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Proteomics

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Title: PowerPoint Presentation Author: mike myers Last modified by: Lenovo User Created Date: 9/15/2003 7:49:23 PM Document presentation format: On-screen Show (4:3) – PowerPoint PPT presentation

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Title: Proteomics


1
Proteomics
What is it? How is it done? Are there
different kinds? Why would you want to do it
(what can it tell you)?
2
The Central Dogma of Molecular Biology
DNA
transcription
RNA
translation
Protein
Key Concept Proteins do the work everything
else is mostly information
Key Concept Proteomics is the high
throughput analysis of proteins.
3
The Central Dogma-omics
4
The Central Dogma-omics
Key Concept Complexity increases the farther
away from information you get
Key Concept Chemical complexity also increases
the farther away you get
5
Protein Machines
The polyAdenylation Machinery
The Proteosome
Key Concept Biochemical functions are carried
out by multi-protein machines
Key Concept A Protein Function can be inferred
by its binding partners
Key Concept Knowledge of a Machines components
is required to understand
how it works and how it is regulated
6
Protein Networks 2 Steps
Key Concept Protein Machines are organized into
larger Networks
7
Proteins are Organized in Super Networks
Key Concept The proteome is HIGHLY Networked
8
Key Concept Proteins vary widely in concentration
9
Major Types of Proteomics
Interactomics Mapping ProteinProtein
Interactions -Yeast 2-hybrid techniques -high
throughput protein identification by Mass
Spectrometry Survey Proteomics Qualitative or
Quantitative Analysis of the protein
component -whole organism, tissue, cell
type, or subcellular compartment -2D gel
electrophoresis -gtMS -typically a few 100
proteins -Multidimensional LC-gtMS/MS -typi
cally a few 1000 proteins Identification of
Biomarkers
10
Information Trade Offs
Vida infra G. MacBeath
11
Proteins are Organized in Super Networks
Key Concept The proteome is HIGHLY Networked
12
Time of Flight Mass Spectrometer
Timed ion selector
Laser
Reflector
Sample plate







Flight tube
Accelerating field
Detectors
Key Concept Mass Spectrometers can only measure
charged species.
Key Concept All Mass Spectrometers have at least
2 parts an ion source and
a mass analyzer
13
A Quadrupole Mass Filter
14
Ion Trap Mass Spectrometer
Key Concept Mass Spectrometers can only measure
ions and more accurately, the behavior of ions in
an electric field
15
A Generic Mass Spectrum
Key Concept A mass spectrum is a graphical
representation of the the data, with intensity
on the y axis and m/z on the x axis
16
Important Properties of a Mass Spectrum
Mass is expressed as m/z (mass/charge). m1000
z1 m/z 1001 m1000 z2 m/z 501 m1000
z3 m/z 334.3 Intensity is usually
normalized to 100 Intensity can be expressed as
ion current or ion counts The intensity is
related to abundance, but only when you are
referring to the same compound. Mass accuracy is
an important parameter that affects downstream
analysis. Mass accuracy is usually expressed as
ppm. EM1000.1 TM1000
ppm (0.1/1000) x 106
100
ppm
17
How to Identify Proteins
Proteolysis
Proteolysis
Proteolysis
Key Concept Each unique protein will give rise
to a unique set of peptides
18
Peptide Mass Fingerprinting
Key Concept A single protein yields many
proteolitic fragments
19
Peptide Mass Fingerprinting
Key Concept A mass spectrometer is a very
accurate scale!
20
Peptide Mass Fingerprinting
21
Peptide Mass Fingerprinting
ENSP00000031135
ENSP00000084795
ENSP00000198939
ENSP00000201886
ENSP00000202773
ENSP00000216019
ENSP00000216038
ENSP00000216520
ENSP00000216832
ENSP00000223129
ENSP00000225430
ENSP00000225792
ENSP00000233078

22
Peptide Mass Fingerprinting
ENSP00000031135
ENSP00000084795
ENSP00000198939
ENSP00000201886
ENSP00000202773
ENSP00000216019
ENSP00000216038
ENSP00000216520
ENSP00000216832
ENSP00000223129
ENSP00000225430
ENSP00000225792
ENSP00000233078

ENSP00000254108
ENSP00000254636
ENSP00000254719
ENSP00000254942
ENSP00000259848
ENSP00000259925
ENSP00000261366
ENSP00000261700
ENSP00000216019
ENSP00000262570
ENSP00000262584
ENSP00000262637
ENSP00000262709

23
Peptide Mass Fingerprinting
ENSP00000031135
ENSP00000084795
ENSP00000198939
ENSP00000201886
ENSP00000202773
ENSP00000216019
ENSP00000216038
ENSP00000216520
ENSP00000216832
ENSP00000223129
ENSP00000225430
ENSP00000225792
ENSP00000233078

