Maldi-tof

Basics on Voyager.....

• Basics on Maldi-Tof• Basics sample preparation

• Resolution• Delayed Extraction

• Guide wire/Beam steering• Reflector

• Instrument tuning• Calibration Theory

Camera

Pumping Pumping

Timed ion selector

Reflector

Lineardetector

Extractiongrids

Reflectordetector

Laser

Sample plate

Components of a Mass Spectrometer

INLET ION SOURCE MASS FILTER DETECTOR

Sample plateHPLCGCSolids probe

MALDIAPI/ElectrosprayIonSprayEI, CI

TOFQuadrupoleIon TrapMagnetic SectorFTMS

“Hybrid”Microchannel PlateElectron Multiplier

Applied BiosystemsMALDI-TOF instruments

Voyager-DE and DE PROVoyager-DE STR

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

Ion Source: MALDI (Matrix Assisted Laser Desorption Ionisation)

+

+

+

+

+Ions of same mass, different velocities

++

Delayed Extraction (DE)

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

0 kV

0 nsec

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

+20 kV+

+++

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

+20 kV ++

+

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

0 kV

150 nsec

++ +

Detector

Ions with different mass, same Kinetic Energy

+

++

Detector

+

++

Flight tube

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

+

+

+

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

+

+

+

Mass Filter: TOF (Time Of Flight) m/z = 2 KE

s2t2

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

Mass Filter: Reflector TOF

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

Sample Preparation

Laboratory Set-Up

Voyager Sample Plates

Hydrophobic surface

SURFACE TENSION

2,4,6-trihydroxy acetophenone (THAP)

-cyano-4-hydroxycinnamic acid2,5-dihydroxybenzoic acid (2,5-DHB)

Dithranol trans-3-indoleacrylic acid

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

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

3-hydroxypicolinic acid (3-HPA)

COOH

OH

N N

OH O OH

HO

CH3O

CH3O

CH CHCOOH

COOH

OHN

OH

OHHO

COCH3

HO

CH C(CN)COOH

C C

COOH

N

H

H

OH

HO

COOH

Peptide (0.1-10 pmol/l)

Protein (0.1-10 pmol/ l)

Oligonucleotide (10-100 pmol/ l)

Polymer (10-4M)

MALDI-TOF Matrices

Matrix Preparation and Crystals

-cyano Sinapinic acidDHB

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

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

Super-DHB10 mg/ml in water or 50% ACN

A = 10 mg/ml DHB in 20% ACNB = 10 mg/ml 5-methoxysalicylic acid in 50% ACNCombine A:B (9:1)

Thin Layer (Acetone)First matrix in acetone (dry)

Then sample (dry)

On-plate washing possibleafter drying

Thin Layer (Nitrocellulose)

NC and matrix solution (dry)

Then sample (dry)

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

Dried DropletFirst sample

Immediately after, matrix in solvent

Sandwich MethodFirst matrix

Then sample

And matrix again(air dry)

Isotopic Resolution

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

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

NOT RESOLVED

S=M

FULLY RESOLVED

M

S=2M

5719.0 5724.6 5730.2 5735.8 5741.4 5747.0

Mass (m/z)

0

2.4E+4

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

Overlaid Traces(Voyager Spec #1[BP = 3658.3, 35657] & <<Insulin Reflector Resolution_0003>> Voyager Spec #1 MC[BP = 3659

Resolution 800

Resolution 20,000

5733.61

5735.61

5731.82

5731.60

5722.39 5727.91

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

C12 : 5730.61

C13

2 x C13

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

Delayed Extraction

NO Delayed extraction

++

Acceleration 25KV

+++

Fraction of second post laser fire

Grid 0% (0KV) Ground

++

Acceleration 25KV +

++

Ground

++

Acceleration 25KV +

++

Grid 0% (0KV) Ground

Delayed extraction

+++++

Ground

+

++

+

+

Fraction of second post laser fire

Ground

+

+Acceleration

+

+

+Ground

+

+Acceleration

+++

Ground

+

+Acceleration

25KV +++

Grid 60% (15KV) Ground

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

Linear mode Reflector mode

10600 10800 11000 11200 11400 11600

m/z

continuousextraction

R=125

delayedextractionR=1,100

m/z

6130 6140 6150 6160 6170

delayedextractionR=11,000

continuousextraction

R=650

Delayed Extraction - Why

The 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

CameraCamera

Sample plateSample plate

PumpingPumping PumpingPumping

Beam guideBeam guide

Timed ionTimed ionselector selector ReflectorReflector

LinearLineardetectordetectorExtraction gridsExtraction grids Reflector detectorReflector detector

LaserLaser

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

Sample plate Ground gridVariable-voltage

grid

slow

fast

fast

slow

Ions must line up at the beginning of the flight tube

Refocusing region - some move farther into reflector, than others

Defocusing region

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

Slow Fast

CALIBRATION THEORY

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 THEORYDefinitions

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 not precise either.

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

Since all of the measurements are close together, they are precise, but since they are not close to the true

value, they are not accurate

• 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/M

CALIBRATION THEORY

Definitions

0

2000

4000

6000

8000

Co

unt

s

2840 2845 2850 2855 Mass (m/z)

Res = 14200

Res = 4500

21 ppm error

28 ppm error

55 ppm error

Res = 18100

CALIBRATION THEORYDefinitions

Iterative CalibrationApproach

1500 2000 2500

3657.9231904.4711

1296.6801

1570.6783

2093.0846

2465.2024

1000 1500 2000 2500 3000 3500

1) Calibrate onstandards

2) Apply calibrationto sample

3) Database search

4) Internally calibrateusing “hits” and resubmit

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

1 20060 4/16 (25%) P15992 YEAST 23748.5 HEAT SHOCK PROTEIN 26. 2 34118 3/16 (18%) P08468 YEAST 94523.6 PET111 PROTEIN PRECURSOR. 2 34931 3/16 (18%) P25301 YEAST 52247.7 DNA REPAIR PROTEIN RAD57. 2 37093 3/16 (18%) P10664 YEAST 38961.1 60S RIBOSOMAL PROTEIN L2A (RP2 37100 3/16 (18%) P49626 YEAST 38931.0 60S RIBOSOMAL PROTEIN L2B (RP2). 2 54802 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 matched ppm start end Peptide Sequence Modifications1274.6610 1274.6017 46.5200 117 126 (K)DIDIEYHQNK(N)1461.8579 1461.7953 42.8103 176 189 (K)ADYANGVLTLTVPK(L)1729.9719 1729.9012 40.8452 160 175 (R)VITLPDYPGVDADNIK(A)1886.0931 1886.0024 48.1161 159 175 (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.

In-Gel Digest Hit List : Automated Close External Calibration

Sample preparation