Mass Spectrometry Basics Guide

http://www.bits.vib.be/training

mass spectrometry basics

lennart martens
lennart.martens@UGent.be

Department of Biochemistry, Ghent University
Department of Medical Protein Research, VIB
Ghent, Belgium
Lennart Martens BITS MS Data Processing – Mass Spectrometry
lennart.m artens@UGent.be Gent, Belgium, 16 December 2011

AMINO ACIDS AND PROTEINS


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 R2 + R1 H O
C N C O H
H
C
C
O + H
N
C
C
O H
N C C
N O
H R2
O O
H H H
H
peptide bond
amino group carboxyl group

R1 H O R3 H O R5 H O R7
N N N O
H2N N N N
O R2 H O R4 H O R6 H OH
amino
terminus carboxyl
residue terminus


A protein sequence

R1 H O R3 H O R5 H O R7
N N N O
H2N N N N
O R2 H O R4 H O R6 H OH

Methionine Glycine Alanine Serine Tyrosine Leucine Arginine

Met Gly Ala Ser Tyr Leu Arg

M G A S Y L R

MGASYLR

MASS SPECTROMETRY:
CONCEPTS AND COMPONENTS


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
laser irradiation high vacuum

h ⋅ν H+ + +
+ + + + +
+ + +

desorption
+
+
+
+

proton transfer
+ +
+ + +
+
+
+ + + +
+

matrix Gas phase
molecule
analyte

target
surface
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

+ +
+
+ +
+
+ +

droplet evaporation and
+
+ +

+ + charge-driven fission
+
3-5 kV +
+ or ion expulsion
+
+ +
+
+

+
+ evaporation only
0
N2 + 0 0 0
0 0 0
0

sam ple 0 0 0 0 0

N2 0 0 0
0 0 0 0
0 0 0

needle
nebulisation
Electospray ionization (ESI) 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 (100–5 µm) (nano)
needle spray
solvent mixer

aqueous solvent
organic solvent
H2O + 0.1% FA A B ACN + 0.1% FA
+ 2–5% ACN

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:

π ⋅ r3
A = 4 ⋅π ⋅ r 2
V =4
3

A 3 6
= =
V r D


Analyzers: time-of-flight (TOF)
sample high vacuum
ions

source detector
field-free tube
extraction (time-of-flight tube)
plate (30 kV) > 1 meter

m ⋅ v2 2 ⋅ Ek
Ek = q ⋅ V Ek = ↔v=
2 m

We can now relate m/q (or the more commonly used
m 2 ⋅V 2 ⋅V 2 ⋅V ⋅ t 2
m/z) to the velocity of the ion, and using Newton’s = 2 = 2
=
q v  x x2
kinematica we can relate the speed to the travel time  
and (known + exactly calibrated) field-free tube length 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
**** *** ** *
field gradient

source
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).

average monoisotopic
mass mass

From: Eidhammer, Flikka, Martens, Mikalsen – Wiley 2007


Analyzers: ion trap (IT)

DC/ACRF
voltage

source detector

capping ring capping
electrode electrode 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.


TANDEM MASS SPECTROMETRY
(TANDEM-MS, MS/MS, MS2)


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 z3 x2 y2 z2 x1 y1 z1
R2 R3

R1 CH2 CH2 R4

NH2 C CO N C CO N C CO N C COOH
H H H H H H H

a1 b1 c1 a2 b2 c2 a3 b3 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 precursor ion
(metastable)

non-activated ion fragment ions
(stable)
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 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.


A CID FRAGMENTATION PRIMER


CID fragmentation: the mobile proton model

H+
Assum ption peptide

H+
Hypothesis peptide X
fragmentation event
14
pK a values
12
R–group
10 NH2–

pKa
8

6

4
–COOH
2

0
H ine

Ty an
e

ne
Ph thio e
id

Pr e
e
Ar n e

yl ne
ne

Le e

re e
yp ne

e
G e

e
C cid
rti e

e
ta tein

n

in
n

rin

lin
in

Is din

in
in
pa gin

Ac

ph

si
si
i

en ni
As ni

ci

Tr oni
A

c

an
an

ol
m

uc

Va
Se
ly

ro
Ly
eu
gi

ti
As ara

ys

ta

to
ic
c

is

al
Al

m

ol
lu

Th
p

e
G

M
lu
G


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

peptide 1+
fragmentation

b-ion y-ion

1+ ?

peptide 2+
fragmentation
1+ 1+
b-ion y-ion


A SPECIAL FLAVOUR OF MS/MS:
MULTIPLE REACTION MONITORING


Multiple Reaction Monitoring (MRM)

mass filter 1 collision mass filter 2
cell

peptide selected fragments selected
mixture peptides of both fragment
peptides

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


MRM analysis

less complex MS1 analysis
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.


MASS SPECTROMETER
CONFIGURATIONS


ESI ion trap

ESI detector
source 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 triple quadrupole

ESI detector
source quadrupole 1 quadrupole 2 quadrupole 3

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 linear
source TOF tube detector
delayed reflectron
ex traction
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 2 linear
source TOF 1 TOF 2 detector
delayed reflectron
ex traction
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
detector

ESI
source
quadrupole TOF

collision cell
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 linear ion trap
source

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.


Thank you!
Questions?

Mass Spectrometry Basics Guide

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