MALDI MS Tutorial

Pierre Chaurand, Lisa Manier

Mass Spectrometry Research CenterVanderbilt University

Mass Range

OrganicChemistry

p+n0

0.000 000 000 000 000 000 000 001 g

AtomsBiochemistrye-

Alchemy

Beginning of the 20th Century

Desorption / Ionization : The Probes

Molecules are brought from a surface into the gas phase (desorbed) and ionized at the same time.

Plasma Desorption (PD)252Cf fission fragments, High energy (MeV) particles or ions

Fast Atom Bombardment (FAB)Low energy atoms (keV), (cesium)

Secondary Ion MSLow energy ions (keV), (cesium, gallium...)

Matrix-assisted Laser Desorption/Ionization (MALDI)UV and IR lasers

Matrix Assisted Laser Desorption-Ionization or MALDI

Mixture between a MATRIX andan analyte molecule (5000 for 1)

Co-crystallization (solid sample)

Laser pulse (106W/cm2)

Desorption-Ionization of the analyte molecule (Molecular ion [M+H]+)

Analysis of the molecular ions bytime-of-flight mass spectrometry

+ ++

Matrix for UV nm laser light (337 & 355 nm)

C C

OCH3

HO

3H COH

H

COOHC C

HO

H

CN

COOHSinapinic ac. (SA)Alpha Cyano-4-hydroxy-

cinnamic ac. (αCHCA)

OH

COOHN

OH

HO COOH

2,5-Dihydroxy- benzoic ac. 3-Hydroxy-picolinic (DHBA) (3HPA)

• Absorb at wave length used• Low vapor pressure (< 10-7 Torr)• Solvent compatible with sample• Co-crystallize with sample• Proton donor (M → [M+H]+)

Matrix for UV laser light (337 & 355 nm)

Matrix PolarityBiomolecule

PeptidesProteinsPolysaccharidesNucleic ac.

SA- αCHCAαCHCA

DHBA3HPA

++

+

+/

SA: 10-20 mg/ml, in 50/50/0.1 - Acetonitrile/H2O/TFAαCHCA: 10 mg/ml, in 50/50/0.1 - Acetonitrile/H2O/TFADHBA: 10 mg/ml, in:100/0.1 - H2O/TFA

50/50/0.1 - EtOH/H2O/TFA

Loading Methods

Droplet method:

1. Pipette 0.3-0.5 µl of sample directly onto the sample plate.

2. Immediately pipette 0.3-0.5 µl of matrix on top of sample drop.

3. Dry in the desiccator under vacuum.

Mixing method:

1. Mix 0.3-0.5 µl sample and 0.3-0.5 µl matrix solution in a small Eppendorf tube.

2. Pipette ~1 µl of mixture onto the sample plate.3. Dry in desiccator under vacuum.

Thin Layer Method

1. Make up a solution of 99% acetone/ 1% 0.1% TFA with 20 mg/ml CHCA or 100% MeOH with 20 mg/ml THAP.

2. Pipette 0.5-1 µl of matrix solution onto sample plate and allow it to spread and dry at room temperature and pressure.

3. For acidic sample solutions (<pH2), add the sample directly on top of dried matrix. For other samples, place 0.5 µl of TFA (0.5-10%) on top of dried matrix and then pipette the sample solution on top of the TFA drop.

4. Dry at room temperature and pressure.

Ref. O. Vorm, et al., Anal. Chem. 1994, 66, 3281.

Matrix Crystallization After DryingαCHCA

rounded

Sinapinic Acid

rhomboid

DHBA, air dried

irregular crystals

3-HPA

fan-like around edge of sample well

Buffers

Suitable for Direct Analysis by MALDI MS

Unsuitable for Direct Analysis by MALDI MS

Ammonium bicarbonate

Ammonium acetate

Bis-Tris

Tris (≤ 100 mM)

Hepes (< 100 mM)

Phosphate buffers

Sulfate buffers

Tris (> 100 mM)

Hepes (>100 mM)

Sample Clean upFor samples containing: Phosphate or sulfate buffers, Salts, Detergents

Washing:

1. Use either 0.1% TFA for buffer and salt removal or 5% isopropanol for removal of nonionic detergents.

2. Pipette 1-5 µl of cleaning solvent onto the dried sample and matrix spot. Wait 5-10 seconds and remove the liquid either by pipette or blowing off with a stream of clean air.

3. Repeat step 2 two to five times.

4. Dry before MALDI analysis.

Cation Exchange: Used for desalting

1. Place ~0.1 mg of cation exchange beads (200 mesh) on a piece of parafilm.

2, Add 5 to 10 µl of sample to the beads.

