Mass spectrometry: how can it be used for medical research? Peter - - PowerPoint PPT Presentation

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Mass spectrometry: how can it be used for medical research? Peter - - PowerPoint PPT Presentation

Oct 20, 2022 398 likes •910 views

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 Proteomics Proteomics aims to describe or

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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

  • f 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