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

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 TOF MS Quad MS Detector Acceleration Field Free Region Quadrupole

11. Selected Examples  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

12. Organic Compound Analysis Single compound or mixture analysis of small (<500 Da) organic compounds by GC/MS:  Identify analytes in sample  Confirming purity

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

14. SNP Genotyping  Measure the mass differences of the incorporated bases after a minisequencing reaction ddA = 297.2 Da ddC = 273.3 Da ddG = 313.2 Da ddT = 288.2 Da  Possibility of multiplexing the analysis

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

16. SNP Genotyping 6600680070007200 Intensity / A.U. m/z m/z= 6674.0 m/z = 6971.1m/z = 6987.0  m = 313.0 ddG = 313.2  m = 297.1 ddA = 297.2 MALDI TOF MS

17. 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)

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

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

20. Posttranslational Modifications Phosporylation – identification of peptides E E PO 3 cleavage PO 3  m = 98 m/z intesity linear MALDI MS m/z intesity reflectron MALDI MS

21. m/z intesity ESI QTOF MS/MS Posttranslational Modifications PO 3 Phosporylation – peptide sequencing m/z intesity ESI QTOF MS

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

23. Edman Protein/peptide Sequencing X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -...-X n MALDI TOF MS phenyl isothiocyanate X 2 -X 3 -X 4 -X 5 -X 6 -...-X n X 3 -X 4 -X 5 -X 6 -...-X n X 4 -X 5 -X 6 -...-X n PC-X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -...-X n PC-X 2 -X 3 -X 4 -X 5 -X 6 -...-X n PC-X 3 -X 4 -X 5 -X 6 -...-X n PC-X 4 -X 5 -X 6 -...-X n... low % phenyl isocyanate

24. Edman Protein/peptide Sequencing MALDI TOF MS m/z intesity EGVNDNE... 1295799114115114129

25. 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