Proteomics Informatics Analysis of mass spectra: signal processing, - - PowerPoint PPT Presentation
Proteomics Informatics Analysis of mass spectra: signal processing, peak finding, and isotope clusters (Week 3) Charge-State Distributions MALDI ESI 1+ 2+ intensity intensity 3+ Peptide 4+ 1+ 2+ mass/charge mass/charge m M nH
Proteomics Informatics – Analysis of mass spectra: signal processing, peak finding, and isotope clusters (Week 3)
Charge-State Distributions
mass/charge intensity MALDI ESI mass/charge intensity 1+ 1+ 2+ 3+ 4+ Peptide Protein 2+
n nH M z m + =
M - molecular mass n - number of charges H – mass of a proton mass/charge intensity mass/charge intensity 1+ 27+ 2+ 3+ 4+ MALDI ESI 5+ 31+
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
n nH M z m + =
M - molecular mass n - number of charges H – mass of a proton
m = 1035 Da m= 1878 Da m = 2234 Da
Isotope Distributions
m/z m/z m/z Intensity
0.015% 2H 1.11% 13C 0.366% 15N 0.038% 17O, 0.200% 18O, 0.75% 33S, 4.21% 34S, 0.02% 36S Only 12C and 13C: p=0.0111 n is the number of C in the peptide m is the number of 13C in the peptide Tm is the relative intensity of the peptide m 13C
12C 14N 16O 1H 32S
+1Da +2Da +3Da
Isotope distributions
Peptide mass Intensity ratio Peptide mass Intensity ratio
m/z monoisotopic mass GFP 29kDa
Resolution
Resolution = minimum peak separation, ∆M, which allows to distinguish two ion species
Relative Intensity m/z
I I I I I
501.5 502.0 500.5 500.0 499.5
500
50 %
Resolution = M/∆M = 500/0.5 = 1000 ∆M = full width at half maximum (FWHM)
R = M ∆M = resolving power
Resolution
a 1600 Da peptide that carries either an acetylation (+ 42.0100) or trimethylation (42.0464 )?
R = M ∆M = resolving power
Resolution
Isotope Clusters and Charge State
m/z Intensity 1+ 1 1 1 m/z Intensity 2+ 0.5 0.5 0.5 m/z Intensity 3+ 0.33 0.33 0.33
Isotope Clusters and Charge State
m/z Intensity Possible to Determine Charge? Yes Yes Maybe No
432.8990 433.2330 433.5671 433.9014 713.3225 713.8239 714.3251 714.8263
What is the Charge State?
∆ between the isotopes is 0.5 Da ∆ between the isotopes is 0.33 Da
Noise
Smoothing
Smoothing
Adaptive Background Correction (Unsharp masking)
+ = − =
w l k w l k
Unsharp masking Original
Adaptive Background Correction
Smoothing and Adaptive Background Correction
Peak Finding
m/z Intensity
∑
+ = − =
=
w l k w l k
k I l S ) ( ) ( Find maxima of The centroid m/z of a peak
∑ ∑
+ = − = + = − =
⋅
w l k w l k w l k w l k
k I k z m k I ) ( ) ( ) (
Peak Finding
m/z Intensity
The signal in a peak can be estimated with the RMSD 2
2 2
/
/ | |
) ) ( (
w
w l k
I k I ∑ > < −
< −
and the signal-to-noise ratio of a peak can be estimated by dividing the signal with the RMSD of the background
Estimating peptide quantity
Peak height Curve fitting Peak area Peak height Curve fitting
m/z Intensity
Time dimension
m/z Intensity
Time
m/z Time
Sampling
Retention Time Intensity
5 10 15 20 25 30 0.8 0.85 0.9 0.95 1
3 points
20 40 60 80 100 120 140 0.8 0.85 0.9 0.95 1
3 points
5%
Acquisition time = 0.05σ
5%
Sampling
0.5 0.6 0.7 0.8 0.9 1 1.1 1 2 3 4 5 6 7 8 9 10
Thresholds (90%) # of points
Sampling
What is the best way to estimate quantity?
Peak height
Peak area
Curve fitting
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
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
Proteomics Informatics – Analysis of mass spectra: signal processing, peak finding, and isotope clusters (Week 3)