Maldi-tof
This presentation is the property of its rightful owner.
Sponsored Links
1 / 37

Maldi-tof PowerPoint PPT Presentation


  • 276 Views
  • Uploaded on
  • Presentation posted in: General

Maldi-tof. Basics on Voyager. Basics on Maldi-Tof Basics sample preparation Resolution Delayed Extraction Guide wire/Beam steering Reflector Instrument tuning Calibration Theory. Sample plate. Extraction grids. Reflector detector. Linear detector. Timed ion selector.

Download Presentation

Maldi-tof

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


Maldi tof

Maldi-tof


Basics on voyager

Basics on Voyager.....

  • Basics on Maldi-Tof

  • Basics sample preparation

  • Resolution

  • Delayed Extraction

  • Guide wire/Beam steering

  • Reflector

  • Instrument tuning

  • Calibration Theory


Maldi tof

Sample

plate

Extraction

grids

Reflector

detector

Linear

detector

Timed ion

selector

Reflector

Laser

Camera

Pumping

Pumping

INLET

ION SOURCE

MASS FILTER

DETECTOR

Sample plate

HPLC

GC

Solids probe

MALDI

API/Electrospray

IonSpray

EI, CI

TOF

Quadrupole

Ion Trap

Magnetic Sector

FTMS

“Hybrid”

Microchannel Plate

Electron Multiplier

Components of a Mass Spectrometer


Maldi tof

Voyager-DE STR

Voyager-DE and DE PRO

Applied BiosystemsMALDI-TOF instruments


Maldi tof

+

+

+

+

Ion Source: MALDI

(Matrix Assisted Laser Desorption Ionisation)

Laser flash produces matrix (M) neutrals, positive, negative ions and sample neutrals.

M M*, MH+, (M-H)-

Sample molecules (A) are ionised by gas phase proton transfer

MH++A AH++M

(M-H)-+A AH-+M


Maldi tof

Ions of same mass, different velocities

+

+

+

+

+

+

+

+

+

+

+

+

0 kV

150 nsec

Detector

2: Expansion of the ion cloud in the absence of an electric field

+20 kV

3: Field applied. Gradient accelerates slow ions more than fast ones.

+

+20 kV

4: Slow ions catch up with faster ones at the detector

Delayed Extraction (DE)

0 kV

0 nsec

1: Laser fired. Formed ions detach from plate in the absence of an electric field


Maldi tof

2 KE

t2

m/z =

s2

Flight tube

+

+

+

+

Detector

Ions with different mass, same Kinetic Energy

+

+

+

+

+

+

Detector

Ions with lighter mass will fly faster and will reach the detector first

+

+

Flying times of the ions are proportional to m/z ratios

Mass Filter: TOF

(Time Of Flight)


Maldi tof

Mass Filter:

Reflector TOF

The electrical field applied within the reflector produces an ion mirror effect directing the ions towards a second detector

  • Improvement in resolution by

    • Increasing the effective flight length of the tube

    • Re-focusing of analogous ions having slight different energy due to initial spread in the ion source


Maldi tof

Sample Preparation


Maldi tof

Laboratory Set-Up


Maldi tof

Hydrophobic surface

Voyager Sample Plates

SURFACE TENSION


Maldi tof

MALDI-TOF Matrices

Sinapinic acid (3,5-Dimethoxy-4-hydroxy cinnamic acid)

-cyano-4-hydroxycinnamic acid

2,5-dihydroxybenzoic acid (2,5-DHB)

Peptide (0.1-10 pmol/l)

Protein (0.1-10 pmol/ l)

Oligonucleotide (10-100 pmol/ l)

Polymer (10-4M)

2,4,6-trihydroxy acetophenone (THAP)

2-(4-hydroxyphenylazo)-benzoic acid (HABA)

trans-3-indoleacrylic acid

Dithranol

3-hydroxypicolinic acid (3-HPA)


Maldi tof

Matrix Preparation and Crystals

Sinapinic acid

DHB

Super-DHB

a-cyano

10 g/L in 30-50% ACN with 0.1% TFA

10 mg/ml in water or 50% ACN

A = 10 mg/ml DHB in 20% ACN

B = 10 mg/ml 5-methoxysalicylic acid in 50% ACN

Combine A:B (9:1)

