Transcript Mass Spectrometry in the Biosciences: Introduction to Mass

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 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
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

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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.
Dm = 313.0
ddG = 313.2
m/z= 6674.0
Dm = 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
S
E
SH
reduction
HS
S
SH
SH
MALDI MS
MALDI MS
intesity
S
intesity
S
peptide mixture
peptide mixture
m/z
m/z
Posttranslational Modifications
Phosporylation – identification of peptides
PO3
E
reflectron MALDI MS
cleavage
m/z
PO3
Dm = 98
Dm = 98
PO3
linear MALDI MS
intesity
E
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
...
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