3. Mix by pipetting up and down about 20 times.

4. Allow the beads to settle for 30 seconds.

5. Remove the supernatant with a clean pipet tip and spot onto the MALDI plate.

Time-of-Flight Mass Spectrometry

Probe (Start)+/- U

+m/z

dsource dtof

Ion detectorIon Source Field free drift tube

ttotal = tsource + ttof

Eqm aFrrr

==

Time-of-flight in Source (constant field acceleration)

Time-of-flight in tube (field-free)

Ekin = qU = ½ mV2

V = t tof

d tof

qUm

V 2t tof == d tof d tof

Kinetic energy:(U, accelerating potential)

Drift time : orV

=d toft tof

ts qEmds2

=

a Eq/m=

V = Eq/m = Vo + (Eq/m)t⌡⌠

~0

Force and acceleration:(a, acceleration; E, electric field in source; q, charge; m, mass)

Velocity and time:(V, velocity; t, time)

t =E

V - Vo (m/q)

d = V dt⌡⌠Position:

(d, position in source)

~0d = do + Vot + ½(Eq/m)t2

~0

a dV/dt=

Total time-of-flight

ttot = ts + ttof

bqmat +=

where a and b are defined by the physical dimensions of the instrument and the operating parameters.

qUmttot 2

+= d tofmds2

qE

~0

Time-of-Flight Mass Spectrometry (TOF-MS)

Linear TOF :

Ionsignals

Ionizing Probe (start)M3 M2 M1

+/- U

Ion detector (MCP)

M3

M2

M1

t3t 2t 1 Time or MStart

t = a M + b

MALDI: Data acquisition Transient Recorder

Ionsignals

Ion detector (MCP)

++

0 200 1000 1200400 600 800

Time [100 ns]

4000 6000 8000 10000

Mass [m/z]

SoftwareData transfer to PC,Calibration

MALDI DE-STR TOF MS from Applied Biosystems

Time-of-Flight Mass Spectrometers, General Organization

Laser

AttenuatorPrism

Camera

Guide Wire

Reflector

Detectors

Ion Selector

Sample Plate

Grids

BA1

TC2

BA2

TC1

Turbo PumpsValves

Inlet

Fore Pump

Instrument Panel

Indicates that the laser is powered on Mayor problem! High voltage is on

Pumps are operating

normal

Computer is controlling the

instrument (software is ready)

Indicates the laser is firing

Select Gauge

Press to Switch on / off Gauge

Pressure Gauges

displays E03 (indicates gauge not connected)

Not usedTC1,TC3,TC4

Less than 10 –2 during operationHigher when loading or ejecting sample plate.

Pressure in sample loading chamberTC2

Less than 5 x10 -8Pressure in mirror chamberBA2

Less than 5 x 10 –7Pressure in main source chamberBA1

Expected PressureMeasuresGauge

[torr]

Isotopic Distribution

1312

100

90

80

70

60

50

40

30

20

10

0

C1

122121120

100

90

80

70

60

50

40

30

20

10

0

C10

1,2061,2041,2021,200

100

90

80

70

60

50

40

30

20

10

0

C100

12,03012,02012,01012,000

100

90

80

70

60

50

40

30

20

10

0

C1000

1,6821,6801,6781,6761,6741,672

1673.9 Average MassNeurotensin 100

90

80

70

60

50

Res. 1’000 40

30

20

10

pQLYENKPRRPYILMW 1672.9

40

1,6821,6801,6781,6761,6741,672

100

90

80

70

60

50

30

20

10

0

[M+H]+

[M+H+1]+

[M+H+2]+

[M+H+3]+

[M+H+4]+

1672.9 Monoisotopic Mass

Res. 10’000

Constant field extraction TOF MS

Sample E1

25 kV

++

Single ion counting (PDMS, SIMS, FAB...)

E1

25 kV

∆ t

++

++

Sample

LD, MALDI

+εE

++

∆ t

+εE, + ∆d∆d

V. Bokelmann, B. Spengler, R. Kaufmann, Eur. Mass Spectrom. 1, 81-93 (1995)

B. SpenglerJ. Mass Spectrom., 32, 1019-1036 (1997)

MALDI ion plume

DHB matrix and Substance P [M+H]+ ions densities measured 1 µs after laser impact.

Bleu: DHBRed: Substance P (MW 1347.7

Delayed Extraction in MALDI TOF MS

[M+H]+

velocitydistribution

(300<v<1000 m/s)

Sam

ple

E1 E20

25 t=0, startUbias=25 kVt=TextUpulse<25 kV

+

+

M/∆M = f(Ubias, Upulse, Text., m)

Compensation of the initial velocity distribution resulting from the MALDI desorption process to time focus the ions on the detector.

