introduction to mass spectrometry
and proteomics
lennart martens
lennart.martens@vib-ugent.be
computational omics and systems biology group
VIB / Ghent University, Ghent, Belgium
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Amino Acids and Proteins
Mass Spectrometry: Concepts and Components
Tandem Mass Spectrometry (MS/MS, MS²)
A CID Fragmentation Primer
Mass Spectrometer Configurations
Proteomics Methods:
2D PAGE
Shotgun (Gel-Free) Approaches
Workhorse Shotgun Method: MudPIT
Amino Acids and Proteins
Mass Spectrometry: Concepts and Components
Tandem Mass Spectrometry (MS/MS, MS²)
A CID Fragmentation Primer
Mass Spectrometer Configurations
Proteomics Methods:
2D PAGE
Shotgun (Gel-Free) Approaches
Workhorse Shotgun Method: MudPIT
Amino Acids and their properties
From: http://courses.cm.utexas.edu/jrobertus/ch339k/overheads-1/ch5-amino-acids.jpg
A protein backbone
H
H
O
side chain
R1
H
C
N
H
C
+
H
C
C
N
O
H
H
H
O
N
H2N
H
O
R2
H
O
residue
C
N
C O H
C
C
O
R2
H
N
O
R7
O
H
R5
N
R4
O
peptide bond
O
N
N
H
H
H
R3
O
H
R1
N
carboxyl group
R1
amino
terminus
O
O
amino group
+
R2
N
R6
H
O
OH
carboxyl
terminus
A protein sequence
N
H2N
O
N
N
R2
O
H
N
N
R4
O
H
R7
O
H
R5
O
H
R3
O
H
R1
O
N
R6
OH
H
Methionine
Glycine
Alanine
Serine
Tyrosine
Leucine
Arginine
Met
Gly
Ala
Ser
Tyr
Leu
Arg
M
G
A
S
Y
L
R
MGASYLR
Amino Acids and Proteins
Mass Spectrometry: Concepts and Components
Tandem Mass Spectrometry (MS/MS, MS²)
A CID Fragmentation Primer
Mass Spectrometer Configurations
Proteomics Methods:
2D PAGE
Shotgun (Gel-Free) Approaches
Workhorse Shotgun Method: MudPIT
Schematic view of a generalized mass spec
sam ple
ion source
m ass analyzer(s)
detector
digitizer
Generalized mass spectrometer
All mass analyzers operate on gas-phase ions using electromagnetic fields. Results
are therefore plotted on a cartesian system with mass-over-charge (m/z) on the X-axis
and ion intensity on the Y-axis. The latter can be in absolute or relative measurements.
The ion source therefore makes sure that (part of) the sample molecules are ionized
and brought into the gas phase. The detector is responsible for actually recording the
presence of ions. Time-of-flight analyzers also require a digitizer (ADC).
Ion sources: MALDI
h ⋅ν
laser irradiation
desorption
analyte
target
surface
matrix
molecule
high vacuum
+
+
+
+
+
+
+
+
+
+
H+
proton transfer
+ +
+ +
+
+
+ +
+
+ + +
+ + +
+
Gas phase
Matrix Assisted Laser Desorption and Ionization (MALDI)
MALDI sources for proteomics typically rely on a pulsed nitrogen UV laser
(υ = 337 nm) and produce singly charged peptide ions. Competitive ionisation occurs.
