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Sigurður V. Smárason, Ph.D. New Technologies Division

Mass Spectrometry in the Biosciences: Introduction to Mass Spectrometry and Its Uses in a Company Like Decode. Sigurður V. Smárason, Ph.D. New Technologies Division. Peptides Proteins Oligonucleotides Oligosaccharides Fatty Acids Phosphoglycerides Ceramides. Steroids Prostaglandins

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Sigurður V. Smárason, Ph.D. New Technologies Division

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  1. Mass Spectrometry in the Biosciences: Introduction to Mass Spectrometry and Its Uses in a Company Like Decode. Sigurður V. Smárason, Ph.D. New Technologies Division

  2. Peptides Proteins Oligonucleotides Oligosaccharides Fatty Acids Phosphoglycerides Ceramides Steroids Prostaglandins Acylglycerols Bile Salts Phospholipids Glycophospholipids Sphingolipids Take your pick !

  3. What is Mass Spectrometry ? A fancy word for a highly precise analytical balance!!! • Analytical balances: 0.001g to 1g ± 0.0001g • Mass spectrometers: 1e-24g to 1e-19g ± 1E-25g Or 1 Da to 100.000 Da ± 0.1 Da

  4. Basic Concept:Play Ping-Pong with Molecules • Accelerates and/or changes the trajectory of a charged particle by employing electric and magnetic fields and based on the observed behavior determines its m/z • how much a particle responds to any outside electromagnetic field is determined by both its mass and charge • Higher mass => Less response • Higher charge => More response • m/z = 2m/2z , m/2z = 0.5m/z

  5. In-house available instrumentation • MALDI TOF MS • Matrix Assisted Laser Desorption Ionization • Time of Flight Mass Spectrometer • ESI QTOF LC/MS/MS • Electrospray Ionization • Quadrupole-Time of Flight Orthogonal Double Mass Spectrometer • Liquid Chromatography Separation Prior to MS Analysis • EI Quad GC/MS • Electron Impact Ionization • Quadrupole Mass Spectrometer • Gas Chromatography Separation Prior to MS Analysis

  6. What They Can Analyze: • The MALDI TOF • (Organic and) Biological Molecules – MS • The ESI QTOF • (Organic and) Biological Molecules – MS/MS • The GC/MS • “Small” Organic Molecules – MS

  7. Why the Extended Acronyms? • Because analytical chemist like to confuse ordinary people.... • ....and mass spectrometry is defined by: • The type of ionization technique employed • The type of mass analyzer(s) employed

  8. Ionization • “Soft” Ionization: MALDI, ESI • Produces intact molecular ions of the analyte • Can be either singly charged (MALDI) or multiply charged (ESI) • “Hard” Ionization: EI • Produces mainly singly charged submolecular ions of the analyte

  9. Mass Analyzers • TOF MS • Greater Sensitivity • Separation obtained by the ions traveling at different speeds • Quadrupole MS • Greater Selectivity • Seperation obtained by filtering which ion can reach the detector

  10. Mass Analyzers – Ion Paths Field Free Region TOF MS Acceleration Detector Quadrupole Quad MS

  11. Organic compound analysis Single compound or mixture analysis of small (<500 Da) organic compounds by GC/MS Single nucleotide polymorphism genotyping Measure the mass differences of the incorporated bases after a minisequencing reaction - MALDI MS Proteins/peptides Postranslational modifications - MALDI MS & ESI QTOF MS/MS Protein-ligand interactions - ESI QTOF MS Peptide sequencing (Edman) – MALDI TOF MS Selected Examples

  12. Organic Compound Analysis GC/MS total ion chromatogram: Intensity Mw= 320.35 Da min Mass spectrum at peak: m/z = 320 Intensity m/z

  13. Non-pinpoint Assay SNP Genotyping Pinpoint Assay ddA = 297.2 Da ddC = 273.3 Da ddG = 313.2 Da ddT = 288.2 Da

  14. Dm = 313.0 ddG = 313.2 Dm = 297.1 ddA = 297.2 SNP Genotyping MALDI TOF MS Intensity / A.U. m/z= 6674.0 m/z = 6971.1 m/z = 6987.0 6600 6800 7000 7200 m/z

  15. SNP Genotyping • Aquisition can be multiplexed at least 5 fold (theoretical limit ~ 30plex) • 4.7-7 sec aquistion time  4000-6000 aquisitions per 8h day  20-30k SNPs/day (5plex analysis)

  16. Higher-order structure elucidation Native vs. denatured protein Protein-protein interactions Protein-ligand interactions Modification characterization Identification Quantification Sequencing Protein/peptide Analysis

  17. peptide mixture peptide mixture S S SH cleavage reduction S HS S SH SH SH MALDI MS MALDI MS intesity intesity m/z m/z Posttranslational Modifications Intra- versus intermolecular disulfide bridges protein E E

  18. PO3 PO3 E E reflectron MALDI MS cleavage Dm = 98 Dm = 98 PO3 PO3 linear MALDI MS intesity intesity m/z m/z Posttranslational Modifications Phosporylation – identification of peptides

  19. ESI QTOF MS intesity ESI QTOF MS/MS m/z PO3 intesity PO3 m/z Posttranslational Modifications Phosporylation – peptide sequencing

  20. intesity intesity intesity m/z m/z m/z Protein-ligand Interactions The pH dependence of the Ras-GTP complex Ras 18.8 kDa Ras-GTP 19.4 kDa pH ~ 4.0 ESI QTOF MS pH ~ 3.4 pH ~ 2.8

  21. Edman Protein/peptide Sequencing MALDI TOF MS phenyl isothiocyanate low % phenyl isocyanate X1-X2-X3-X4-X5-X6-...-Xn PC-X1-X2-X3-X4-X5-X6-...-Xn X2-X3-X4-X5-X6-...-Xn PC-X2-X3-X4-X5-X6-...-Xn X3-X4-X5-X6-...-Xn PC-X3-X4-X5-X6-...-Xn X4-X5-X6-...-Xn PC-X4-X5-X6-...-Xn ...

  22. ... E G V N D N E Edman Protein/peptide Sequencing MALDI TOF MS 129 114 115 114 99 57 129 intesity m/z

  23. Conclusions • Mass spectrometers can do everything.... including making coffee or • Mass spectrometry can play an important role in almost any biological oriented research... ...if you let it

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