Mass spectrometry based proteomics (1)
Kenny Helsens
Department of Biochemistry, Ghent University Department of Medical Protein Research, VIB Ghent, Belgium
Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 AMINO ACIDS AND PROTEINS
Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 Amino Acids and their properties
From: http://courses.cm.utexas.edu/jrobertus/ch339k/overheads-1/ch5-amino-acids.jpg
Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 A protein backbone
H H O side chain R1 H O R1 R2 + N C C C O H C H O H C C H O + C N N C N O R2 O H O H H H H pep de bond amino group carboxyl group
R1 H O R3 H O R5 H O R7 N N N O N H2N N N O R2 H O R4 H O R6 H OH amino terminus carboxyl residue terminus
Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 A protein sequence
R1 H O R3 H O R5 H O R7 N N N O N H2N 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
Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 MASS SPECTROMETRY:
CONCEPTS AND COMPONENTS
Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 Schematic view of a generalized mass spec
sample ion source mass analyzer(s) detector digi zer
Generalized mass spectrometer
All mass analyzers operate on gas-phase ions using electromagne c fields. Results are therefore plo ed on a cartesian system with mass-over-charge (m/z) on the X-axis and ion intensity on the Y-axis. The la er can be in absolute or rela ve 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 digi zer (ADC).
Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 Ion sources: MALDI
laser irradia on high vacuum
h⋅ν + H + + + + + + + + + + + + + + + + + + + + + + + desorp on + + proton transfer +
matrix Gas phase molecule analyte
target surface Matrix Assisted Laser Desorp on and Ioniza on (MALDI)
MALDI sources for proteomics typically rely on a pulsed nitrogen UV laser (υ = 337 nm) and produce singly charged pep de ions. Compe ve ionisa on occurs.
The term ‘MALDI’ was coined by Karas and Hillenkamp (Anal. Chem., 1985) and Koichi Tanaka received the 2002 Nobel Prize in Chemistry for demonstra ng MALDI ioniza on of biological macromolecules (Rapid Commun. Mass Spectrom., 1988)
Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 Ion sources: ESI
www.sitemaker.umich.edu/mass-spectrometry/sample_prepara on m/z analyzer inlet + + + + + + + + + + + droplet evapora on and + + charge-driven fission + + or ion expulsion + 3-5 kV + + + + + + + evapora on only 0 + 0 0 0 0 N 0 0 0 2 0 0 0 0 sample 0 0 0 0 0 0 0 0 0 0 N2 0 needle nebulisa on Electospray ioniza on (ESI) barrier
ESI sources typically heat the needle to 40°to 100°to facilitate nebulisa on and evapora on, and typically produce mul ply charged pep de ions (2+, 3+, 4+)
John B. Fenn received the 2002 Nobel Prize in Chemistry for demonstra ng ESI ioniza on of biological macromolecules (Science, 1989) – ESI is also used in fine control thrusters on satellites and interstellar probes…
Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 ESI – online LC and solvents
(nano) RP column (100–5 µm) (nano) needle spray solvent mixer
aqueous solvent organic solvent H O + 0.1% FA 2 A B ACN + 0.1% FA + 2–5% ACN
Nanospray ESI sources (5-10 µm diameter needle) achieve a higher sensi vity, probably due to the higher surface-to-volume ra o. For a spherical droplet this ra o is:
π ⋅r3 A = 4⋅π ⋅r 2 V = 4 3
A 3 6 = = V r D
Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 Analyzers: time-of-flight (TOF)
high vacuum sample ions
source detector field-free tube extrac on ( me-of-flight tube) plate (30 kV) > 1 meter
2 m⋅v 2⋅ Ek Ek = q⋅V E = ↔ v = k 2 m
We can now relate m/q (or the more commonly used m/z) 2 m 2⋅V 2⋅V 2⋅V ⋅t to the velocity of the ion, and using Newton’s kinema ca = 2 = 2 = 2 q v ⎛ x ⎞ x we can relate the speed to the travel me and (known + ⎜ ⎟ exactly calibrated) field-free tube length ⎝ t ⎠
Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 Analyzers: quadrupole (Q)
+ (U +V ⋅cosωt) permi ed m/z ejected m/z
− (U +V ⋅cosωt)
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.
Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 Analyzers: ion trap (IT)
DC/ACRF voltage
source detector
capping ring capping electrode electrode electrode
Ion traps operate by effec vely trapping the ions in an oscilla ng electrical field. Mass separa on is achieved by tuning the oscilla ng 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.
Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 Fourier transform ion cyclotron resonance (FTICR)
electrodes ion orbit
strong magne c field
An FT-ICR is essen ally a cyclotron, a type of par cle accelerator in which electrons are captured in orbits by a very strong magne c 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 superposi on of sine waves is obtained. A Fourier transforma on is therefore required to tease out the individual ion frequencies.
Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 Fourier transform ion cyclotron resonance (FTICR)
Movie h p://medicine.yale.edu/keck/proteomics/technologies/mass_spectrometry/ icrvideo.aspx
Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 Orbitrap
outer electrode inner electrode
From: h p://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 electrosta c field in which the ions form an orbitally harmonic oscilla on along the axis of the field. The frequency of the oscilla on is inversely propor onal 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.
Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 Resolution and why it matters
Resolu on in mass spectrometry is usually defined as the width of a peak at a given height (there is an alterna ve defini on based on percent valley height). This width can be recorded at different heights, but is most o en recorded at 50% peak height (FWHM).
average monoisotopic mass mass
From: Eidhammer, Flikka, Martens, Mikalsen – Wiley 2007
Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 Detectors: electron multiplier
single ion in
40V
20V 80V
60V
120V
100V
106 electrons out
Different varia ons of electron mul plier (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.
Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 TANDEM MASS SPECTROMETRY
(TANDEM-MS, MS/MS, MS2)
Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 Tandem-MS: the concept
source detector ion selector fragment mass analyzer fragmenta on
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 func on of ion selector, by selec vely allowing only ions of a given m/z to pass through. The second mass analyzer is situated a er fragmenta on is triggered (see next slides) and is used in its normal capacity as a mass analyzer for the fragments.
Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 Why tandem-MS?
pep de 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 la er are fragments that no longer contain an intact terminus. These are harder to use for ‘ladder sequencing’, but can s ll 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.
Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 Creating fragments (unimolecular)
laser ( ( ) ) ac vated ion precursor ion (metastable)
non-ac vated ion fragment ions (stable) 200 400 600 800 1000 1200 1400 1600 m/z
This fragmenta on method is called post-source decay (PSD) and relies on a single unimolecular event, in which a highly energe c (metastable) ion spontaneously fragments. PSD typically causes backbone fragmenta on. y and b ions are by far the most prevalent fragment types, although TOF-TOF instruments (see later) specifically also yield substan al internal fragments.
Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 Creating fragments (bimolecular I)
gas inlet collision cell collision gas (atom or molecule)
selected pep de
ΔV
This fragmenta on method is called collision-induced dissocia on (CID) and relies on a series of bimolecular events (collisions) to provide the pep de precursor with sufficient energy to fragment. CID typically causes backbone fragmenta on. 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.
Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 Creating fragments (bimolecular II)
electron source - fragmenta on cell electron - - - - -
selected pep de
ΔV
This fragmenta on method is called electron-capture dissocia on (ECD) or electron-transfer dissocia on (ETD) and relies on a single impact of an electron on a pep de precursor. This high- speed impact immediately imparts sufficient energy to fragment the precursor (non-ergodic process). Like CID, ETD and ECD typically cause backbone fragmenta on, but they typically result
in c and z ions. ECD is only workable in FT-ICR mass spectrometers, whereas ETD is used in traps.
Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 Ionization and fragmentation
pep de 1+
fragmenta on
b-ion y-ion
1+ ?
pep de 2+
fragmenta on 1+ 1+ b-ion y-ion
Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 MASS SPECTROMETER
CONFIGURATIONS
Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 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 rela vely poor however, and the resolu on is lacking as well (unable to dis nguish isotopes, hampering charge state determina on).
Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 ESI triple quadrupole
ESI detector source quadrupole 1 quadrupole 2 quadrupole 3
Simple instrument, that recently a racted a en on because it is well-suited for Mul ple Reac on 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.
Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 MALDI TOF-TOF
reflectron detector
MALDI source 2 linear source detector TOF 1 TOF 2 delayed reflectron extrac on voltage gate for precursor selec on
A modern version of the MALDI DE RE-TOF, the TOF-TOF relies on two TOF tubes in tandem. The second TOF is fi ed with a reflectron. Mass accuracy and resolu on are very high and the instrument can perform MS and MS/MS, both for pep des 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.
Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 ESI linear ion trap FT-ICR or Orbitrap
C-trap
ESI linear ion trap source
FT-ICR Orbitrap
The combina on of a linear ion trap and a high-resolu on, 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 resul ng fragments with high accuracy.
Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011