Increase in resolution

MALDI TOF MS of Cytochrome C, MW 12360.1

Constant field extraction Delayed extraction

12000 12200 12400 12600 12800 13000Mass (m/z)

12000 12200 12400 12600 12800 13000Mass (m/z)

[M+H]+ [M+H]+

[M+SA+H]+

[M+SA+H]+

M/∆M = 300 M/∆M = 1100

MALDI TOF MS of Protein Mixture

2000 7000 12000 17000 22000 27000Mass (m/z)

0

20

40

60

80

100%

Inte

nsity

5734

.6[M

+H]+

Insu

lin

1695

2.5

[M+H

]+M

yogl

obin

1236

1.1

[M+H

]+C

ytoc

hrom

e C

2399

2.8

[M+H

]+T

ryps

inog

en

8476

.8, [

M+2

H]2+

Myo

.

6181

.4, [

M+2

H]2+

Cyt

.C

1199

6.8,

[M+2

H]2+

Try

p.

2865

.4, [

M+2

H]2+

Ins.

MALDI TOF MS of Protein Mixture, Ubias= 25 keV

5550 5650 5750 5850 5950 6050 23100 23540 23980 24420 24860 25300

5550 5650 5750 5850 5950 6050Mass (m/z)

23100 23540 23980 24420 24860 25300

M/∆M = 800

M/∆M = 350 M/∆M = 500

M/∆M = 220[M+H]+

[M+H]+

[M+H]+

[M+H]+

Upulse = 23625 V

delay = 150+εns

Upulse = 22750 V

delay = 250+εns

[M+SA+H]+

[M+SA+H]+

[M+SA+H]+

[M+2SA+H]+

Delayed extraction in linear MALDI TOF MS

Usample = 10 kVText = 1050 ns

Sub P, MW 1347Melittin, MW 2848Insulin, MW 57342Ins., MW 11467

Reflex Time-of-Flight Mass Spectrometry

Increase in resolutionTandem mass spectrometry

Detector

+U

+U1, (U1>U)

Ion source Reflectron

++

E

E+ε

+

+

+U1+U1

Single stage Two-stage Curved-field

+U1+ U1

Reflex MALDI TOF Mass Spectrometer

LaserOptics

Nitrogen Laser (337 nm)

TOFAnalyzer

MicrochannelDetector

MALDITarget

Ion MirrorIon

Grid

(1)

Reflex MALDI TOF Mass Spectrometer

LaserOptics

Nitrogen Laser (337 nm)

TOFAnalyzer

MicrochannelDetector

MALDITarget

Ion MirrorIon

Grid

(2)

Time-of-Flight variation f(energy variation)

Two-stage electrostatic reflectron

(20 keV)(+600 eV)

(-600 eV)

U, Nominal energy∆U, Energy variations acquired during the desorption processT, Nominal time-of-flight∆ T, Time-of-flight variations

1672

.92

Cou

nts

1665 1670 1675 1680 Mass (m/z)

1672

.92

1670 1675 1680 1685

M/∆M = 4000 M/∆M = 12700

∆M = 1 u∆M = 1 u

DE Linear Mode DE Reflex Mode

+U +U +U1

MALDI TOF MS of Neurotensin, MW 1671.92

MALDI TOF MS of Bovine Insulin, MW 5733.5

DE Linear Mode DE Reflex Mode

+U

5725 5730 5735 5740 5745 Mass (m/z)

∆M = 1 u

M/∆M ≥ 10,000

5725 5730 5735 5740 5745

M/∆M ≥ 1000

(FWHM)

C254 H377 N65 O75 S6

Calculated(FWHM 4000)

Measured

+U +U1

Delayed extraction in reflex MALDI TOF MS

+U +U1

Laser irradiance less critical under DE conditions

Sub P, MW 1182

Ion detection: Microchannel plate detector

R2 R2R3 R4-2 kV

+

+

Gain / MCP ~ 103-4

e-

e-

Anode

Single Ion counting, n < 1(PDMS, SIMS…)

Time to Digital Converter+ Histogram PC card

Nb ions > 10, (n1000)(LD, MALDI…)

Transient Recorder

(20 keV)

MCP

1 < Nb ions < 10TDC + charge coder,or Multi Anode Detector

~106-8 e-

Electronic emission from a CsI coated surface

Mass (Da)

γe

γe ~ M0.6 f(V)(1-4)

2000 7600 13200 18800 24400 30000

27000 41600 56200 70800 85400 100000

80000 164000 248000 332000 416000 500000Mass (m/z)

