mass spectrometry basics

lennart martens
lennart.martens@vib-ugent.be
computational omics and systems biology group
VIB / Ghent University, Ghent, Belgium

www.compomics.com
@compomics
Advertorial 
Amino Acids and Proteins

Mass Spectrometry: Concepts and Components

Ion sources
Analyzers
Detectors
FT-ICR and Orbitrap

Tandem Mass Spectrometry (MS/MS, MS²)

A CID Fragmentation Primer

Amino Acids and Proteins

Mass Spectrometry: Concepts and Components

Ion sources
Analyzers
Detectors
FT-ICR and Orbitrap

Tandem Mass Spectrometry (MS/MS, MS²)

A CID Fragmentation Primer

Amino Acids and their properties

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
Amino Acids and Proteins

Mass Spectrometry: Concepts and Components

Ion sources
Analyzers
Detectors
FT-ICR and Orbitrap

Tandem Mass Spectrometry (MS/MS, MS²)

A CID Fragmentation Primer

Schematic view of a generalized mass spec

sam ple ion source m ass analyzer(s) detector digitizer

Generalized mass spectrometer

All mass analyzers use electromagnetic fields to manipulate gas-phase ions.

Results are plotted as a spectrum, with mass-over-charge (m/z) on the X-axis and
ion intensity on the Y-axis. The latter can be absolute (counts) or relative. The ion
source ensures that (a part of) the sample molecules are ionized and brought into
the gas phase. The detector is responsible for actually recording the presence of
ions. Digitizers (analog to digital converters; ADC) transform the continuous,
analog detector signal into a digital, discretized spectrum.
Amino Acids and Proteins

Mass Spectrometry: Concepts and Components

Ion sources
Analyzers
Detectors
FT-ICR and Orbitrap

Tandem Mass Spectrometry (MS/MS, MS²)

A CID Fragmentation Primer

 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
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
Amino Acids and Proteins

Mass Spectrometry: Concepts and Components

Ion sources
Analyzers
Detectors
FT-ICR and Orbitrap

Tandem Mass Spectrometry (MS/MS, MS²)

A CID Fragmentation Primer

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: 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. This creates
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.
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

Amino Acids and Proteins

Mass Spectrometry: Concepts and Components

Ion sources
Analyzers
Detectors
FT-ICR and Orbitrap

Tandem Mass Spectrometry (MS/MS, MS²)

A CID Fragmentation Primer

Detectors: electron multiplier

single ion in

40V

20V
80V

60V

120V

100V

10 6 electrons out

Different variations of electron multiplier (EM) detectors are used, and these are
the most common type of detector. An EM relies on several Faraday cup dynodes
with increasing charges to produce an electron cascade from a few incident ions.
The detector signal strength is assumed to
represent the abundance of the analyte

1 over 2

1 over 1

2 over 1
However, while 1/1 ratios are quite reliable,
outlying ratios quickly deviate from a line

Gevaert, Proteomics, 2007

The errors also rise quickly for outlying ratios

Vaudel, Proteomics, 2010

And the effects remain quite visible,
even on modern instruments (LTQ-OrbiTrap)
Amino Acids and Proteins

Mass Spectrometry: Concepts and Components

Ion sources
Analyzers
Detectors
FT-ICR and Orbitrap

Tandem Mass Spectrometry (MS/MS, MS²)

A CID Fragmentation Primer

Fourier transform ion cyclotron resonance (FT-ICR)

electrodes
ion orbit

strong magnetic field

An FT-ICR is essentially a cyclotron, a type of particle accelerator in which ions

are captured in orbits by a very strong magnetic field, while being accelerated by an
applied voltage. The cyclotron frequency is related to the m/z of the ions. Because
many ions are detected simultaneously, the result is a complex superposition of sine
waves. Fourier transformation is then used 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

Ion sources
Analyzers
Detectors
FT-ICR and Orbitrap

Tandem Mass Spectrometry (MS/MS, MS²)

A CID Fragmentation Primer

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). A single

ion trap can also perform tandem-MS. The first mass analyser 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 (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.
Amino Acids and Proteins

Mass Spectrometry: Concepts and Components

Ion sources
Analyzers
Detectors
FT-ICR and Orbitrap

Tandem Mass Spectrometry (MS/MS, MS²)

A CID Fragmentation Primer

 pKa values
Hypothesis
Assumption

pKa
Al

0
2
4
6
8
10
12
14
an
i
Ar n e
gi
As ni
p ne
As ara
pa gin
rti e
c
A
C cid
G ys
lu
ta tein
m e
ic
G A
lu cid
ta
m
in
G e
ly
c
NH2–

H ine
is
ti
Is din
ol e
eu
ci
n
Le e
uc
in
e
Ly
M s
peptide

peptide

e in
Ph thio e
en ni
yl ne
al
an
R–group

in
Pr e
ol
in
e
Se
Th in r
CID fragmentation: the mobile proton model

re e
Tr oni
yp ne
X

to
ph
Ty an
ro
H+

–COOH

si
ne
Va
lin
e
H+

fragmentation event
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