introduction to maldi tof ms l.
Download
Skip this Video
Loading SlideShow in 5 Seconds..
Introduction to MALDI-TOF MS PowerPoint Presentation
Download Presentation
Introduction to MALDI-TOF MS

Loading in 2 Seconds...

play fullscreen
1 / 42

Introduction to MALDI-TOF MS - PowerPoint PPT Presentation


  • 436 Views
  • Uploaded on

Introduction to MALDI-TOF MS. Sandler Mass Spectrometry User’s Group University of California San Francisco May 20, 2003. Features of MALDI-TOF MS. Soft ionization - analyze intact biomolecules and synthetic polymers Broad mass range - analyze a wide variety of biomolecules

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'Introduction to MALDI-TOF MS' - MikeCarlo


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
introduction to maldi tof ms

Introduction to MALDI-TOF MS

Sandler Mass Spectrometry User’s Group

University of California San Francisco

May 20, 2003

slide2

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
maldi matrix assisted laser desorption ionization
MALDI: Matrix Assisted Laser Desorption Ionization

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+.

AH+

Variable Ground

Grid Grid

+20 kV

slide4

Time-of-flight mass analyzer

Source

Drift region (flight tube)

+

+

detector

+

+

V

  • Ions are formed in pulses.
  • Small ions reach the detector before large ones.
  • Measures the time for ions to reach the detector.
calibration of the mass scale
Calibration of the mass scale

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

t = Drift time

L = Drift length

m = Mass

K = Kinetic energy of ion

z = Number of charges on ion

voyager de str maldi tof
Voyager-DE STR MALDI TOF

Sample

Linear

Extraction

plate

Reflector

detector

grids

Timed ion

detector

Attenuator

selector

Reflector

Prism

Laser

Collision cell

Camera

Pumping

Pumping

maldi tof hardware
MALDI TOF Hardware

Laser, Attenuator and Prism

Nitrogen 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 Stage

An accelerating voltage is applied to the sample plate in the range

15-25 kV.

Variable Voltage Grid

A grid 1-2 mm above the sample plate with an additional voltage to fine- tune ion acceleration

Ground Grid

Grounded surface defines end of acceleration region

Grounded Aperture

Entrance to flight tube

maldi tof hardware8
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.

ions are detected with a microchannel plate
Ions are detected with a microchannel plate

primary ion

-

1000V

+

-

e

-

e

L

-

e

-

e

-

100V

D

L >> D

slide11

High current detector schematic

Used in linear mode to enhance signal from high mass molecules

Fast scintillator

Microchannel plate

Condenser

Photomultiplier tube

signal

1kV

15kV

maldi tof hardware12
MALDI TOF Hardware

Reflector

A 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 detector

Collision Cell

Gas cell for collision induced dissociation (CID) to enhance fragmentation in PSD analysis

Reflector Detector

Measures ions reflected by the mirror. In the DE-STR this is a 6-10 mm pore size micro-channel plate.

Timed Ion Selector

A 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.

voyager de str maldi tof13
Voyager-DE STR MALDI TOF

Sample

Linear

Extraction

plate

Reflector

detector

grids

Timed ion

detector

Attenuator

selector

Reflector

Prism

Laser

Collision cell

Camera

Pumping

Pumping

slide14

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

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.

Sample + matrix on target

Ions of same mass, different velocities

+

+

+

slide15

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

Delayed Extraction

Reflector TOF Mass Analyzer

delayed extraction de improves performance
Delayed Extraction (DE) improves performance

Ions of same mass, different velocities

0 V.

+

+

+

0 V.

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

+

20 kV.

+

+

0 V.

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

+

20 kV.

+

+

0 V.

Step 3: Slow ions catch up with faster ones.

slide17

What is a reflector TOF analyzer?

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

Detector

Ion Source

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.

resolution mass accuracy on mellitin

Resolution = 18100

8000

15 ppm error

6000

Resolution = 14200

4000

Counts

24 ppm error

Resolution = 4500

2000

0

55 ppm error

2840

2845

2850

2855

Mass (m/z)

Resolution & mass accuracy on mellitin
fundamentals of post source decay psd
Fundamentals of Post Source Decay(PSD)
  • PSD refers to a method of detecting and measuring the masses of fragment ions that are formed from a selected precursor ion.
  • Fragment ions are mainly formed by unimolecular decomposition after the precursor ions are fully accelerated (after they exit the source—hence post-source decay)
  • Fragment ions are separated and detected in the reflector.
decomposition occurs in the flight tube
Decomposition occurs in the flight tube

Lineardetector

Reflector detector

Laser

Decay can occur at any point along here

Reflector

Source

internal energy of precursor ions
Internal energy of precursor ions

No of ions

Internal energy

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

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

increasing psd fragmentation
Increasing PSD Fragmentation
  • 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
psd fragment ion velocities are the same as their precursors

+

+

+

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.

timed ion selector tis
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

+

-

timed ion selector operation
Timed Ion Selector operation

TIS off

“Gate open”

TIS on

“Gate closed”

before fragmentation
Before fragmentation

The intact molecular ion has translational kinetic energy equal to:

KE = 1/2 Mv2

where:

KE = kinetic energy (= zeV)

M = mass

v = velocity

post source fragmentation
Post source fragmentation

The translational kinetic energy of a fragment ion is

where

KEM = precursor kinetic energy

KEm = fragment kinetic energy

M = precursor mass

m = fragment mass

precusor and psd fragment ions take different paths in the normal reflector

+

+

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

Reflectordetector

Intact precursor ion

Fragment ion formed by PSD

0 V.

+20 kV

Reflector

slide32

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.

psd mirror ratio setting

MH+ AH+ + B

MH+ A + BH+

PSD mirror ratio setting

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

at mirror ratio 1 00
At mirror ratio = 1.00

MH+ ( 1,000) correctly focused

AH+ (700) Poorly focused

BH+ (300) Poorly focused

AH+

BH+

MH+

at mirror ratio 0 7
At mirror ratio = 0.7

BH+

AH+

MH+

MH+ ( 1,000) not focused

AH+ (700) correctly focused

BH+ (300) Poorly focused

at mirror ratio 0 3
At mirror ratio = 0.3

BH+

AH+ & MH+

MH+ ( 1,000) not focused

AH+ (700) not focused

BH+ (300) correctly focused

resolution decreases as the fragment ions penetrate less into the mirror
Resolution decreases as the fragment ions penetrate less into the mirror

MR=0.80

MR=0.71

MR=0.61

(MR= mirror ratio)

slide39

PSD Spectrum of Angiotensin I, MH+ = 1296.7 Da

Composite of the focused mass regions from several spectra acquired with different mirror ratios

characteristics of cid collison induced dissociation
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.
cid to distinguish between ile and leu
CID to distinguish between Ile and Leu

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