application of mass spectrometry burgers… · = ½.m.v2. u pot = e kin m.g.h = ½.m v2 m.g.h =...
TRANSCRIPT
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Mass spectrometry: how can it be used for medical research?
Peter Burgers
Laboratory of Neuro-Oncology & Clinical and Cancer Proteomics
Department of Neurology Erasmus Medical Center, Rotterdam
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Proteomics
Proteomics aims to describe or explain biological phenomena
in terms of qualitative and/or quantitative changes in proteins
of cells and extra cellular biological materials
Why is proteomics important?
Neurodegenerative disorders and brain tumors
• Alzheimer’s Disease
• Parkinson’s Disease
• Glioma
• Multiple sclerosis
Proteins play a key role
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Mass spectrometry
A mass spectrometer is a device which converts neutral
molecules into ions and then sorts these ions according
to their masses
mass
abundance
m1
m2
m3
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h m
Gravitational field
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h m Upot = m.g.h
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Ekin = ½.m.v2
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Upot = Ekin
m.g.h = ½.m v2
m.g.h = ½.m v2
g.h = ½. v2
There is no m in this equation
√ 2.h g t =
Galileo Galileï (1564-1642)
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m +
- - - - - - - - - - - - - - - - V
h
Electric field
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m +
- - - - - - - - - - - - - - - - V
Upot = e.V h
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+ - - - - - - - - - - - - - - - - V
Ekin = ½.m.v2
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Upot = Ekin
e.V = ½.m v2
Now there is an m in this equation!!!
√ m
2eV t = h
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+ +
Time-of-Flight
½mv2 = eV = constant
Mass 1,000: v = 7 x 104 m/s or 250,000 km/hr
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Founder of Mass Spectrometry: Sir J.J. Thomson
1905
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Calutron F.O.M. (Amsterdam) c. 1950
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Control panels and operators for calutrons at Oak Ridge
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AEI MS902 c. 1972
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Era of mass spectrometry
1930 - 1950 nuclear physics
1950 - 1990 chemistry: GC-MS
1990 - life sciences: ESI and MALDI
2002 Nobel prize: Fenn, Tanaka "for their development of soft desorption ionisation methods for mass
spectrometric analyses of biological macromolecules"
Physics chemistry life sciences
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Forensic sciences (murder cases)
Doping (Olympic games)
Art (forgeries)
Historical issues (Shroud of Turin)
Life sciences (proteomics, metabolomics)
Nuclear physics (isotope enrichment)
Enriching 28Si for quantum computing
Sniffing smuggled money
Flavors ans fragrances (coffee, perfume)
Bacterial identification
Antibiotic resistance screening
Applications of Mass Spectrometry
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Volume 2
Issue 8
Table of Contents:
How the Legal and Medical Systems failed Patricia and Ryan Stallings
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How mass spectrometry freed a mother convicted of baby murder
The Patricia Stallings case
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The Stallings Case
July 7 1989 Ryan becomes ill
July 9 1989 Ryan is hospitalized
July 12 1989 Ethylene glycol found in Ryan’s blood
HO-CH2-CH2-OH
How measured ? Gas chromatography : retention index
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July 17 1989 Ryan discharged from hospital foster home
Sept 1 1989 Mrs Stallings feeds Ryan by bottle
Sept 4 1989 Ryan dies
Mrs Stallings charged with murder
Sentenced to life imprisonment
The Stallings Case, ctd
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The Stallings Case, an unexpected turn of events
April 1990 David Jr. is born foster home
April 1990 Diagnosis: Methylmalonic Acidemia (MMA)
Byproducts: organic acids: CH3CH2COOH
