1 I. Introduction 1.Definition: Protein Characterization/Proteomics i.Classical Proteomics...
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Transcript of 1 I. Introduction 1.Definition: Protein Characterization/Proteomics i.Classical Proteomics...
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I. Introduction
I. Introduction1. Definition: Protein Characterization/Proteomics
i. Classical Proteomicsii. Functional Proteomics
2. Mass spectrometeryI. 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
“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
Protein Characterization• one protein at a time
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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 massin 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: ionization and transfer ofmolecules into the gas phase
• mass analyzer: separation of the moleculesdue to their mass
ion source
mass analyzer detector
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II. Mass Spectrometry
II. Mass Spectrometry1. Analytical Parameters/Definitions
i. Molecular weightii. Mass Accuracyiii. Chemical Background vs. Peakiv. 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 O# M# Mass Relative
abundanceAverage mass
C 6 12 12.000000 98.90 12.011
13 13.003355 1.10
H 1 1 1.007825 99.985 1.00794
2 2.014102 0.015
O 8 16 15.994915 99.762 15.9994
17 16.999131 0.038
18 17.999159 0.200
N 7 14 14.003074 99.634 14.0067
15 15.000109 0.366
S 16 32 31.972072 95.02 32.066
33 32.971459 0.75
34 33.967868 4.21
36 35.967079 0.02
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II.1.i. Molecular weight
Expected mass:Acetic acid: C2H4O2
Isotopes: 12C, 13C, 1H, 2H,16O,17O, 18O
Monoisotopic: 12C21H4
16O2
90 possible formulas6 formulas with significant abundances
composition mass relative abundance12C2
1H416O2 60.02113 100.000
12C13C1H416O2 61.02448 2.224
12C21H4
16O17O 61.02534 0.07612C2
1H32H16O2 61.02741 0.060
12C21H4
16O18O 62.02538 0.40113C2
1H416O2 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
• 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-enkephalinC28H38N5O7
556.28557.28558.28559.29
100.0034.04
6.890.89
monoisotopic 556.64 average mass
PNGF fragmentC89H140N27O26
2003.052004.052005.052006.052007.052008.06
88.61100.0060.1425.26
7.781.68
monoisotopicbase peak 2004.26
average mass
Ubiquitin C378H630N105O118S
8560.628561.638562.638563.638564.638565.648566.648567.648568.658569.658570.658571.66
4.1419.0247.2778.4798.75
100.0086.5863.6240.3922.09
8.813.19
monoisotopic
base peak8565.89
average mass
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II.1.iii. Mass Accuracy
Mass accuracy
• mass accuracy = ΔM(calculated-observed)
• 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 ppmTOF high ~ 10-50 ppmIon-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
• estimating unit resolution: M/0.5
• estimating complete isotopic (M, M’) resolution:([M+M’]/2)/([M-M’] x 0.5)
ΔM
m/z
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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)