Transcript Bioc158

I.
I.
Introduction
Introduction
1. Definition: Protein Characterization/Proteomics
i. Classical Proteomics
ii. Functional Proteomics
2. Mass spectrometery
I. Advantages in Studying Proteins
II. General configuration
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I.
Introduction
Why is proteomics necessary?
(Pandey, A., Mann, M., Nature, 405, 837-846, 15June2000)
• complete sequences of genomes is not sufficient to elucidate
biological function
• existence of an open reading frame in genomic data doesn’t imply
the existence of a functional gene (8% error in annotations for 340
genes from theMycoplasma genitalium genome) verification of gene
product is an important first step in genome annotating
• modifications of proteins are not apparent from the DNA sequence
(isoforms, post-translational modifications)
• mRNA may or may not correlate with protein level
• localization of gene product can be determined
• protein-protein interaction and molecular composition of cellular
structures such as organelles can be determined only at the
protein level
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I.1.
Definition:
Protein Characterization/Proteomics
Protein Characterization
• one protein at a time
“Proteome” (Wilkins and Williams)
• entire protein complement of a given genome
“Proteomics”
•
•
•
naturally: study of the proteome
catalog and characterize these proteins
large scale analysis of proteins within a single experiment
(or series thereof)
Proteomics is classified into two disciplines
 classical proteomics
 functional proteomics
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I.2.
Mass Spectrometry
Definition/Requirement: Mass Spectrometry
•
technique to determine the relative weight of
atoms and molecules by separation of charged
atoms and molecules based (ions) on their mass
in the gas phase.
(first mass spectrometer 1910, Ne-isotope 20/22)
•
molecules need to be in the vapor phase
•
molecules need to be ionized
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I.2.i.
Advantages in Studying Proteins
 High mass accuracy
• 10 ppm: 1000 Da ± 0.01 Da (UNC)
 Identification of proteins via database searching
 Detection of post-translational modifications
 High sensitivity
• femto-mol (=50 pg of 50 kDa protein) (UNC)
 Study proteins at physiological level
 Provides sequence information
 Identification of modification sites
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I.2.ii. General configuration
ion
source
mass analyzer
detector
•
ion source:
ionization and transfer of
molecules into the gas phase
•
mass analyzer:
separation of the molecules
due to their mass
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II.
II.
Mass Spectrometry
Mass Spectrometry
1. Analytical Parameters/Definitions
i. Molecular weight
ii. Mass Accuracy
iii. Chemical Background vs. Peak
iv. Mass Resolution
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II.1.i. Molecular weight
Mono-isotopic molecular weight:
•
mass of the molecule which elementary
composition possesses only the most natural
abundant isotopes
(12C, 1H,16O,14N, etc.)
Average-isotopic molecular weight:
•
calculated mass of the molecule out of a
elementary composition possesses isotopes in the
proportion corresponding to their natural
abundances
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II.1.i. Molecular weight
Masses and Abundance of isotopes of natural elements:
Element
C
H
O
N
S
O#
M#
6
12
12.000000
98.90
13
13.003355
1.10
1
1.007825
99.985
2
2.014102
0.015
16
15.994915
99.762
17
16.999131
0.038
18
17.999159
0.200
14
14.003074
99.634
15
15.000109
0.366
32
31.972072
95.02
33
32.971459
0.75
34
33.967868
4.21
36
35.967079
0.02
1
8
7
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Mass
Relative
abundance
Average mass
12.011
1.00794
15.9994
14.0067
32.066
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II.1.i. Molecular weight
Expected mass:
Acetic acid:
Isotopes:
Monoisotopic:
C 2 H 4 O2
12C, 13C, 1H, 2H,16O,17O, 18O
90 possible formulas
6 formulas with
significant abundances
12C 1H 16O
2
4
2
composition
mass
relative abundance
12C 1H 16O
2
4
2
60.02113
100.000
12C13C1H 16O
4
2
61.02448
2.224
12C 1H 16O17O
2
4
61.02534
0.076
12C 1H 2H16O
2
3
2
61.02741
0.060
12C 1H 16O18O
2
4
62.02538
0.401
13C 1H 16O
2
4
2
62.02784
0.012
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II.1.ii. Chemical Background vs. Peak
Definition:
•
peak: must be at least twice the baseline; S/N > 2
•
peak: more than one data point is needed to define
a peak
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chemical background:
chemical must be evaluated to show peak comes
from sample
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II.1.ii. Chemical Background vs. Peak
Molecular weight peak width increases with mass:
Peptide
Mass MH+
Rel. abundance
Leu5-enkephalin
C28H38N5O7
556.28
557.28
558.28
559.29
100.00
34.04
6.89
0.89
monoisotopic
PNGF fragment
C89H140N27O26
2003.05
2004.05
2005.05
2006.05
2007.05
2008.06
88.61
100.00
60.14
25.26
7.78
1.68
monoisotopic
base peak
Ubiquitin
C378H630N105O118S
8560.62
8561.63
8562.63
8563.63
8564.63
8565.64
8566.64
8567.64
8568.65
8569.65
8570.65
8571.66
4.14
19.02
47.27
78.47
98.75
100.00
86.58
63.62
40.39
22.09
8.81
3.19
monoisotopic
base peak
556.64
average mass
2004.26
average mass
8565.89
average mass
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II.1.iii. Mass Accuracy
Mass accuracy
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mass accuracy = ΔM(calculated-observed)
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•
in Da, amu, ppm (parts per million)
ppm: [(m/zobs-m/zcalc)/m/z calc] x 106
Mass spectrometer
Mass accuracy for peptides
FTICR
highest
~ 1-5 ppm
TOF
high
~ 10-50 ppm
Ion-trap/quadrupoles
moderate/low
> 500 ppm
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II.1.iv. Mass Resolution
Definition:
•
Resolution = R = M/ΔM
•
ΔM : width at half height
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estimating unit resolution: M/0.5
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estimating complete isotopic (M, M’) resolution:
ΔM
m/z
([M+M’]/2)/([M-M’] x 0.5)
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II.1.iv. Mass Resolution
Example acetic acid (see slide 14):
•
“unit” resolution:
60 Da and 61 Da
 R=60/0.5=120
•
“complete” isotopic resolution:
61.02448 Da and 61.02534 Da
 R=61.02491/0.00043=141,918
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II.1.iv. Mass Resolution
Resolution illustrated: angiotensin II C50H73N13O12
(from K.G. Owens, M.M. Vestling “Fundamentals and Applications of MALDI-TOF-MS”,
A Short Course Spnosored by the American Society for Mass Spectrometry)
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II.1.iv. Mass Resolution
Resolution illustrated: ubiquitin C378H630N105O118S
(from K.G. Owens, M.M. Vestling “Fundamentals and Applications of MALDI-TOF-MS”,
A Short Course Spnosored by the American Society for Mass Spectrometry)
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