The term ‘MALDI’ was coined by Karas and Hillenkamp (Anal. Chem., 1985) and Koichi Tanaka received the 2002 Nobel
Prize in Chemistry for demonstrating MALDI ionization of biological macromolecules (Rapid Commun. Mass Spectrom., 1988)
MALDI matrix molecules
Matrix properties:
• Small organic molecules
• Co-crystallize with the analyte
• Need to be soluble in solvents compatible with analyte
• Absorb the laser wavelength
• Cause co-desorption of the analyte upon laser irradiation
• Promote analyte ionization
MALDI – spotting on target
+
matrix
sample
matrix + sample
1-2 mm
spot on target
and let air-dry
stainless steel,
gold, teflon, …
matrix crystals
Ion sources: ESI
m/z analyzer inlet
www.sitemaker.umich.edu/mass-spectrometry/sample_preparation
+
+
+
3-5 kV
+
+
+
+
+
+
+
+
+
+
+ +
+
+
+
+
droplet evaporation and
charge-driven fission
or ion expulsion
+
+
sam ple
N2
N2
+
0
needle
nebulisation
Electospray ionization (ESI)
+
0
0
evaporation only
+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
barrier
ESI sources typically heat the needle to 40°to 100°to facilitate nebulisation
and evaporation, and typically produce multiply charged peptide ions (2+, 3+, 4+)
John B. Fenn received the 2002 Nobel Prize in Chemistry for demonstrating ESI ionization of biological macromolecules
(Science, 1989) – ESI is also used in fine control thrusters on satellites and interstellar probes…
ESI – online LC and solvents
(nano) RP column
solvent mixer
aqueous solvent
H2O + 0.1% FA
+ 2–5% ACN
A
B
(100–5 µm) (nano)
needle
spray
organic solvent
ACN + 0.1% FA
Nanospray ESI sources (5-10 µm diameter needle) achieve a higher sensitivity,
probably due to the higher surface-to-volume ratio. For a spherical droplet this ratio is:
A = 4 ⋅π ⋅ r
V =4
2
A 3 6
= =
V r D
π ⋅ r3
3
Analyzers: time-of-flight (TOF)
high vacuum
sample
ions
source
extraction
plate (30 kV)
Ek = q ⋅ V
detector
field-free tube
(time-of-flight tube)
> 1 meter
2 ⋅ Ek
m ⋅ v2
↔v=
Ek =
2
m
We can now relate m/q (or the more commonly used
m/z) to the velocity of the ion, and using Newton’s
kinematica we can relate the speed to the travel time
and (known + exactly calibrated) field-free tube length
m 2 ⋅V
2 ⋅V
2 ⋅V ⋅ t 2
= 2 =
=
2
q
v
x2
 x
 
t
Analyzers: time-of-flight – reflectron
Ekin
> Ekin
ion mirror
reflectron
detector
with reflectron
Analyzers: time-of-flight: delayed extraction
source
extraction plate (0 kV)
source
extraction plate (0 kV)
field gradient
**** ***
**
*
source
extraction plate (30 kV)
time
t0
tx
t x + 100 ns
Resolution and why it matters
Resolution in mass spectrometry is usually defined as the width of a peak
at a given height (there is an alternative definition based on percent
valley height). This width can be recorded at different heights, but is most
often recorded at 50% peak height (FWHM).
monoisotopic
mass
average
mass
From: Eidhammer, Flikka, Martens, Mikalsen – Wiley 2007
Analyzers: ion trap (IT)
DC/ACRF
voltage
source
detector
capping
electrode
ring
electrode
capping
electrode
Ion traps operate by effectively trapping the ions in an oscillating electrical field. Mass
separation is achieved by tuning the oscillating fields to eject only ions of a specific
mass. Big advantages are the ‘archiving’ during the analysis, allowing MSn.
Wolfgang Paul and Hans Georg Dehmelt received the 1989 Nobel Prize in Physics for the development of the ion trap.
Analyzers: quadrupole (Q)
+ (U + V ⋅ cos ωt )
permitted m/z
ejected m/z
− (U + V ⋅ cos ωt )
ejected m/z
Quadrupole mass analyzers also use a combined RF AC and DC current. They thus
create a high-pass mass filter between the first two rods, and a low-pass mass filter
between the other two rods. The net result is a filter that can be fine-tuned to overlap
(and thus permit) only in a specific m/z interval; ions of all other m/z values will be ejected.
Detectors: electron multiplier
single ion in
40V
20V
80V
60V
120V
100V
10 6 electrons out
Different variations of electron multiplier (EM) detectors are in use, and they are
the most common type of detector. An EM relies on several Faraday cup dynodes
with increasing charges to produce an electron cascade based on a few incident ions.
Fourier transform ion cyclotron resonance (FTICR)
electrodes
ion orbit
strong magnetic field
An FT-ICR is essentially a cyclotron, a type of particle accelerator in which electrons
are captured in orbits by a very strong magnetic field, while being accelerated by an
applied voltage. The cyclotron frequency is then related to the m/z. Since many ions are
detected simultaneously, a complex superposition of sine waves is obtained. A Fourier
transformation is therefore required to tease out the individual ion frequencies.
