Title: Introduction to MALDITOF
1(No Transcript)
2Introduction to MALDI-TOF
- Jennifer Krone Ph.D.
- Senior Field Application Specialist
3Basic Components of a Mass Spectrometer
Vacuum Envelope
Ionization
Mass Sorting (filtering)
Detection
Ion Source
Ion Detector
Mass Analyzer
Forms ions (charged molecules)
Sort Ions by Mass (m/z)
Detect ions
Data System
Mass Spectrum
Relative Abundance
Sample Inlet
Sample/Matrix dried on MALDI Plate
2000
1000
m/z
4Mass Spectrometer Schematic
5Ion Sources make ions from sample molecules
- For biological applications, ion sources convert
neutral molecules to ions by adding (taking away)
one or more protons. - Ions may be singly or multiply charged.
- Ions are easier to control in the mass
spectrometer than neutral molecules. - Ions are easier to detect than neutral molecules.
- In Voyager MALDI-TOF MS ions are formed in pulses
at 3 20 cycles per second.
6Ion Sources Make IonsFrom Sample Molecules
- For biological applications, ion sources convert
neutral molecules to ions by adding or taking
away one or more protons. - Ions may be singly or multiply charged.
- Ions are easier to control in the mass
spectrometer than neutral molecules. - Ions are easier to detect than neutral molecules.
M H energy MH
M2H2
7Primary Ionization Techniques for Biological
Molecules
- Electrospray Ionization (ESI)
- Includes high (ml) and low (nl) flow rate liquid
transfer - NanosprayTM Ion Source
- ElectrosprayTM Source
- Micro Ion SprayTM Source
- Turbo Ion Spray TM Source
- Matrix Assisted Laser Desorption Ionization
(MALDI) - Sample is dissolved with an energy transferring
compound or matrix - This is spotted on to a metal plate and allowed
to crystallize - When a laser is applied matrix crystals transfer
energy facilitating ionization
8Electrospray Ionization (ESI)
Vacuum Interface
charged droplets form
High Voltage
Sample Flow
Nebulizer Gas
Ions released
Spray
Broad range of implementations based on flow
rate and polarity of compound class
9ESI Spectrum of Trypsinogen (MW 23983)
M 15 H
1599.8
M 16 H
M 14 H
1499.9
1714.1
Relative Abundance
M 13 H
1845.9
1411.9
1999.6
2181.6
m/z
Mass-to-charge ratio
10Matrix Assisted Laser Desorption Ionization
(MALDI)
Laser
Sample plate
hn
1. Sample (A) is mixed with excess matrix (M)
and dried on a MALDI plate. 2. Laser flash
ionizes matrix molecules. 3. Sample molecules
are ionized by proton transfer from matrix MH
A ? M AH.
Variable Ground Grid Grid
20 kV
11MALDI/TOF Mass Spectrum of IgG
MH
Relative Abundance
(M2H)2
(M3H)3
50000
100000
150000
200000
m/z
12Mass Analyzers Measure The Mass of Ions
- Operate under high vacuum (keeps ions from
bumping into gas molecules) - Actually measure mass-to-charge ratio of ions
(m/z) - Key specifications are resolution, mass accuracy,
sensitivity and mass range.
13Another parameter to rememberIsotopes
Most elements have more than one stable
isotope. For example, most carbon atoms have a
mass of 12 Da, but in nature, 1.1 of C atoms
have an extra neutron, making their mass 13 Da.
Mass spectrum of peptide with 94 C-atoms (19
amino acid residues)
Monoisotopic mass
No 13C atoms (all 12C)
One 13C atom
Two 13C atoms
14Mass Analyzers Employed in Modern Proteomics
- Quadrupole (including multiple Quadrupoles)
- Unit resolution capabilities using frequencies
to separate ions. Low mass accuracy and
resolution. Limited mass range. - Time of Flight (including multiple TOF)
- High resolution and accurate mass capabilities
using time and distance to separate ions.
Unlimited mass range. - Ion Trap
- Unit and higher resolution capabilities using
frequency to separate ions. Moderate mass
accuracy, limited mass range. - Fourier Transform (FT)
- High resolution and mass accuracy using frequency
to separate ions. - Hybrid
- Combination of different types of analyzers to
achieve specific application requirements.
15Quadrupole Mass Analyzer
Uses a combination of Radio Frequency(RF) and
Direct Current(DC) voltages to operate as a mass
filter.
- Has four parallel metal rods.
- Lets one mass pass through at a time.
- Can scan through all masses or sit at one fixed
mass.
16QTRAP Hybrid Mass Spectrometer
3D Trap Full Scan Sensitivity MS3 (or greater)
Q Trap
Tandem in SPACE TIME
Tandem in TIME
QqQ MRM Sensitivity Neutral Loss Precursor Scan
Full Scan Sensitivity MS3 MRM Sensitivity Neutral
Loss Precursor Scan Novel Scan Types
Tandem in SPACE
17MS/MS Tandem Mass Spectrometry Provides
information about the basic structure of the
compound
Applying two or more steps of mass analysis
separated in space or time (in the same
instrument) to select an analyte (ion) of
interest from a mixture and generate fragments
from it to give structural information.
