Lecture 9 Mass Spectrommetry Techniques

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Lecture 9
Mass Spectrometry
Oct 2011 SDMBT
Objectives
 Describe the principle of Mass Spectrometry
 In particular, the ionisation methods
(a) MALDI
(b) ESI
Oct 2011 SDMBT
General workflow for proteomic analysis
Sample
Sample preparation
Protein mixture
Sample separation and visualisation
Comparative analysis
Digestion
Peptides
Mass spectrometry
MS data
Database search
Protein identification
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Protein Identification
MALDI-TOF
ESI-MS
Molecular weight of tryptic peptides
Molecular weight of protein
MS/MS
Molecular weight of peptide fragments
Oct 2011 SDMBT
Mass spectrometry workflow
Typical process
ionisation
detection
acceleration
(Medical College of Georgia)
sorting
Oct 2011 SDMBT
Mass-tocharge ratio
(m/z)
The need for ionisation
Analyte has to be converted into gas-phase ions – only
charged ions can be detected by MS
Movement of gas-phase ions can be precisely controlled
by electromagnetic fields
Difficulty of generating gas-phase ions results in complex
instruments and high costs – need high vacuum
Gas phase ions can be generated by
e.g. an electron beam (electron ionisation)
collision with inert gas molecules (MS-MS)
a laser beam (MALDI-TOF)
a charged nozzle (ESI-MS)
Oct 2011 SDMBT
Mass Spectrum
A plot mass of ions (m/z) (x-axis) versus the intensity of the signal
(roughly corresponding to the number of ions) (y-axis)
Mass spectrum of water – ionised by electron ionisation
Notice that the water molecule fragments upon ionisation
H2O+
HO+
O+
H+
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HCHO+
CHO+
CO+
HCHO2+
HCH+
CH+
C+
Notice: the horizontal axis is mass/charge ratio of ions (m/z)
Oct 2011 SDMBT
The need for ionisation
Proteins are large macromolecules – electron ionisation
works only for volatile compounds – also electron
ionisation is a ‘hard’ ionisation technique – molecule
fragments into smaller ions
Need to find an efficient method to convert proteins from
liquid phase to gas-phase ions
Soft ionisation methods like electrospray ionisation (ESI)
and matrix-assisted laser-desorption ionisation (MALDI)
Oct 2011 SDMBT
Soft ionisation
Ionise peptides into gas-phase ions
Too much energy (hard ionisation) breaks peptides into
smaller fragments
Oct 2011 SDMBT
(Prof. Jose-Luis Jimenez)
Soft ionisation
MALDI-TOF mass spectrum
Peptide does not fragment
Maldi TOF/TOF mass spectroscopic spectre for UCH-L3 digested Ecotin-Ubiquitin-FLS in the m/z range 799–4013. The peaks
at 2683 and 1342 represent the single and double protonation states of the FLS peptide, respectively .
Oct 2011 SDMBT
MALDI
Peptides are mixed with a matrix, which crystallise on a
metal plate
Peptides protonated by matrix and solvent
Pulses of laser transfer energy to matrix
Matrix vaporises with peptide samples
Oct 2011 SDMBT
MALDI
Oct 2011 SDMBT
MALDI plate
(University of Pittsburgh BRSF)
(Bruker Daltonics)
Oct 2011 SDMBT
Matrix
Contains ring structures to absorb energy from UV laser
Contains acid group to protonate peptides
OH
Matrix solution – DHB or CHCA dissolved in
acetonitrile and trifluoroacetic acid
HOOC
OH
2,5-dihydroxybenzoic acid
COOH
CN
HO
a-cyano-4-hydroxycinnamic acid
Sinapinic acid
Oct 2011 SDMBT
Characteristics of MALDI
Efficient protonation of peptides
Ions generated in discrete packets due to laser pulses
Combined with time-of-flight (TOF) mass analysis to give
very high sensitivity
More tolerant than ESI to contaminants (urea, SDS), thus
HPLC separation is not required
Oct 2011 SDMBT
Mw of cytochrome C 12000
Mw of ubiqutin 8500
Mw of myoglobin
17000
MALDI-TOF of mixture of cytochrome, ubiquitin, myoglobin
(without PSD) – note single peaks (except cytochrome) – can
tell Mw of 3 proteins in one spectrum
Oct 2011 SDMBT
Trypsin digested peptides
From last lecture: one spot in 2D-gel represents one protein – this protein
is digested with trypsin into smaller peptides.
