# pptx - Fenyolab.org

AI and Robotics

Nov 24, 2013 (4 years and 6 months ago)

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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%
2
H

1.11%
13
C

0.366%
15
N

0.038%
17
O, 0.200%
18
O,

0.75%
33
S, 4.21%
34
S, 0.02%
36
S

Only
12
C and
13
C:

p=0.0111

n is the number of C in the peptide

m is the number of
13
C in the peptide

T
m

is the relative intensity of

the
peptide m
13
C

𝑇

=


𝑝

(
1

𝑝
)


12
C

14
N

16
O

1
H

32
S

+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

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

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

Unsharp

)

w
l
k
w
l
k
k
I
w
d
w
d
l
I
)
(
1
2
)
,
,
(
'
Unsharp

Original

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

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 = 0.05
s

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

-

resistant to interference

-

poor statistics

Peak area

-

better statistics

-

more sensitive to interference

Curve fitting

-

better statistics

-

needs to know the peak shape

-

slow

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 or http://10.193.36.219

Proteomics Informatics

Analysis of mass spectra: signal processing, peak
finding, and isotope clusters

(Week 3)