5 mg/ml in 50% ACN 0,1% TFA


Maldi tof

Dried Droplet

First sample

Immediately after, matrix in solvent

Sandwich Method

First matrix

Then sample

And matrix again

(air dry)

Thin Layer (Nitrocellulose)

NC and matrix solution (dry)

Then sample (dry)

TFA on top, blow off with an air supply; repeat

Thin Layer (Acetone)

First matrix in acetone (dry)

Thensample (dry)

On-plate washing possible

after drying


Maldi tof

Isotopic Resolution


Resolution 1

Resolution - 1

  • What benefit is high resolution

    • Improved identification of peptides

    • Indication of potential modification

    • Greater degree of mass accuracy

  • Resolution is defined as :

    Mass / (peak width at half peak height)


  • Resolution 2

    S=DM

    S=2DM

    DM

    NOT RESOLVED

    FULLY RESOLVED

    Resolution - 2

    R=M/DM, where M is the mass to of peak and DM is the peak width at half maximum. S= peak separation.


    Consequence of high resolution

    Consequence of High Resolution

    • The Grey lines indicate the isotopic distribution of the peptide.

    • The Red line indicates the centroid mass data for each.

      • Resolution 1000 = 1297.000 (Average Mass)

      • Resolution 3000 = 1296.680 (Monoisotopic Mass)

        RESULT = Better Mass Accuracy


    High resolution too much data monoisotopic resolution of insulin

    2 x C13

    C13

    C12 : 5730.61

    High Resolution - Too much data?Monoisotopic resolution of Insulin

    In compounds with more than 100 carbon atoms the height of the 13C isotope peak exceeds the height of the 12C peak


    Maldi tof

    Delayed Extraction


    No delayed extraction

    Fraction of second post laser fire

    Acceleration 25KV

    +

    +

    +

    +

    +

    Grid 0% (0KV)

    Ground

    Acceleration 25KV

    +

    Acceleration 25KV

    +

    +

    +

    +

    +

    +

    +

    +

    +

    Grid 0% (0KV)

    Ground

    Ground

    NO Delayed extraction


    Delayed extraction

    +

    +

    +

    +

    +

    Ground

    Fraction of second post laser fire

    +

    +

    +

    +

    +

    +

    Acceleration

    Acceleration

    25KV

    Acceleration

    +

    +

    +

    +

    +

    +

    +

    +

    +

    +

    +

    +

    +

    +

    Grid 60% (15KV)

    Ground

    Ground

    Ground

    Ground

    Delayed extraction


    Principle of delayed extraction

    Principle of Delayed Extraction

    When ions are accelerated they exhibit a broad energy spread. When forming ions in a weak electric field then applying a high voltage pulse after a time delay, this energy spread can be minimized. A potential gradient is formed in the ionization region by the voltages applied to the sample plate and the variable voltage grid.

    Ref: W.C.Wiley and I.H.McLaren Rev.Sci.Instrum 1953,26,1150-1157


    Delayed extraction why

    continuous

    extraction

    R=650

    delayed

    extraction

    R=11,000

    delayed

    extraction

    R=1,100

    continuous

    extraction

    R=125

    6130

    6140

    6150

    6160

    6170

    10600

    10800

    11000

    11200

    11400

    11600

    m/z

    m/z

    Delayed Extraction - Why

    Reflector mode

    Linear mode


    Maldi tof

    The Reflector


    Benefits of using a reflector

    Benefits of Using a Reflector

    • Provides higher performance - resolution and mass accuracy

      • Increases separation due to longer flight time

      • Filters out neutral molecules

      • Corrects time dispersion due to initial kinetic energy

      • distribution

    • Capability for PSD experiment


    Schematic of voyager de pro and de str systems

    Schematic of Voyager DE-PRO and DE-STR Systems

    Linear

    detector

    Reflector detector

    Sample plate

    Extraction grids

    Timed ion

    selector

    Reflector

    Laser

    Beam guide

    Camera

    Pumping

    Pumping


    What is the reflector

    What is the reflector?

    • The reflector is an electrical mirror with a voltage potential applied across the sides.

