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Introduction to MALDI-TOF

Introduction to MALDI-TOF

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Introduction to MALDI-TOF. Features of MALDI-TOF MS. Soft ionization - analyze intact biomolecules and synthetic polymers Broad mass range - analyze a wide variety of biomolecules Simple mixtures are okay Relatively tolerant of buffers and salts Fast data acquisition - PowerPoint PPT Presentation

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Page 1: Introduction to MALDI-TOF

Introduction to MALDI-TOF

Page 2: Introduction to MALDI-TOF

Features of MALDI-TOF MS

• Soft ionization - analyze intact biomolecules and synthetic polymers

• Broad mass range - analyze a wide variety of biomolecules

• Simple mixtures are okay

• Relatively tolerant of buffers and salts

• Fast data acquisition

• Easy to use and maintain, no water or gas hook ups required

• High sensitivity, superior mass resolution and accuracy

Page 3: Introduction to MALDI-TOF

MALDI: Matrix Assisted Laser Desorption Ionization

h

Laser

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+.

AH+

+20 kV

Variable Ground Grid Grid

Sample plate

Page 4: Introduction to MALDI-TOF

Time-of-flight mass analyzer

+

+

+

+

Source Drift region (flight tube)

dete

ctor

V

•Ions are formed in pulses.

•Small ions reach the detector before large ones.

•Measures the time for ions to reach the detector.

Page 5: Introduction to MALDI-TOF

Calibration of the mass scale

The mass-to-charge ratio of an ion is proportional to the square of its drift time.

t = Drift timeL = Drift lengthm = MassK = Kinetic energy of ionz = Number of charges on ion

2

22

L

Kt

z

m

Page 6: Introduction to MALDI-TOF

Voyager-DE STR MALDI TOF

Camera

Laser

Sample plate

Pumping Pumping

Timed ion selector Reflector

Linear detector

Extractiongrids

ReflectordetectorAttenuator

Prism

Collision cell

Page 7: Introduction to MALDI-TOF

MALDI TOF HardwareLaser, Attenuator and PrismNitrogen laser at 337 nm, 3 ns wide pulses, 20 Hz. Laser attenuator varies the intensity of the laser hitting the sample. Prism deflects the laser beam into the ion source.Sample Plate and Sample StageAn accelerating voltage is applied to the sample plate in the range 15-25 kV.Variable Voltage GridA grid 1-2 mm above the sample plate with an additional voltage to fine- tune ion accelerationGround GridGrounded surface defines end of acceleration regionGrounded ApertureEntrance to flight tube

Page 8: Introduction to MALDI-TOF

MALDI TOF Hardware

Vacuum System

High vacuum is required to avoid ion collisions

Flight tube

A field free region where ions drift at a velocity inversely proportional to the square root of their mass/charge.

Linear Detector

Measures the ion abundance in linear mode (no reflector used) and sends a signal to the digitizer.

Page 9: Introduction to MALDI-TOF

+e-

primary ion

e-

e- e-L

D

-1000V

-100V

L >> D

Ions are detected with a microchannel plate

Page 10: Introduction to MALDI-TOF

Microchannel Plate (MCP)

Page 11: Introduction to MALDI-TOF

High current detector schematic

Microchannel plate

Fast scintillator

Condenser

Photomultiplier tube

signal

1kV 15kV

Used in linear mode to enhance signal from high mass molecules

Page 12: Introduction to MALDI-TOF

MALDI TOF HardwareReflectorA single stage gridded ion mirror that subjects the ions to a uniform repulsive electric field to reflect them. It is tilted by 1° in the DE-STR to focus the ions on to the detectorCollision CellGas cell for collision induced dissociation (CID) to enhance fragmentation in PSD analysisReflector DetectorMeasures ions reflected by the mirror. In the DE-STR this is a 6-10 m pore size micro-channel plate. Timed Ion SelectorA velocity selector that allows a single precursor ion of a selected mass and their fragment ions to pass to the detector. A Bradbury-Neilson gate is used.