Orbitrap
From: http://www.univ-lille1.fr/master-proteomique/proteowiki/index.php/Orbitrappe
An OrbiTrap is a special type of trap that consists of an outer and inner coaxial electrode,
which generate an electrostatic field in which the ions form an orbitally harmonic oscillation
along the axis of the field. The frequency of the oscillation is inversely proportional to the
m/z, and can again be calculated by Fourier transform.
The OrbiTrap delivers near-FT-ICR performance, but is cheaper, much more robust, and
much simpler in maintenance. It is a recent design, only a few years old.
Amino Acids and Proteins
Mass Spectrometry: Concepts and Components
Tandem Mass Spectrometry (MS/MS, MS²)
A CID Fragmentation Primer
Mass Spectrometer Configurations
Proteomics Methods:
2D PAGE
Shotgun (Gel-Free) Approaches
Workhorse Shotgun Method: MudPIT
Tandem-MS: the concept
source
detector
ion selector
fragm ent
m ass analyzer
fragm entation
Tandem-MS is accomplished by using two mass analyzers in series (tandem)
(note that a single ion trap can also perform tandem-MS). The first mass analyzer
performs the function of ion selector, by selectively allowing only ions of a given m/z to
pass through. The second mass analyzer is situated after fragmentation is triggered
(see next slides) and is used in its normal capacity as a mass analyzer for the fragments.
Why tandem-MS?
peptide structure
x3
y3
R1
NH2
C
H
a1
CO
b1
N
H
c1
z3
x2
y2
z2
R2
R3
CH2
CH2
C
H
CO
a2
b2
N
H
c2
C
H
x1
z1
R4
CO
a3
y1
b3
N
H
C
H
COOH
c3
There are several other ion types that can be annotated, as well as
‘internal fragments’. The latter are fragments that no longer contain an intact
terminus. These are harder to use for ‘ladder sequencing’, but can still be interpreted.
This nomenclature was coined by Roepstorff and Fohlmann (Biomed. Mass Spec., 1984) and Klaus Biemann (Biomed.
Environ. Mass Spec., 1988) and is commonly referred to as ‘Biemann nomenclature’. Note the link with the Roman alphabet.
Creating fragments (unimolecular)
laser
(( ))
activated ion
(metastable)
non-activated ion
(stable)
precursor ion
fragment ions
200 400 600 800 1000 1200 1400 1600 m/z
This fragmentation method is called post-source decay (PSD) and relies
on a single unimolecular event, in which a highly energetic (metastable)
ion spontaneously fragments. PSD typically causes backbone fragmentation.
y and b ions are by far the most prevalent fragment types, although TOF-TOF
instruments (see later) specifically also yield substantial internal fragments.
Creating fragments (bimolecular I)
gas inlet
collision cell
collision gas
(atom or molecule)
selected
peptide
∆V
This fragmentation method is called collision-induced dissociation (CID) and relies
on a series of bimolecular events (collisions) to provide the peptide precursor with
sufficient energy to fragment. CID typically causes backbone fragmentation.
y and b ions are by far the most prevalent fragment types.
The collision gas is typically an inert noble gas (e.g.: Ar, He, Xe), or N2.
Creating fragments (bimolecular II)
electron source
fragm entation cell
-
-
-
-
-
electron
-
selected
peptide
∆V
This fragmentation method is called electron-capture dissociation (ECD) or electrontransfer dissociation (ETD) and relies on a single impact of an electron on a peptide
precursor. This high-speed impact immediately imparts sufficient energy to fragment the
precursor (non-ergodic process). Like CID, ETD and ECD typically cause backbone
fragmentation, but they typically result in c and z ions. ECD is only workable in FT-ICR
mass spectrometers, whereas ETD is used in traps.
