

482.72

483.28

485.23

486.73

487.86

484.07

0

482.5

483.0

483.5

484.0

484.5

485.0

485.5

486.0

486.5

487.0

487.5

488.0

m/z


Ratio1

Ratio

The ratio can be calculated from:

2. Making extracted ion chromatograms (XIC) of the peptides and calculating the area under the curve and taking the ratio of those areas. This is the safer methods as intensities might change over the elution time and peptides might not co-elute


Quantification

Name: Matrikelnumber:

Exercise 1

In the quantification raw file go to scan number 8874

Zoom into the peak at m/z 428

This is a light labeled peptide. The Heavy labeled peptide contains on Lys(4)

Determine the charge state of the peptide

Look for the Heavy labeled peptide

Measure the intensity of all the isotopes for both light and heavy peptide

Calculate the ratio


Quantification

Name: Matrikelnumber:

Exercise 2

In the quantification raw file go to scan number 12996

Zoom into the peak at m/z 818

This is a light labeled peptide. The Heavy labeled peptide contains on Arg(6)

Determine the charge state of the peptide

Look for the Heavy labeled peptide

Make an extracted ion chromatogram of the two peaks

Overlay and calculate the peak area

Calculate the ratio


Automated quantification

Automated quantification

  • MaxQuant performs automatic quantification and identification of proteins by finding the ratios between light and heavy labeled peptides. Protein ratio’s are then derived from the median of the peptide ratio’s.

  • Perseus is a software that has statistical tools that allow you to look at the data


Perseus

Perseus

  • Load the file: AffinityPurification_SILAC.txt into Perseus

  • Log2 transform the Intensity and Ratio columns

  • Make a scatterplot

  • Should the data be normalized???

    Which proteins are changing significantly

    Processing Significance B

    (use for truncation P-value)


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