Inte

nsity

Protein extract from mouse anterior prostate

Signal Optimization

Accelerating VoltageGuide Wire Voltage%Shots/SpectrumLow Mass Gate

Signal-to-Noise Ratio

Delay TimeGuide Wire Voltage%Grid Voltage%

ResolutionLaser intensitySignal intensityAdjust…To Optimize…

Instrument Control

1. Select method2. Select data path3. File name4. Choose sample spot5. Enter comment

Instrument Control

1. Select method2. Select data path3. File name4. Choose sample spot5. Enter comment

Instrument Parameters

Instrument Parameters

Peptides – linear mode 0.5 – 1 ns 200 mV – 1000 mV

Proteins – linear mode5 to 20 kDa, 4 – 10 ns 200 mV> 20 kDa 10 – 20 ns 50 – 200 mV

Peptides – reflex mode 1 – 2 ns 50 – 200 mv

Bin size Vertical scaleExperiment

Substance P in HCCA

0

10000

20000

30000

40000C

ount

s

1340 1350 1360 1370 1380 1390 1400 Mass (m/z)

[M+H]+

[Mox+H]+[M+Na]+

[M+K]+

[M+H]+

[Mox+H]+ +18 u[M+Na]+ +22 u[M+K]+ +38 u

Bovine Insulin in Sinapinic AcidC

ount

s

5500 5600 5700 5800 5900 6000 6100 6200

0

10000

20000

30000

40000

50000

2000 4000 6000 8000 10000 12000 14000 16000 18000 Mass (m/z)

[M+H

]+

[M+2

H]2+

[2M

+H]+

[3M

+H]+

0

2000

4000

6000

8000

10000

+206

+224

[M+Matrix+H]+

0

2000

4000

6000

8000C

ount

s

5000 10000 15000 20000 25000 Mass (m/z)

Ins+

Cyt.C+

Trg+Cyt.C2+

Ins2+ Trg2+

2Cyt

.C+

Bovine Insulin, MW 5733.5Cytochrome C, MW 12360.1Trypsinogen, MW 23981

Mixture of 3 Proteins in SA

Mixture of Trypsin and Trypsinogen

0

10000

20000

30000

40000C

ount

s

10000 20000 30000 40000 50000 Mass (m/z)

Trp+Trg+

Trp2+Trg2+ Trp+, Trg+

[Trp+Trg]+

Trp, MW 23293Trg, MW 23981

Mixture of Trypsin and Trypsinogen

0

10000

20000

30000

40000C

ount

s

21000 22000 23000 24000 25000 26000 27000 Mass (m/z)

[M+H]+Trp+Trg+

Trp, MW 23293Trg, MW 23981

[M+Matrix+H]+

[M+2Matrix+H]+

[M+3Matrix+H]+

MALDI TOF MS of Glucose 6 Phosphate Dehydrogenase

10000 28000 46000 64000 82000 1000000

20

40

60

80

% In

tens

ity

56500 56940 57380 57820 58260

57432, [M+H]+

57630, [M+SA+H]+

57827,[M+2SA+H]+

[M+H]+

[M+2H]2+

100

Mass (m/z)

Influence of the Choice of Matrix

20000 40000 60000 80000 100000 120000 Mass (m/z)

Rel

ativ

e in

tens

ities

[M+H

]+4+

5+

6+

[2M

+H]+

3+

2+

2M3+

2M5+

Sinapinic Ac.

α-Cyano-4OH-cinnamic Ac

0

5000

10000

15000

20000

Cou

nts

50000 60000 70000 80000 90000 100000 Mass (m/z)

BSA+ Cox2+, 73 070 +/- 200 Da

8050

0

6820

0

Cox2 in Sinapinic Acid (Glycoprotein)

IgG in Sinapinic Acid

0

1000

2000

3000

4000

Cou

nts

50000 100000 150000 200000 250000 300000 Mass (m/z)

[M+3H]3+

[M+2H]2+

[M+H]+

[2M+H]+

Igg, 1.5 pmole on target

MALDI-TOF Mass SpectrometryPerformances

Biological materials : Peptides, Proteins, Polysaccharides, Polynucleotides...

Mass accuracy : 1000 ≤ Mass ≤ 30,000 Da ⇒ ≤ 10-4

Mass > 30,000 Da ⇒ ≥ 10-4

Sample amounts : Mass analysis ⇒ a few fentomoles toa few picomoles.Structure analysis ⇒ high femtomole range

AdvantagesRapid, easy sample preparation, Large mass scan (Routine, up to 150,000 Da), Mixture analysis, Tolerance towards impurities (buffers, salts...).

Disadvantages

No on-line couplingpossibilities

Getting Started…

• Fill out form• Check pressure gauges and instrument status• Start Voyager software• Load sample plate

Before you leave…

• Eject plate (and press ‘load no plate’)• (Close Voyager software)• Complete form• Report problems with the instrument to a

MSRC member