Amino Acids and Proteins
Mass Spectrometry: Concepts and Components
Tandem Mass Spectrometry (MS/MS, MS²)
A CID Fragmentation Primer
Mass Spectrometer Configurations
Proteomics Methods:
2D PAGE
Shotgun (Gel-Free) Approaches
Workhorse Shotgun Method: MudPIT
Al
an
i
Ar n e
gi
As ni
ne
p
As ara
pa gin
rti e
c
A
C cid
y
G
lu ste
ta
m ine
ic
G Ac i
lu
d
ta
m
in
G e
ly
c
H ine
is
ti
Is din
ol
e
eu
ci
n
Le e
uc
in
e
Ly
si
M
n
e
Ph thio e
en ni
yl ne
al
an
in
Pr e
ol
in
e
Se
Th rin
re e
Tr oni
yp ne
to
ph
Ty an
ro
si
ne
Va
lin
e
CID fragmentation: the mobile proton model
Assum ption
peptide
Hypothesis
peptide
pK a values
12
10
pKa
4
2
NH2–
H+
X
H+
fragmentation event
14
R–group
8
6
–COOH
0
Illustration of the MPM hypothesis
The necessary collision energy to achieve a given fragmentation efficiency is highest and equal for
both 1+ peptides on the left (the R present in both sequesters the charge), and is higher for
PPGFSPFR for the 2+ peptides. This is because the latter has an N-terminal P, which has a relatively
high pKa for the N-terminus; so the second available charge is located at either end of the peptide.
The other peptide has no real main contender (in pKa) for the second charge, and therefore
fragments more easily. On the right, having two R’s in the sequence negates the effect of double
charge (both charges are sequestered by an R).
From: Wysocki et al, J. Mass Spectrom., 2000
Ionization and fragmentation
1+
peptide
fragmentation
b-ion
y-ion
1+ ?
2+
peptide
fragmentation
1+
b-ion
y-ion
1+
Amino Acids and Proteins
Mass Spectrometry: Concepts and Components
Tandem Mass Spectrometry (MS/MS, MS²)
A CID Fragmentation Primer
Mass Spectrometer Configurations
Proteomics Methods:
2D PAGE
Shotgun (Gel-Free) Approaches
Workhorse Shotgun Method: MudPIT
ESI ion trap
ESI
source
ion trap
detector
Very simple and reliable instrument, that can perform MS and CID MS/MS thanks to
the ion trap. Mass accuracy is relatively poor however, and the resolution is lacking
as well (unable to distinguish isotopes, hampering charge state determination).
ESI triple quadrupole
ESI
source
quadrupole 1 quadrupole 2 quadrupole 3
detector
Simple instrument, that recently attracted attention because it is well-suited for
Multiple Reaction Monitoring (MRM). It can perform MS and MS/MS, where the
first quadrupole is the ion selector, the second quadrupole a collision cell and the
third quadrupole a mass analyzer. Due to the ‘wastefulness’ of a quadrupole as mass
analyzer, it is not very popular for general MS/MS analysis, however.
MALDI DE RE-TOF
reflectron
detector
M ALDI
source
delayed
ex traction
TOF tube
linear
detector
reflectron
voltage gate
for precursor
selection
Relatively simple instrument, that can measure intact masses quite accurately
and can perform fragmentation analysis through PSD (unimolecular fragmentation),
but the yield of fragmentation is poor. Cheap and reliable, but out of date nowadays.
MALDI TOF-TOF
reflectron
detector
M ALDI
source
delayed
ex traction
linear
detector
source 2
TOF 1
TOF 2
reflectron
voltage gate
for precursor
selection
A modern version of the MALDI DE RE-TOF, the TOF-TOF relies on two TOF tubes in
tandem. The second TOF is fitted with a reflectron. Mass accuracy and resolution are
very high and the instrument can perform MS and MS/MS, both for peptides as well as
whole proteins. The archiving nature of the MALDI targets allows the instruments to scan
a single sample more thoroughly. TOF-TOF instruments are also well-suited for profiling.
ESI quadrupole time-of-flight (Q-TOF)
pusher
ESI
source
quadrupole
collision cell
detector
TOF
reflectron
One of the first hybrid mass spectrometers, the Q-TOF combines the excellent precursor
selection characteristics of the quadrupole with the mass resolution and accuracy of the
TOF tube. The ESI source produces multiply charged ions, and can be coupled to an
online (nano-) LC separation. A Q-TOF is a very good instrument when high-quality data
are more important than high-throughput, and is well suited for protein characterisation.
