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

Proteins

Oligonucleotides

Oligosaccharides

Fatty Acids

Phosphoglycerides

Ceramides

Steroids

Prostaglandins

Acylglycerols

Bile Salts

Phospholipids

Glycophospholipids

Sphingolipids

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

Or1 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 = 2m/2z , m/2z = 0.5m/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 and) Biological Molecules – MS

The ESI QTOF (Organic and) Biological Molecules – MS/MS

The GC/MS “Small” Organic Molecules – MS

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

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 MS Greater Selectivity Seperation obtained by filtering which ion

can reach the detector

Mass Analyzers – Ion Paths

TOF MS

Quad MS

DetectorAcceleration

Field Free Region

Quadrupole

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

Organic Compound Analysis

m/z = 320

m/z

min

Inte

nsity

Inte

nsity

GC/MS total ion chromatogram:

Mass spectrum at peak:

Mw= 320.35 Da

SNP Genotyping

Pinpoint Assay

ddA = 297.2 Da ddC = 273.3 Da

ddG = 313.2 Da ddT = 288.2 Da

Non-pinpoint Assay

SNP Genotyping

6600 6800 7000 7200

Inte

nsity

/ A.

U.

m/z

m/z= 6674.0

m/z = 6971.1 m/z = 6987.0

m = 313.0 ddG = 313.2

m = 297.1 ddA = 297.2

MALDI TOF MS

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)

Protein/peptide Analysis

Higher-order structure elucidation

Native vs. denatured protein

Protein-protein interactions

Protein-ligand interactions

Modification characterization

Identification

Quantification

Sequencing

Posttranslational ModificationsIntra- versus intermolecular disulfide bridges

SS

SH

EE

protein

SS

SH

peptide mixture

cleavageSH

HS

SH

peptide mixture

reduction

m/z

inte

sity

MALDI MS

m/z

inte

sity

MALDI MS

Posttranslational Modifications

Phosporylation – identification of peptides

EE

PO3

PO3

cleavage

PO3

PO3

m = 98 m = 98

m/z

inte

sity

linear MALDI MS

m/z

inte

sity

reflectron MALDI MS

m/z

inte

sity

ESI QTOF MS/MS

Posttranslational Modifications

PO3

PO3

Phosporylation – peptide sequencing

m/z

inte

sityESI QTOF MS

Protein-ligand InteractionsThe pH dependence of the Ras-GTP complex

m/z

inte

sity

m/z

inte

sity

m/z

inte

sity

pH ~ 4.0

pH ~ 3.4

pH ~ 2.8

Ras-GTP 19.4 kDaRas 18.8 kDa

ESI Q

TOF

MS

Edman Protein/peptide Sequencing

X1-X2-X3-X4-X5-X6-...-Xn

MALDI TOF MSphenyl isothiocyanate

X2-X3-X4-X5-X6-...-Xn

X3-X4-X5-X6-...-Xn

X4-X5-X6-...-Xn

PC-X1-X2-X3-X4-X5-X6-...-Xn

PC-X2-X3-X4-X5-X6-...-Xn

PC-X3-X4-X5-X6-...-Xn

PC-X4-X5-X6-...-Xn

...

low % phenyl isocyanate

Edman Protein/peptide SequencingMALDI TOF MS

m/z

inte

sity

EGVNDNE...

1295799114115114129

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