Page 13: Introduction to MALDI-TOF

Voyager-DE STR MALDI TOF

Camera

Laser

Sample plate

Pumping Pumping

Timed ion selector Reflector

Linear detector

Extractiongrids

ReflectordetectorAttenuator

Prism

Collision cell

Page 14: Introduction to MALDI-TOF

The problem: Peaks are inherently broad in MALDI-TOF spectra (poor mass resolution).

++

+

Sample + matrix on target

Ions of same mass, different velocities

The cause: Ions of the same mass coming from the target have different speeds. This is due to uneven energy distribution when the ions are formed by the laser pulse.

Page 15: Introduction to MALDI-TOF

Can we compensate for the initial energy spread of ions of the same mass to produce narrower peaks?

Delayed Extraction

Reflector TOF Mass Analyzer

Page 16: Introduction to MALDI-TOF

Step 1: No applied electric field. Ions spread out.

++ +

Ions of same mass, different velocities

Step 2: Field applied. Slow ions accelerated more than fast ones.

0 V.

0 V.

++ +

Step 3: Slow ions catch up with faster ones.

20 kV.

20 kV.

0 V.

0 V.+++

Delayed Extraction (DE) improves performance

Page 17: Introduction to MALDI-TOF

Detector

Ion Source

What is a reflector TOF analyzer?

Reflector (Ion Mirror)

The reflector or ion mirror compensates for the initial energy spread of ions of the same mass coming from the ion source, and improves

resolution.

A single stage gridded ion mirror that subjects the ions to a uniform repulsive electric field to reflect them.

Page 18: Introduction to MALDI-TOF

0 V. +20 kV

A reflector focuses ions to give better mass resolution

++

Page 19: Introduction to MALDI-TOF

Reflector

Page 20: Introduction to MALDI-TOF

Resolution & mass accuracy on mellitin

0

2000

4000

6000

8000

Cou

nts

2840 2845 2850 2855

Mass (m/z)

Resolution = 14200

Resolution = 4500

Resolution = 18100 15 ppm error

24 ppm error

55 ppm error

Page 21: Introduction to MALDI-TOF

1. PSD refers to a method of detecting and measuring the masses of fragment ions that are formed from a selected precursor ion.

2. Fragment ions are mainly formed by unimolecular decomposition after the precursor ions are fully accelerated (after they exit the source—hence post-source decay)

3. Fragment ions are separated and detected in the reflector.

Fundamentals of Post Source Decay(PSD)

Page 22: Introduction to MALDI-TOF

Laser

ReflectorSource

Lineardetector

Reflector detector

Decay can occur at any point along here

Decomposition occurs in the flight tube

Page 23: Introduction to MALDI-TOF

No of ions

Internal energy

Only a small fraction of the precursor ions have enough energy to fragment during their lifetimes.

Internal energy of precursor ions

For peptides the efficiency of PSD fragmentation is amino acid composition and sequence dependent.

Page 24: Introduction to MALDI-TOF

There are two ways to increase the amount of fragmentation: both act to increase the precursor ions’ internal energy.

•Use higher laser intensity

•Use a collision cell

Increasing PSD Fragmentation

Page 25: Introduction to MALDI-TOF

PSD fragment ion velocities are the same as their precursors

+

+All three of these species travel at the same velocity in the flight tube until they reach the reflector.

Why? Velocity is determined by initial acceleration. Initial energy = 20 keV. Bond energies = ~ 10 eV, so breaking a bond has a very minor effect on velocities.

+

Page 26: Introduction to MALDI-TOF

Timed Ion Selector (TIS)

The TIS is a Bradbury-Neilson gate, which is a type of velocity selector. It allows only selected precursor ions and their fragments to pass through to the reflector.