ESI quadrupole linear ion trap
source
detector
capping quadrupole capping
electrode
electrode
Linear ion traps store ions in a fixed linear trajectory rather than in a ‘blob’.
The major advantage is the increased capacity for trapping ions. This allows for
a broader dynamic range and better quantitation performance. The linear ion trap is
these days often encountered as the workhorse mass spectrometer in many labs, and
can be extended with the highly accurate orbitrap or FT-ICR mass analyzers/detectors.
ESI linear ion trap FT-ICR or Orbitrap
C-trap
ESI
source
linear ion trap
FT-I CR
Orbitrap
The combination of a linear ion trap and a high-resolution, high-accuracy FT
analyzer allows for a broad dynamic range and highly accurate mass measurements.
Since the ion trap can be used as a collision cell, the FT analyzer can also measure the
resulting fragments with high accuracy.
Amino Acids and Proteins
Mass Spectrometry: Concepts and Components
Tandem Mass Spectrometry (MS/MS, MS²)
A CID Fragmentation Primer
Mass Spectrometer Configurations
Proteomics Methods:
2D PAGE
Shotgun (Gel-Free) Approaches
Workhorse Shotgun Method: MudPIT
2D-PAGE separation of proteins (Est. 1975)
P rinciple
cell lysis
protein ex traction
cells
Protein A
Protein C
Protein B
Protein D
protein m ix ture
pI
Chem istry
toolbox
Mr
2D-PAGE
2D-PAGE separation of proteins (Est. 1975)
protein
complex protein mixture
extraction
http://www.akh-wien.ac.at/biomed-research/htx/platweb1.htm
2D-PAGE separation
MS/MS analysis
100
pI
%
0
100
300
500
700
900
1100
1300
1500
1700
1900
2100
m/z
fragmentation
100
MS analysis
tryptic
%
0
digest
300
400
500
600
700
800
900
1000
1100
m/z
MW
Amino Acids and Proteins
Mass Spectrometry: Concepts and Components
Tandem Mass Spectrometry (MS/MS, MS²)
A CID Fragmentation Primer
Mass Spectrometer Configurations
Proteomics Methods:
2D PAGE
Shotgun (Gel-Free) Approaches
Workhorse Shotgun Method: MudPIT
Overall gel-free proteomics workflow
protein
extraction
complex protein mixture
http://www.akh-wien.ac.at/biomed-research/htx/platweb1.htm
Data-dependent M S/ M S analyses
100
100
100
%
%
%
0
100
300
500
700
900
1100
1300
1500
1700
1900
2100
m/z
0
100
300
500
700
900
1100
1300
1500
1700
1900
2100
m/z
0
100
300
500
700
900
1100
1300
1500
1700
enzymatic
digest
extremely complex
peptide mixture
1900
2100
m/z
MS analysis
separation
selection
less complex
peptide fractions
Going gel-free in the new millennium
• ICAT (Gygi et al., 1999)
• MudPIT (Washburn et al., 2001)
• Accurate Mass Tags for proteome analysis (Conrads et al., 2000)
• Signature Peptides approach for proteomics (Ji et al., 2000)
• AA-based covalent chromatography peptide selection (Wang & Regnier, 2001)
• Affinity-based enrichment of phosphopeptides (Oda et al., 2001)
• ICAT for phosphopeptides (Zhou et al., 2001)
• Reversible biotinylation of Cys-peptides (Spahr et al., 2000)
• COFRADIC (Gevaert et al., 2002)
Amino Acids and Proteins
Mass Spectrometry: Concepts and Components
Tandem Mass Spectrometry (MS/MS, MS²)
A CID Fragmentation Primer
Mass Spectrometer Configurations
Proteomics Methods:
2D PAGE
Shotgun (Gel-Free) Approaches
Workhorse Shotgun Method: MudPIT
MudPIT: that which we call a rose…
Strong cation
ex changer
SCX
R everse-phase
resin
RP
ESI-based MS
• Orthogonal, 2D separation of peptides
• 2D analogon: pI = SCX, Mr = RP
But what about the complexity?
e.g., Escherichia coli
4,349 predicted proteins
if 100% expressed
109,934 detectable tryptic peptides
if 50% expressed
54,967 detectable tryptic peptides
Sample complexity increased one order of magnitude!
Thank you!
Questions?