    • The ions are sequentially slowed down through the reflector


    Velocity focusing in reflector mode

    Velocity Focusing in Reflector Mode

    Ions must line up at the beginning of the flight tube

    Slow Fast

    slow

    slow

    fast

    fast

    Variable-voltage

    grid

    Defocusing region

    Ground grid

    Sample plate

    This initial focus is refocused by the reflector which can be fine tuned for second order velocity focusing.

    Refocusing region - some move farther into reflector, than others


    Calibration theory

    CALIBRATION THEORY


    Calibration theory definitions

    CALIBRATION THEORYDefinitions

    Accuracy, Precision & Resolution

    • a. Precision. This is a measure of repeatability, i.e. the degree of agreement between individual measurements of a set of measurements, all of the same quantity.

    • b. Accuracy. This is a measure of reliability, and is the difference between the True Value of a measured quantity and the Most Probable Value which has been derived from a series of measures. The True Value is, of course, never known.

    • c. Resolution. This is the smallest interval measurable by an instrument


    Calibration theory1

    CALIBRATION THEORY

    None of the darts are close to the true value (bull’s eye) : the measurements are not accurate. Also, since the darts are not very close to each other, the set of measurements is notprecise either.

    Since all of the measurements are close together, they areprecise, but since they are not close to the true value, they are not accurate

    The measurements are all close to the true value, so they are accurate. Also, the measurements are all close to each other, so they are precise


    Calibration theory definitions1

    CALIBRATION THEORYDefinitions

    • Accuracy : in ppm (or Da, or %)

      • 100 ppm = 0,01%

      • 10 ppm = 0,001%

      • 1 ppm = 0,0001%

  • Resolution : in FWHM (Full Width at Half Maximum) - No unit = M/DM


  • Maldi tof

    Res = 18100

    8000

    21 ppm error

    6000

    Res = 14200

    Counts

    4000

    28 ppm error

    Res = 4500

    2000

    55 ppm error

    0

    2840

    2845

    2850

    2855

    Mass (m/z)

    CALIBRATION THEORYDefinitions


    Maldi tof

    Iterative CalibrationApproach

    2) Apply calibration

    to sample

    3) Database search

    4) Internally calibrate

    using “hits” and resubmit

    1500

    2000

    2500

    1296.6801

    1) Calibrate on

    standards

    2093.0846

    2465.2024

    904.4711

    1570.6783

    3657.9231

    1000

    1500

    2000

    2500

    3000

    3500


    Maldi tof

    In-Gel Digest Hit List :Automated Close External Calibration

    Rank Digest # # (%) SwissProt Species MW (Da) Protein Name

    120060 4/16 (25%) P15992YEAST 23748.5 HEAT SHOCK PROTEIN 26.

    2341183/16 (18%) P08468 YEAST 94523.6 PET111 PROTEIN PRECURSOR.

    234931 3/16 (18%) P25301 YEAST 52247.7 DNA REPAIR PROTEIN RAD57.

    237093 3/16 (18%) P10664 YEAST 38961.1 60S RIBOSOMAL PROTEIN L2A (RP

    237100 3/16 (18%) P49626 YEAST 38931.0 60S RIBOSOMAL PROTEIN L2B (RP2).

    254802 3/16 (18%) P38863 YEAST 96825.4 HYPOTHETICAL 96.8 KD PROTEIN

    Detailed Results

    1. 4/16 matches (25%). YEAST. HEAT SHOCK PROTEIN 26. ( 23748.5 Da)

    submitted matchedppmstartendPeptide Sequence Modifications

    1274.66101274.601746.5200117126(K)DIDIEYHQNK(N)

    1461.85791461.795342.8103176189(K)ADYANGVLTLTVPK(L)

    1729.97191729.901240.8452160175(R)VITLPDYPGVDADNIK(A)

    1886.09311886.002448.1161159175(K)RVITLPDYPGVDADNIK(A)

    12 unmatched masses: 888.3 998.6 1139.6 1211.8 1225.8 1288.7 1314.8 1350.7 1537.3 1788.0 1820.0 2041.1

    The matched peptides cover 19% (41/213 AA's) of the protein.


    Maldi tof

    Sample preparation


  • Login