+

+

-

-

Gate closed: alternating potentials on wires

Gate open: wires at ground potential

Ions

Page 27: Introduction to MALDI-TOF

Timed Ion Selector operation

TIS off

TIS on

“Gate open”

“Gate closed”

Page 28: Introduction to MALDI-TOF

Effect of the timed ion selector

Page 29: Introduction to MALDI-TOF

The intact molecular ion has translational kinetic energy equal to:

KE = 1/2 Mv2

where:

KE = kinetic energy (= zeV)

M = mass

v = velocity

Before fragmentation

Page 30: Introduction to MALDI-TOF

The translational kinetic energy of a fragment ion is

Post source fragmentation

M

mKEKE Mm

where

KEM = precursor kinetic energy

KEm = fragment kinetic energy

M = precursor mass

m = fragment mass

Page 31: Introduction to MALDI-TOF

Precusor and PSD fragment ions take different paths in the “normal” reflector

Reflectordetector

Reflector+20 kV0 V.

Fragment ion formed by PSD

Intact precursor ion

++

Page 32: Introduction to MALDI-TOF

How are PSD fragment ions that are traveling at the same speed as the

precursor ion but contain reduced kinetic energy made to arrive at the detector so

that they are focused?

By varying the “steepness” of the voltage gradient in the reflector across

the fragment ion mass range.

Page 33: Introduction to MALDI-TOF

Consider an ion (MH+) that can decompose into two fragments, A and B.

Either of the following reactions can occur:

Assume MH+ = 1,000 Da, AH+ = 700 Da, and BH+ = 300 Da

PSD mirror ratio setting

MH+ AH+ + B

MH+ A + BH+

Page 34: Introduction to MALDI-TOF

MH+

BH+

AH+

MH+ ( 1,000) correctly focused

AH+ (700) Poorly focused

BH+ (300) Poorly focused

At mirror ratio = 1.00

Page 35: Introduction to MALDI-TOF

MH+

BH+

At mirror ratio = 0.7

MH+ ( 1,000) not focused

AH+ (700) correctly focused

BH+ (300) Poorly focused

AH+

Page 36: Introduction to MALDI-TOF

BH+

AH+ & MH+

MH+ ( 1,000) not focused

AH+ (700) not focused

BH+ (300) correctly focused

At mirror ratio = 0.3

Page 37: Introduction to MALDI-TOF

MR=0.61

MR=0.71

MR=0.80

(MR= mirror ratio)

Resolution decreases as the fragment ions penetrate less into the mirror

Page 38: Introduction to MALDI-TOF

A PSD spectrum is taken in “stitches”

Page 39: Introduction to MALDI-TOF

591 753 915 1077 1239 1401

Mass (m/z)

0

4.7E+4

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

Stitched PSD[BP = 354.1, 59562]

784.3

1181.7756.3

619.31182.8

1296.7767.4650.3

646.3 739.3

742.2 1137.7

1164.8881.5 1000.7 1046.6 1229.2

59.0 164.2 269.4 374.6 479.8 585.00

6.0E+4

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

Stitched PSD[BP = 354.1, 59562]354.1

269.2(-His)

(-Tyr)(-Pro) 255.1

382.2513.2

112.1(-Val)364.1

506.3416.1115.1 235.1 534.0156.2138.1 326.1 343.1272.2 400.1 426.1 489.3

133.0 217.2 313.059.0 95.0 251.1 337.181.0 195.1 370.2140.1

PSD Spectrum of Angiotensin I, MH+ = 1296.7 DaComposite of the focused mass regions from several spectra acquired with different

mirror ratios

Page 40: Introduction to MALDI-TOF

Characteristics of CID (collison induced dissociation)

• Immonium ion signals are enhanced with collision gas; use routinely below fragment mass 200.

•Collisions can induce fragmentation of ions that do not decompose under normal PSD conditions.

•Side chain fragmentation may allow one to distinguish between Leu and Ile.

Page 41: Introduction to MALDI-TOF

Effect of CID on immonium ions

Page 42: Introduction to MALDI-TOF

(GRF Lys-C peptide KLLQDILSR; MH+ = 1085.667)

CID to distinguish between Ile and Leu