The peptide mixture is spotted on the MALDI plate
Arginine or Lysine
(ExPasy PeptideCutter)
Proline
Oct 2011 SDMBT
MALDI spectrum of Tryptic digest of β-casein
Major peaks at:
646
742
748
780
830
2186
Each peak represents one peptide
digested by trypsin from casein
Represent expected sizes of tryptic peptides
Soft ionisation ensures no further
College
fragmentation of peptides King’s
London
(Pierce)
Oct 2011 SDMBT
Tryptic digest of β-casein
Most abundant peak  base peak,
abundance set to 100%
MS is seldom quantitative, only tells
relative abundance
Ideally every peptide is King’s
ionised
to same
College
London
extent  100%
(Pierce)
Oct 2011 SDMBT
Electrospray Ionisation (ESI)
Peptides in acidic solution are pushed out of a fine capillary
A high positive charge at the capillary results in the formation
of a Taylor cone
Peptides protonated in charged droplets and ionised
Gas-phase ions repelled from capillary into instrument
Nitrogen aids in evaporation of solvent from charged droplets
Oct 2011 SDMBT
Electrospray Ionisation (ESI)
Oct 2011 SDMBT
(Royal Society of Chemistry)
Electrospray Ionisation (ESI)
High voltage results in accumulation of positive charges on
droplets
Increased charge density results in instability of droplets
Droplets break down successively into smaller sizes
Oct 2011 SDMBT
(New Objective, Inc)
Characteristics of ESI
Acidic conditions result in protonation of all basic sites in
peptides
Basic side groups of lysine, arginine, histidine
Produces peptide ions that are multiply protonated
Advantageous for peptides digested by trypsin as doubly
charged peptides are formed
Oct 2011 SDMBT
Characteristics of ESI
Efficient ionisation process results in sensitivity of ESI
experiments
General compatibility of ESI with reversed-phase high
performance liquid chromatography (RP-HPLC)  LC/MS-MS
Oct 2011 SDMBT
ESI-MS - often see doubly, triply, multiply charged ions
Oct 2011 SDMBT
ESI of myoglobin
Compare with slide 17
More complicated
Mw=1413.8x12-12 = 16953.6
Different scale on horizontal axis
Oct 2011 SDMBT
Mw – need to know how to interpret by assigning charges
Compare with slide 17
More complicated
Different scale on horizontal axis
ESI of cytochrome C
Oct 2011 SDMBT
Compare with slide 17
More complicated
Different scale on horizontal axis
7+
6+
5+
ESI of ubiquitin
Mw = 8484.5
Oct 2011 SDMBT
MALDI-TOF – 3 peaks
Each of each protein
ESI – complicated
if sample was a mixture
– 3 superimposed
spectra
Oct 2011 SDMBT
Comparison of ionisation methods
MALDI-TOF
Sample
ESI
Solution but ends up
embedded in crystalline
matrix
Solution
eg can come straight from
HPLC
Sample tolerant to salts
Results can be affected by
salts eg phosphates
Singly charged ions
Adduct-formation not so
Common.
Appearance – one peak
Oct 2011 SDMBT
Multiply-charged ions
Adducts with salts common
May be difficult to interpret
spectra
Appearance – many peaks
due to multiple charges
Comparison of ionisation methods
MALDI-TOF
ESI
Ionisation
Laser
Charged spray nozzle
Mass analyser
TOF
Quad
Fragmentation is made
possible by
PSD (Post-Source decay)
MS-MS or Tandem MS
“Pseudo-MS-MS”
Ions fragmented by
collision with inert gas
Mainly peptides
Peptides and proteins
Use
Oct 2011 SDMBT
Components of a mass spectrometer
ionisation
Atmosphere
Sample
Inlet
Ionisation
Method
acceleration
detection
Vacuum System
Mass
Analyser
MALDI
TOF
ESI
Quadrupole
Detector
Data
System
Ion trap
Combination
Oct 2011 SDMBT
(adapted from Thermo Finnigan)
The need for a mass analyser
Gas-phase ions has to be filtered/arranged in order to
allow selection of specific ions for further analysis.
