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
Take your pick ! ® Peptides ® Steroids ® Proteins ® Prostaglandins ® Oligonucleotides ® Acylglycerols ® Oligosaccharides ® Bile ® Fatty ® Phospholipids Acids Salts ® Phosphoglycerides ® Glycophospholipids ® Ceramides ® Sphingolipids
What is Mass Spectrometry ? A fancy word for a highly precise analytical balance!!! ® Analytical balances: 0. 001 g to 1 g ± 0. 0001 g ® Mass spectrometers: 1 e-24 g to 1 e-19 g ± 1 E-25 g Or 1 Da to 100. 000 Da ± 0. 1 Da
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 = 2 m/2 z , m/2 z = 0. 5 m/z
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 ®
What They Can Analyze: ® The MALDI TOF ® (Organic ® The ESI QTOF ® (Organic ® The and) Biological Molecules – MS/MS GC/MS ® “Small” Organic Molecules – MS
Why the Extended Acronyms? ® Because analytical chemist like to confuse ordinary people. . ®. . and ® The mass spectrometry is defined by: type of ionization technique employed ® The type of mass analyzer(s) employed
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
Mass Analyzers ® TOF MS ® Greater Sensitivity ® Separation obtained by the ions traveling at different speeds ® Quadrupole ® Greater MS Selectivity ® Seperation obtained by filtering which ion can reach the detector
Mass Analyzers – Ion Paths Field Free Region TOF MS Acceleration Detector Quadrupole Quad MS
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 & QTOF MS/MS ® Protein-ligand interactions - ESI QTOF MS ® Peptide sequencing (Edman) – MALDI TOF MS ESI
Organic Compound Analysis Intensity GC/MS total ion chromatogram: Mw= 320. 35 Da min Intensity Mass spectrum at peak: m/z = 320
SNP Genotyping Pinpoint Assay Non-pinpoint Assay dd. A = 297. 2 Da dd. G = 313. 2 Da dd. C = 273. 3 Da dd. T = 288. 2 Da
SNP Genotyping Intensity / A. U. MALDI TOF MS Dm = 313. 0 dd. G = 313. 2 m/z= 6674. 0 Dm = 297. 1 dd. A = 297. 2 m/z = 6971. 1 m/z = 6987. 0 6600 6800 m/z 7000 7200
SNP Genotyping ® Aquisition can be multiplexed at least 5 fold (theoretical limit ~ 30 plex) ® 4. 7 -7 sec aquistion time 4000 -6000 aquisitions per 8 h day 20 -30 k SNPs/day (5 plex analysis)
Protein/peptide Analysis ® Higher-order ® Native structure elucidation ® Identification vs. denatured protein ® Protein-ligand ® Modification interactions characterization ® Quantification ® Sequencing
Posttranslational Modifications Intra- versus intermolecular disulfide bridges protein peptide mixture S SH reduction S S E SH SH HS SH MALDI MS intesity E cleavage intesity S m/z
Posttranslational Modifications Phosporylation – identification of peptides PO 3 E reflectron MALDI MS cleavage m/z PO 3 Dm = 98 PO 3 intesity E intesity PO 3 linear MALDI MS m/z
Posttranslational Modifications Phosporylation – peptide sequencing PO 3 ESI QTOF MS intesity PO 3 m/z intesity ESI QTOF MS/MS m/z
Protein-ligand Interactions The p. H dependence of the Ras-GTP complex intesity ESI QTOF MS Ras 18. 8 k. Da Ras-GTP 19. 4 k. Da p. H ~ 4. 0 m/z p. H ~ 3. 4 m/z p. H ~ 2. 8 m/z
Edman Protein/peptide Sequencing MALDI TOF MS phenyl isothiocyanate X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -. . . Xn low % phenyl PC-X 1 isocyanate 5 -X 6 -. . . -X 2 -X 3 -X 4 -X Xn X 2 -X 3 -X 4 -X 5 -X 6 -. . . -Xn PC-X 3 -X 4 -X 5 -X 6 -. . . -Xn PC-X 4 -X 5 -X 6 -. . . -Xn . . .
Edman Protein/peptide Sequencing MALDI TOF MS. . . E N D N V G E intesity 129 114 115 114 99 57 129 m/z
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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