How measured: Gas chromatography – mass spectrometry
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Mass spectrometer
Mass spectrum
Identification
Gas Chromatography – Mass Spectrometry
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31
45
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Ion source extraction mass analysis
Mass spectrometry (MS)
Mass spectrometry in life sciences
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Ionization techniques
for biomolecules
• Ablation (Matrix-assisted laser desorption/ionization)
MALDI
Crystal gas-phase
• Nebulization (Electrospray Ionization) ESI
Liquid gas-phase
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•High resolution
•Very sensitive
•Low throughput
•High speed sequencing
LC-ESI-Orbitrap
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Mass measurements
Mass accuracy: < + or - 0.1 ppm systematic error
Mass precision: ± 0.5 ppm experimental uncertainty
Measured mass 1633.7234 ± 0.0008 (30 measurements)
Real mass 1633.7236
Accurate : telling the truth
Precise : telling the same story over and over again
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http://www.mathsisfun.com/accuracy-precision.html
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Low Accuracy
High Precision
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Low Accuracy
High Precision
High Accuracy
Low Precision
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Low Accuracy
High Precision
High Accuracy
High Precision High Accuracy
Low Precision
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The principle of protein
identification by MS
1. Peptide Mass Fingerprinting (PMF)
Objective: generate a unique set of numbers for any protein
Protein sequence
MRRAALWLWLCRALALRL
QPVDERGIVEECCFRATG
ASQRTGASQSLLDRLVAF
MLYRMWLKVGLIFAVCLVL
EEPKQANGGAYQKPRDE
GSYSLEEPKQANGGAYQK
Enzyme specific peptides
ALALR
GIVEECCFR
TGASQSLLDR
LVAFMLYRMWLK
LEEPKQANGGAYQKPR
DEGSYSLEEPKQANGGAYQKPTK
Peptide masses
563.326
1053.457
1187.607
1323.856
1953.185
2. Peptide fragmentation (MS/MS)
GIVEECCFR
Sequencing: b-ions
y-ions
Enzymatic
hydrolysis
GIVEECCFR
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No virus
Influenza
24hrs
A549 cell line
1180.6237
1198.7098
1189.6154
1186.6815
mass
intensity
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The principle of protein
identification by MS
1. Peptide Mass Fingerprinting (PMF)
Objective: generate a unique set of numbers for any protein
Protein sequence
MRRAALWLWLCRALALRL
QPVDERGIVEECCFRATG
ASQRTGASQSLLDRLVAF
MLYRMWLKVGLIFAVCLVL
EEPKQANGGAYQKPRDE
GSYSLEEPKQANGGAYQK
Enzyme specific peptides
ALALR
GIVEECCFR
TGASQSLLDR
LVAFMLYRMWLK
LEEPKQANGGAYQKPR
DEGSYSLEEPKQANGGAYQKPTK
Peptide masses
563.326
1053.457
1187.607
1323.856
1953.185
2. Peptide fragmentation (MS/MS)
GIVEECCFR
Sequencing: b-ions
y-ions
Enzymatic
hydrolysis
GIVEECCFR
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b ions (N-term)
(N-term residue +1) + Σ(residues)
y ions (C-term)
(C-term residue + 19) + Σ(residues)
Calculating b and y ions
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Calculate b and y ions for
HPDYSVVLLLR MH+ = 1311.7419
b ions
y ions
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Calculate b and y ions for
HPDYSVVLLLR MH+ = 1311.7419
b ions
138
235
350
513
600
699
798
911
1024
1137
1293
(+ 18 = 1311)
y ions
175
288
401
514
613
712
799
962
1077
1174
1311
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Conclusions
Mass spectrometry has evolved
A lot of challenges
Development methodology
1 10-12
albumin cytokine
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fM pM nM µM mM M
glucose drugs nucleic
acids
proteins
Abundance of proteins relative
albumin cytokine
Physiological
salt
water
aM zM yM
1 molecule / L 1025 molecules / L
......................
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fmol pmol nmol µmol
glucose drugs nucleic
acids
proteins
Abundance of proteins absolute
albumin cytokine
Physiological
salt
amol zmol ymol
1 molecule (1/N)
......................
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Proteomics and mass spectrometry: a marriage made in heaven