Movement of gas-phase ions precisely controlled by use
of electromagnetic fields in a mass analyser
Main types of mass analyser
-TOF (time-of-flight analyser)
-Quadrupole analyser
-Fourier-Transform Ion Cyclotron Resonance
-Orbitrap
Oct 2011 SDMBT
TOF
Time-of-flight (TOF) analyser is the simplest mass
analyser
Peptide ions accelerated into flight-tube, and maintains a
velocity due its given kinetic energy
TOF analyzer requires that ions are introduced in a pulse
 well-suited for MALDI
Resolution increases with length of TOF tube
Oct 2011 SDMBT
Linear TOF
(John Lennon, University of Washington)
Oct 2011 SDMBT
MALDI- TOF
with reflectron
Reflectron - This turns ions around in an electric field, sending
them towards the detector – improves mass resolution
Oct 2011 SDMBT
Quadrupole
Made up of 4 parallel gold bars
Complex electromagnetic field
set up which allows only ions of a
a set mass/charge ratio through
(NASA)
Oct 2011 SDMBT
Flight path of an ion through a
quadrupole
Correct m/z ratio
Larger/smaller m/z ratio
Ions that spiral out
of control crash into
the rods or casing
Oct 2011 SDMBT
Fourier Transform Ion cyclotron
resonance (FT-ICR)
Magnetic fields applied to trapping plates constrain the ions to move around in a circle
in between the plates. The circular motion induces an alternating current.
The frequency of the AC is related to the m/z ratio.
Main advantage of FT-ICR
is very high mass resolution
See this website
http://www.chm.bris.ac.uk/ms/theory/fticr-massspec.html
Oct 2011 SDMBT
Orbitrap
Similar to FT-ICR
Electric field applied to trapping plates constrain the ions to move around in a circle
in between the plates. The circular motion induces an alternating current.
The frequency of the AC is related to the m/z ratio.
Outer electrode
Main advantage of Orbitrap
is very high mass resolution
Inner electrode
Image from thermo.com
Oct 2011 SDMBT
Tandem MS (MS/MS)
MALDI and ESI are soft ionisation techniques – peptides or proteins
do not fragment – useful for molecular weight determination
However sometimes fragmentation is useful because it give useful information
about the amino acid sequence of peptides (next lecture)
Fragmentation can achieved by a number of ways
-Post source decay (PSD)
-Collision induced dissociation (CID)
-Infrared multiphoton dissociation
-Electron capture dissociation (ECD)
-Electron transfer dissociation (ETD)
Oct 2011 SDMBT
Post-Source Decay (PSD)
- Method of fragmenting intact peptides - get amino acid
sequence information (see next lecture)
- Use higher laser power or introduce inert gas to collide
with ions so that ions fragments between source and TOF
- Electronics to select ions to go into the TOF
Oct 2011 SDMBT
http://abrf.org/ABRFNews/1995/December1995/dec95maldi.html
Ion trap
All other dissociation techniques involve the use of an ion trap
Ion trap essentially a quadrupole where the magnetic field is set such that
a particular ion is trapped in the space between the electrodes
(NASA)
(K. Yoshinari, Rapid Commun. Mass Spectrom. 14, 215-223, 2000)
Oct 2011 SDMBT
Ion trap
-Collision induced dissociation (CID) – inert gas (e.g. Xe, Ar, N2 or He) is
introduced into trap, collisions will cause peptides to fragment usually the C-N peptide
bond to produce b and y ions (see later)
-Infrared multiphoton dissociation (IRMPD) – IR laser is fired into ions to excite the
vibrations of peptides.
-Electron capture dissociation (ECD) – electron beam is fired into ions to produce c
and z ions
-Electron transfer dissociation (ETD) – singly charged anion, fluoranthracene is
introduced. An electron is transferred to peptide. Results in c and z fragments
Oct 2011 SDMBT
(March, JMS Vol. 32, 351-369, 1997 )
Hybrid mass analysers
Basically a combination of two types of mass analysers
or many of the same type
Each analyser performs a different function
Used to perform tandem mass spectrometry, using
collision induced dissociation (CID)
Oct 2011 SDMBT
Tandem quadrupoles (QQQ/TSQ)
Scanning of all
m/z of daughter
ions
Select specific
parent ion
CID with inert
gas
Video
Quantitative Chemical Analysis
Oct 2011 SDMBT
Tandem mass spectrometry
A) MS spectrum of HSP27. The peptide whose MS/MS spectrum is shown in panel B is indicated. B) MS/MS spectrum of the peptide
ion m/z 1163 obtained in CID mode.
Perroud et al. Molecular Cancer 2006 5:64
Oct 2011 SDMBT
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