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Proteomics Informatics – Analysis of mass spectra: signal processing, peak finding, and isotope clusters (Week 3)

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Proteomics Informatics – Analysis of mass spectra: signal processing, peak finding, and isotope clusters  (Week 3). Charge-State Distributions. MALDI. ESI. 1+. 2+. 3+. intensity. intensity. Peptide. 4+. 1+. 2+. mass/charge. mass/charge. M - molecular mass n - number of charges - PowerPoint PPT Presentation

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Page 1: Proteomics Informatics –

Proteomics Informatics – Analysis of mass spectra: signal processing, peak finding, and isotope clusters (Week 3)

Page 2: Proteomics Informatics –

Charge-State Distributions

mass/charge

inte

nsi

tyMALDI ESI

mass/charge

inte

nsi

ty

1+

1+ 2+

3+

4+

Peptide

Protein

2+

nnHM

zm M - molecular mass

n - number of chargesH – mass of a proton

mass/charge

inte

nsi

ty

mass/charge

inte

nsi

ty 1+ 27+2+

3+

4+

MALDI ESI

5+

31+

Page 3: Proteomics Informatics –

Charge-State

Example:

peptide of mass 898 carrying 1 H+ = (898 + 1) / 1 = 899 m/z

carrying 2 H+ = (898 + 2) / 2 = 450

m/z carrying 3 H+ = (898 + 3) / 3 = 300.3

m/z

nnHM

zm M - molecular mass

n - number of chargesH – mass of a proton

Page 4: Proteomics Informatics –

m = 1035 Da m = 1878 Da m = 2234 Da

Isotope Distributions

m/z m/z m/z

Inte

nsi

ty

0.015% 2H1.11% 13C 0.366% 15N0.038% 17O, 0.200% 18O, 0.75% 33S, 4.21% 34S, 0.02% 36S

Only 12C and 13C:p=0.0111n is the number of C in the peptidem is the number of 13C in the peptideTm is the relative intensity of the peptide m 13C

𝑇𝑚=( 𝑛𝑚)𝑝𝑚(1−𝑝)𝑛−𝑚

12C14N16O1H32S

+1Da

+2Da

+3Da

Page 5: Proteomics Informatics –

Isotope distributions

Peptide mass

Inte

nsi

ty r

atio

Peptide mass

Inte

nsi

ty r

atio

m/z

monoisotopicmass

GFP 29kDa

Page 6: Proteomics Informatics –

Resolution

Resolution = minimum peak separation, M, which allows to distinguish two ion species

Rela

tive Inte

nsi

ty

m/z

I II II501.5 502.0500.5500.0499.5

500

50 %

Resolution = M/M = 500/0.5 = 1000

M = full width at half maximum (FWHM)

R = M

M= resolving power

Page 7: Proteomics Informatics –

Resolution

Page 8: Proteomics Informatics –

• What resolution do we need to differentiate a 1600 Da peptide that carries either an acetylation (+ 42.0100) or trimethylation (42.0464 )?

• R = 1600/0.0364 = 43,956

R = M

M= resolving power

Resolution

Page 9: Proteomics Informatics –

Isotope Clusters and Charge State

m/z

Inte

ns

ity

1+1

1

1

m/z

Inte

ns

ity

2+0.5

0.5

0.5

m/z

Inte

ns

ity

3+0.33

0.33

0.33

Page 10: Proteomics Informatics –

Isotope Clusters and Charge State

m/z

Inte

ns

ity

Possible to Determine Charge?

Yes

Yes

Maybe

No

Page 11: Proteomics Informatics –

432.8990

433.2330

433.5671

433.9014

713.3225

713.8239

714.3251

714.8263

What is the Charge State?

D between the isotopes is 0.5

Da

D between the isotopes is 0.33

Da

Page 12: Proteomics Informatics –

Noise

Page 13: Proteomics Informatics –

Smoothing

Page 14: Proteomics Informatics –

Smoothing

Page 15: Proteomics Informatics –

Adaptive Background Correction (Unsharp masking)

wlk

wlk

kIw

dwdlI )(

12),,('

Unsharp masking

Original

Page 16: Proteomics Informatics –

Adaptive Background Correction

Page 17: Proteomics Informatics –

Smoothing and Adaptive Background Correction

Page 18: Proteomics Informatics –

Peak Finding

m/z

Inte

ns

ity

wlk

wlk

kIlS )()(

Find maxima of

The centroid m/z of a peak

wlk

wlk

wlk

wlk

kI

kzm

kI

)(

)()(

Page 19: Proteomics Informatics –

Peak Finding

m/z

Inte

ns

ity

The signal in a peak can beestimated with the RMSD

22

2

//||

))((w

wlkIkI

and the signal-to-noise ratio of a peak can be estimated by dividing the signal with the RMSD of the background

Page 20: Proteomics Informatics –

Estimating peptide quantity

Peak heightCurve fittingPeak area

Peak heightCurve fitting

m/z

Inte

ns

ity

Page 21: Proteomics Informatics –

Time dimension

m/z

Inte

ns

ity

Tim

e

m/z

Tim

e

Page 22: Proteomics Informatics –

Sampling

Retention Time

Inte

nsi

ty

Page 23: Proteomics Informatics –

0

5

10

15

20

25

30

0.8 0.85 0.9 0.95 1

3 points

0

20

40

60

80

100

120

140

0.8 0.85 0.9 0.95 1

3 points

5%

Acquisition time = 0.05s

5%

Sampling

Page 24: Proteomics Informatics –

0.5

0.6

0.7

0.8

0.9

1

1.1

1 2 3 4 5 6 7 8 9 10

Th

res

ho

lds

(90

%)

# of points

Sampling

Page 25: Proteomics Informatics –

What is the best way to estimate quantity?

Peak height - resistant to interference- poor statistics

Peak area - better statistics - more sensitive to

interference

Curve fitting - better statistics- needs to know the peak

shape- slow

Page 26: Proteomics Informatics –

Web Tool

http://10.193.36.101/plot-filter-cgi/plot_filter.pl or http://10.193.36.219/plot-filter-cgi/plot_filter.pl

Page 27: Proteomics Informatics –

Web Tool

http://10.193.36.101 or http://10.193.36.219

Page 28: Proteomics Informatics –

Proteomics Informatics – Analysis of mass spectra: signal processing, peak finding, and isotope clusters (Week 3)