ENSP00000263698
ENSP00000263746
ENSP00000264258
ENSP00000264293
ENSP00000269349
ENSP00000269576
ENSP00000270799
ENSP00000281154
ENSP00000283179
ENSP00000216019
ENSP00000290341
ENSP00000294823
ENSP00000296490
ENSP00000254108
ENSP00000254636
ENSP00000254719
ENSP00000254942
ENSP00000259848
ENSP00000259925
ENSP00000261366
ENSP00000261700
ENSP00000216019
ENSP00000262570
ENSP00000262584
ENSP00000262637
ENSP00000262709

24
Peptide Mass Fingerprinting
ENSP00000031135
ENSP00000084795
ENSP00000198939
ENSP00000201886
ENSP00000202773
ENSP00000216019
ENSP00000216038
ENSP00000216520
ENSP00000216832
ENSP00000223129
ENSP00000225430
ENSP00000225792
ENSP00000233078

ENSP00000263698
ENSP00000263746
ENSP00000264258
ENSP00000264293
ENSP00000269349
ENSP00000269576
ENSP00000270799
ENSP00000281154
ENSP00000283179
ENSP00000216019
ENSP00000290341
ENSP00000294823
ENSP00000296490
ENSP00000254108
ENSP00000254636
ENSP00000254719
ENSP00000254942
ENSP00000259848
ENSP00000259925
ENSP00000261366
ENSP00000261700
ENSP00000216019
ENSP00000262570
ENSP00000262584
ENSP00000262637
ENSP00000262709

ENSP00000202773
ENSP00000216019
ENSP00000216038
ENSP00000216520
ENSP00000216832
ENSP00000223129
ENSP00000225430
ENSP00000225792
ENSP00000233078
ENSP00000233084
ENSP00000233468
ENSP00000240851
Key Concept Each mass pulls out 11,000
candidate proteins
25
Peptide Mass Fingerprinting
ENSP00000031135
ENSP00000084795
ENSP00000198939
ENSP00000201886
ENSP00000202773
ENSP00000216019
ENSP00000216038
ENSP00000216520
ENSP00000216832
ENSP00000223129
ENSP00000225430
ENSP00000225792
ENSP00000263698
ENSP00000263746
ENSP00000264258
ENSP00000264293
ENSP00000269349
ENSP00000269576
ENSP00000270799
ENSP00000281154
ENSP00000283179
ENSP00000216019
ENSP00000290341
ENSP00000294823
ENSP00000254108
ENSP00000254636
ENSP00000254719
ENSP00000254942
ENSP00000259848
ENSP00000259925
ENSP00000261366
ENSP00000261700
ENSP00000216019
ENSP00000262570
ENSP00000262584
ENSP00000262637
ENSP00000202773
ENSP00000216055
ENSP00000216038
ENSP00000216019
ENSP00000216832
ENSP00000223129
ENSP00000225430
ENSP00000225792
ENSP00000233078
ENSP00000233084
ENSP00000233468
ENSP00000240851
ENSP00000244357



Key Concept Ideally only one protein should be
uniquely identified
26
Key Concept Fragments can be matched to a
database of proteins. The number of indentified
proteins is related to the mass accuracy.

27
Peptide Mass Fingerprints often FAIL to
give Significant Results
-Protein Mixtures confuse the statistical
algorithms -Not enough high quality peaks -Too
many popular masses -Possible to play with
the search parameters to give a statistically
meaningful result.
Can be overcome by performing peptide
fragmentation (AKA MS/MS)
28
Thermo Demo Movie
29
What is MS/MS
-MS/MS is a method in which a peptide is
fragmented and the masses of the fragment ions
measured.
30
What is MS/MS
-MS/MS is a method in which a peptide is
fragmented and the masses of the fragment ions
measured.
31
How do you interpret an MS/MS spectra??
A
M

A
S
R
R
E
M
P
S
L
E
P
L
A
M
R
S
P

E
L
R
A
E
M

S
L
P
R

A
E
P
M
S
L
32
A
M
R
S
P

E
L
33
How do you interpret an MS/MS spectra??
  • Steps for Manual Interpretation
  • Label the precursor masses
  • Label any obvious water losses
  • (m/z with D18)
  • Look for any peaks with D28
  • (a b ion pairs)
  • Look up the mass of the first b ion
  • in the dipeptide chart.
  • Begin building the b ion series and
  • use the y ion series for confirmation using
  • the tables.
  • Extend till the you reach the presurser mass.
  • (the last amino acid in the b ion series
    should
  • be a Lys or Arg and will be 18 relative to the
  • mass in the table.)