18MS/MSTandem Mass Spectrum
Product ion spectrum of a compound m 1
Tandem Mass Spectrometers Triple Quadrupoles,
hybrid Quadrupole/trap, hybrid Quadrupole/TOF,
tandem time-of-flight
19Mass Spectrometers used in proteome research
Aebersold and Mann (2003) Nature 422, 198-207
20Voyager System Components
1. Sample matrix dried on target plate
4. Ions are accelerated by an electric field to
the same kinetic energy they separate
according to mass as they drift thru the
field-free region of the flight tube
20 - 25 kV
Flight tube
2. Target is introduced into high vacuum
chamber
High vacuum
High voltage
5. Ions strike the detector at different
times depending on the mass/charge ratio
3. Sample is irradiated with laser, clock starts
to measure time-of-flight
Pulsed laser
.
6. A data system controls instrument
parameters, acquires signal vs. time, and
processes the data
21Mass analyzers measure the mass of ions
- Operate under high vacuum (keeps ions from
bumping into gas molecules), typically 10-7
10-8 torr - Actually measure mass-to-charge ratio of ions
(m/z) - Several kinds exist for bioanalysis, quadrupole
and time-of-flight (TOF) are most used. - Ions are detected by devices that convert the
arrival of ions into emitted electrons or photons
and amplify this signal, e.g., Microchannel
Plates, Photomultipliers.
22MALDI Matrix Assisted Laser Desorption
Ionization
Sample plate
hn
- Sample (M) is mixed with excess matrix (X) and
dried on a MALDI plate. - The plate is loaded onto the sample stage in the
Ion Source
to Mass Analyzer
Sample Matrix
Ground Grid
23MALDI Matrix Assisted Laser Desorption
Ionization
Laser
hn
- Laser flash produces matrix neutrals (X), matrix
ions (XH), (X-H)- , and sample neutrals (M). - 3. Sample molecules are ionized by proton
transfer from matrix ions - XH M ? X MH.
- X-H- M ? X M-H-
24MALDI Matrix Assisted Laser Desorption
Ionization
hn
- Ion Extraction High voltage is applied
to the sample plate, accelerating ions out of the
Ion Source into the Flight Tube. -
MH
20 kV
25Time-of-Flight Mass Analyzer
Flight Tube
Ion Source
20-25 kV
Detector
Principle If ions are accelerated with the same
potential at a fixed point and a fixed initial
time and are allowed to drift, the ions will
separate according to their mass to charge ratios.
26Time-of-Flight Mass Analyzer
Flight Tube
Ion Source
Detector
The ions enter the flight tube with the lighter
ions travelling faster than the heavier ions to
the detector
27Time-of-Flight Mass Analyzer
Flight Tube
Ion Source
Detector
The lighter ions strike the detector before the
heavier ions. This time of flight (TOF) can be
converted to mass
28Ions are detected with a Microchannel Plate
-1000 V
-100 V
D 6-25 u
Primary Ion from Flight Tube
L
29Ions are detected with a Microchannel Plate
-1000 V
-100 V
D 6-25 u
L
30Ions are detected with a Microchannel Plate
-1000 V
-100 V
D 6-25 u
e-
L
31Ions are detected with a Microchannel Plate
-1000 V
-100 V
D 6-25 u
e-
e-
e-
e-
L
32Ions are detected with a Microchannel Plate
-1000 V
-100 V
D 6-25 u
e-
e-
e-
e-
L
33Ions are detected with a Microchannel Plate
-1000 V
-100 V
D 6-25 u
e-
e-
e-
e-
103 Amplification
L
34High Current Detector schematic Used in Linear
Mode
Fast scintillator
Microchannel plate
Lens
Photomultiplier tube
Signal out to Digitizer
Ion in Flight Tube
35What is a Reflector TOF analyzer?
Reflector Detector
Flight Tube
Ion Source
Reflector (Ion Mirror)
The reflector acts as an ion mirror, extending
the flight length without increasing the
instrument size. The reflector also compensates
for the initial energy spread of ions having the
same mass. The result is improved resolution.
36Voyager Data System Options
37Calibration of the mass scale
The mass-to-charge ratio of an ion is
proportional to the square of its time of flight
in the analyzer (drift time).
t Drift time L Drift length m Mass K Kinet
ic energy of ion z Number of charges on ion
38Important performance factors
Mass accuracy How accurate is the mass
measurement? Resolution How well separated are
the peaks from each other? Sensitivity How
small an amount can be detected / analyzed?
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40Higher Resolution means better Mass Accuracy
15 ppm error
24 ppm error
55 ppm error
41Voyager Product Line
- Voyager-DE Biospectrometry Workstation
- Benchtop linear with Delayed Extraction
- Voyager-DE PRO Biospectrometry Workstation
- Benchtop with reflector for increased performance
- Voyager-DE STR Biospectrometry Workstation
- Floor standing with reflector for ultimate
performance and flexibility
42Voyager DE MALDI-TOF Applications
43Features of MALDI-TOF MS
- Soft ionization - biomolecules remain intact
- Broad mass range - analyze a wide variety
of biomolecules (40
Da to gt 400 KDa) - Mixtures are OK - little need for purification
- High sensitivity
- Data is easy to interpret
- Buffers and salts have small effect
- Fast
- Easy to use and maintain
44US Technical Support
Mass Spec 508-383-7779 Michelle Chen, Jing
DeNore e-mail tsupport_at_appliedbiosystems.com
Tech Support Fax 508-383-7212 or
508-383-7855 Front Desk 508-383-7700 Sales/Servi
ce 800-831-6844, option 1 (Foster City,
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