http://www.bits.vib.be/training
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
mass spectrometry basics
lennart martens
Department of Biochemistry, Ghent University Department of Medical Protein Research, VIB
Ghent, Belgium
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
AMINO ACIDS AND PROTEINS
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
From: http://courses.cm.utexas.edu/jrobertus/ch339k/overheads-1/ch5-amino-acids.jpg
Amino Acids and their properties
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
NH2
NN
NN
NN
R1
O
H
R2
O
H
R3
O
H
R4
O
H
R5
O
H
R6
O
H
R7
OH
O
residue
amino terminus carboxyl
terminus
NC
C
R1
O
O
H
H
H
NC
C
R2
O
O
H
H
H
NC
C
R1
OH
H NC
C
R2
O
OH
H
amino group carboxyl group
side chain
+
peptide bond
OHH
+
A protein backbone
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
NH2
NN
NN
NN
R1
O
H
R2
O
H
R3
O
H
R4
O
H
R5
O
H
R6
O
H
R7
OH
O
Methionine Glycine Alanine Serine Tyrosine Leucine Arginine
Met Gly Ala Ser Tyr Leu Arg
M G A S Y L R
MGASYLR
A protein sequence
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
MASS SPECTROMETRY:
CONCEPTS AND COMPONENTS
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
sample ion source mass 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).
Schematic view of a generalized mass spec
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
laser irradiation
target surface
desorption proton transfer
analyte
matrix molecule
H+
+
Ion sources: MALDI
ν⋅h
+
+ +
+
+ +
+
+
+ +
+
+ + +
+
+
+ + + +
+ +
+
+ +
Gas phase
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)
high vacuum
Matrix Assisted Laser Desorption and Ionization (MALDI)
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
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 matrix molecules
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
matrix sample matrix + sample
+
spot on target and let air-dry
matrix crystals
1-2 mm
stainless steel, gold teflon, …
MALDI – spotting on target
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
0
+ 0 +
0 0 0 0
0 0
0
0
0
+
+
+ +
+
+
+
+
+ + +
+ + +
+
+ +
+ +
+ +
needle
barrier
+
3-5 kV
droplet evaporation and charge-driven fission
or ion expulsion
m/z analyzer inlet
0
0
0
0
0
0
0
0
0 0
0
0
evaporation only
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 sources typically heat the needle to 40°to 100°to facilitate nebulisation and evaporation, and typically produce multiply charged peptide ions (2+, 3+, 4+)
nebulisation
www.sitemaker.umich.edu/mass-spectrometry/sample_preparation
N2
N2 sample
Ion sources: ESI
Electospray ionization (ESI)
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
A B
solvent mixer
(100–5 µm) needle
(nano) RP column (nano) spray
aqueous solvent H2O + 0.1% FA + 2–5% ACN
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:
ESI – online LC and solvents
34
3rV ⋅=
π24 rA ⋅⋅= π
DrVA 63
==
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
source
extraction plate (30 kV)
detector field-free tube (time-of-flight tube)
> 1 meter
high vacuum
Analyzers: time-of-flight (TOF)
VqEk ⋅=mEvvmE k
k⋅
=↔⋅
=2
2
2
2
2
22
222x
tV
txV
vV
qm ⋅⋅
=
⋅=
⋅=
sample ions
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
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
Ekin > Ekin
ion mirror reflectron
detector
with reflectron
Analyzers: time-of-flight – reflectron
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
extraction plate (0 kV)
source
time t0 tx tx + 100 ns
extraction plate (0 kV)
source
extraction plate (30 kV)
source
field gradient **** *** ** *
Analyzers: time-of-flight: delayed extraction
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
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).
From: Eidhammer, Flikka, Martens, Mikalsen – Wiley 2007
average mass
monoisotopic mass
Resolution and why it matters
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
ring electrode
capping electrode
capping electrode
source detector
Wolfgang Paul and Hans Georg Dehmelt received the 1989 Nobel Prize in Physics for the development of the ion trap.
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.
DC/ACRF voltage
Analyzers: ion trap (IT)
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
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.
permitted m/z
ejected m/z
ejected m/z
)cos( tVU ω⋅+−
)cos( tVU ω⋅++
Analyzers: quadrupole (Q)
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
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.
single ion in
106 electrons out
20V
60V
100V
40V
80V
120V
Detectors: electron multiplier
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
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.
electrodes
strong magnetic field
ion orbit
Fourier transform ion cyclotron resonance (FTICR)
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
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.