34
99.1
213.1
35
99.1
213.1
36
99.1
213.1
AA Code MW AA Code MW AA Code MW AA Code MW
G 57.02 T 101.04 D 115.02 H 137.05
A 71.037 C 103.01 Q 128.05 F 147.06
S 87.03 L 113.08 K 128.09 R 156.10
P 97.05 I 113.08 E 129.04 Y 163.06
V 99.06 N 114.04 M 131.04 W 186.08
37
V
99.1
213.1
AA Code MW AA Code MW AA Code MW AA Code MW
G 57.02 T 101.04 D 115.02 H 137.05
A 71.037 C 103.01 Q 128.05 F 147.06
S 87.03 L 113.08 K 128.09 R 156.10
P 97.05 I 113.08 E 129.04 Y 163.06
V 99.06 N 114.04 M 131.04 W 186.08
38
V
N
114
AA Code MW AA Code MW AA Code MW AA Code MW
G 57.02 T 101.04 D 115.02 H 137.05
A 71.037 C 103.01 Q 128.05 F 147.06
S 87.03 L 113.08 K 128.09 R 156.10
P 97.05 I 113.08 E 129.04 Y 163.06
V 99.06 N 114.04 M 131.04 W 186.08
39
Q
V
N
128
114
AA Code MW AA Code MW AA Code MW AA Code MW
G 57.02 T 101.04 D 115.02 H 137.05
A 71.037 C 103.01 Q 128.05 F 147.06
S 87.03 L 113.08 K 128.09 R 156.10
P 97.05 I 113.08 E 129.04 Y 163.06
V 99.06 N 114.04 M 131.04 W 186.08
40
I/L
113
Q
V
N
128
114
AA Code MW AA Code MW AA Code MW AA Code MW
G 57.02 T 101.04 D 115.02 H 137.05
A 71.037 C 103.01 Q 128.05 F 147.06
S 87.03 L 113.08 K 128.09 R 156.10
P 97.05 I 113.08 E 129.04 Y 163.06
V 99.06 N 114.04 M 131.04 W 186.08
41
I/L
Q
G
V
N
I
S
E
I/L
T
K
AA Code MW AA Code MW AA Code MW AA Code MW
G 57.02 T 101.04 D 115.02 H 137.05
A 71.037 C 103.01 Q 128.05 F 147.06
S 87.03 L 113.08 K 128.09 R 156.10
P 97.05 I 113.08 E 129.04 Y 163.06
V 99.06 N 114.04 M 131.04 W 186.08
42
I/L
Q
I/L
N
Q
V
G
V
N
I
S
E
I/L
T
K
AA Code MW AA Code MW AA Code MW AA Code MW
G 57.02 T 101.04 D 115.02 H 137.05
A 71.037 C 103.01 Q 128.05 F 147.06
S 87.03 L 113.08 K 128.09 R 156.10
P 97.05 I 113.08 E 129.04 Y 163.06
V 99.06 N 114.04 M 131.04 W 186.08
43
584.10
826.25
175.00
417
44
157.04
45
Proteolysis
Key Concept A sample analyzed by MALDI can
generate between 10-20 MS/MS
spectra before it is consumed.
46
How to Identify Proteins
C
E
Protein Bands are cut from gel, trypsinized and
analyzed by mass spectrometry -direct
sequencing -search a database (probability
based ID)
Key Concept A few MS/MS spectra or even MS/MS
spectra from a single
protein can be manually interpreted
47
MS/MS allows for Shot Gun Proteomics
Proteolysis
Key Concept Shot Gun Proteomics lets you look
at many things at once
48
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49
Protein ID by Mass Spectrometry
50
Protein ID by Mass Spectrometry
10,000 MS/MS per hour
Key Concept LC-MS/MS data is time restricted by
the elution profile of the peptides. Maldi is
restricted by sample consumption.
51
I/L
Q
G
V
N
I
S
E
I/L
T
K
AA Code MW AA Code MW AA Code MW AA Code MW
G 57.02 T 101.04 D 115.02 H 137.05
A 71.037 C 103.01 Q 128.05 F 147.06
S 87.03 L 113.08 K 128.09 R 156.10
P 97.05 I 113.08 E 129.04 Y 163.06
V 99.06 N 114.04 M 131.04 W 186.08
Key Concept de Novo interpretation is
computationally intensive. Most
software programs do it by Spectral Matching
52
Spectral Matching
100
acquired spectrum
0
1
Theoretical spectrum (y/b ions)
0
53
Spectra matched
100
acquired spectrum
x
0
1
theoretical spectrum (y/b ions)
0
54
Protein ID by Mass Spectrometry
55
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56
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