Mass Spectrometry in the

Biosciences:
Introduction to Mass
Spectrometry and Its Uses in
a Company Like Decode.
Sigurur V. Smrason, Ph.D.
New Technologies Division

Take your pick !

Peptides

Steroids

Proteins

Prostaglandins

Oligonucleotides

Acylglycerols

Oligosaccharides

Bile Salts

Fatty Acids

Phospholipids

Phosphoglycerides

Glycophospholipids

Ceramides

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

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 = 2m/2z , m/2z = 0.5m/z

In-house available instrumentation

MALDI TOF MS

ESI QTOF LC/MS/MS

Matrix Assisted Laser Desorption Ionization

Time of Flight Mass Spectrometer
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

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 nucleotide polymorphism genotyping

Single compound or mixture analysis of small (<500 Da)

organic compounds by GC/MS
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

Intensity

GC/MS total ion chromatogram:

Mw= 320.35 Da
min

Mass spectrum at peak:

Intensity

m/z = 320

m/z

SNP Genotyping
Pinpoint Assay

Non-pinpoint Assay

ddA = 297.2 Da
ddG = 313.2 Da

ddC = 273.3 Da
ddT = 288.2 Da

SNP Genotyping
MALDI TOF MS

Intensity / A.U.

m = 313.0
ddG = 313.2

m/z= 6674.0

m = 297.1
ddA = 297.2

m/z = 6971.1

6600

6800

m/z

m/z = 6987.0

7000

7200

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 Modifications
Intra- versus intermolecular disulfide bridges
protein
cleavage

E
SH

SH

reduction

HS

SH

SH

MALDI MS

MALDI MS
intesity

intesity

peptide mixture

peptide mixture

m/z

m/z

Posttranslational Modifications
Phosporylation identification of peptides
PO3

reflectron MALDI MS

cleavage

m/z

PO3

m = 98

m = 98

PO3

linear MALDI MS

intesity

intesity

PO3

m/z

Posttranslational Modifications
Phosporylation peptide sequencing
PO3

ESI QTOF MS

intesity

PO3

m/z

intesity

ESI QTOF MS/MS

m/z

Protein-ligand Interactions
The pH dependence of the Ras-GTP complex
Ras-GTP 19.4 kDa
pH ~ 4.0

intesity

m/z

pH ~ 3.4
m/z

intesity

ESI QTOF MS

intesity

Ras 18.8 kDa

pH ~ 2.8
m/z

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

...

Edman Protein/peptide Sequencing

MALDI TOF MS
...

V G

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