From: http://www.univ-lille1.fr/master-proteomique/proteowiki/index.php/Orbitrappe
Orbitrap
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
TANDEM MASS SPECTROMETRY
(TANDEM-MS, MS/MS, MS2)
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
source detector
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.
ion selector fragment mass analyzer
fragmentation
Tandem-MS: the concept
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
NH2 C H
CO COOH N H
CH2 R1
R2
C H
CO N H
C H
CO N H
C H
CH2
R3
R4
x3 y3 z3 x2 y2 z2 x1 y1 z1
a1 b1 c1 a2 b2 c2 a3 b3 c3
peptide structure
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.
Why tandem-MS?
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
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.
) ) ( ( activated ion (metastable)
non-activated ion (stable)
precursor ion
fragment ions
200 400 600 800 1000 1200 1400 1600 m/z
laser
Creating fragments (unimolecular)
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
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.
collision cell
∆V
gas inlet
collision gas (atom or molecule)
selected peptide
Creating fragments (bimolecular I)
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
This fragmentation method is called electron-capture dissociation (ECD) or electron-transfer 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.
fragmentation cell
∆V
electron source
electron
selected peptide
- -
- - -
-
Creating fragments (bimolecular II)
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
A CID FRAGMENTATION PRIMER
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
peptide X
peptide H+
0
2
4
6
8
10
12
14
Alanine
Arginin
e
Aspara
gine
Aspart
ic Acid
Cystei
ne
Glutam
ic Acid
Glutam
ine
Glycine
Histidi
ne
Isoleu
cine
Leuc
ineLy
sine
Methion
ine
Pheny
lalan
ine
Proline
Serine
Threon
ine
Tryptop
han
Tyrosin
eVali
ne
NH2–
–COOH
R–group
pKa
H+
fragmentation event
Assumption
Hypothesis
pKa values
CID fragmentation: the mobile proton model
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
From: Wysocki et al, J. Mass Spectrom., 2000
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).
Illustration of the MPM hypothesis
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
peptide 1+
fragmentation
1+ ?
b-ion y-ion
peptide 2+
fragmentation
b-ion y-ion
1+ 1+
Ionization and fragmentation
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
A SPECIAL FLAVOUR OF MS/MS:
MULTIPLE REACTION MONITORING
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
mass filter 1 mass filter 2 collision cell
selected peptides
fragments of both peptides
selected fragment
MRM removes noise, yielding better signal-to-noise ratio MRM removes ‘contaminating’ peaks, aiding targeted identification
MRM works well with proteotypic peptides MRM can be performed with Q-Q-Q, Q-LIT and IT instruments
peptide mixture
Multiple Reaction Monitoring (MRM)
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
MS1 analysis less complex peptide fractions
MS2 analysis
fragment selection
Multiple Reaction Monitoring is the most often used name for this method, even though the IUPAC draft standard for mass spectrometry does not endorse this term, and prefers Single Reaction Monitoring (SRM) instead. You can find both terms in use.
MRM analysis
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
MASS SPECTROMETER
CONFIGURATIONS
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
ESI source
detector ion trap
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 ion trap
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
ESI source
detector quadrupole 1
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.
quadrupole 2 quadrupole 3
ESI triple quadrupole
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
MALDI source
linear detector TOF tube
delayed extraction
reflectron
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.
voltage gate for precursor
selection
reflectron detector
MALDI DE RE-TOF
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
MALDI source
linear detector TOF 1
delayed extraction
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.
voltage gate for precursor
selection
reflectron
reflectron detector
TOF 2
source 2
MALDI TOF-TOF
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
ESI source
detector
quadrupole
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.
collision cell reflectron
TOF
pusher
ESI quadrupole time-of-flight (Q-TOF)
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
quadrupole capping electrode
capping electrode
source detector
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 quadrupole linear ion trap
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
ESI source
linear ion trap
C-trap
FT-ICR 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.
ESI linear ion trap FT-ICR or Orbitrap
BITS MS Data Processing – Mass Spectrometry Gent, Belgium, 16 December 2011
Lennart Martens [email protected]
Thank you!
Questions?