MALDI MS Tutorial

Pierre Chaurand, Lisa Manier

Mass Spectrometry Research Center

Vanderbilt University
 Mass Range
Organic
Chemistry n0
p+ e-
Alchemy Biochemistry Atoms

0.000 000 000 000 000 000 000 001 g

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.
Q Plasma Desorption (PD)
252Cf fission fragments, High energy (MeV) particles or ions
Q Fast Atom Bombardment (FAB)
Low energy atoms (keV), (cesium)
Q Secondary Ion MS
Low energy ions (keV), (cesium, gallium...)
Q Matrix-assisted Laser Desorption/Ionization (MALDI)
UV and IR lasers
Matrix Assisted Laser Desorption-Ionization or MALDI

Mixture between a MATRIX and

an 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 by

time-of-flight mass spectrometry
Matrix for UV nm laser light (337 & 355 nm)
OCH3

HO HO

CN H
C C H3CO C C
H COOH H COOH

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

cinnamic ac. (αCHCA)

OH OH

HO COOH N 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)

Biomolecule Matrix Polarity

Peptides αCHCA +
Proteins SA- αCHCA +
Polysaccharides DHBA +
Nucleic ac. 3HPA /+

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

αCHCA: 10 mg/ml, in 50/50/0.1 - Acetonitrile/H2O/TFA
DHBA: 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 Drying
αCHCA Sinapinic Acid
rounded rhomboid

DHBA, air dried 3-HPA

irregular crystals fan-like around edge of
sample well
 Buffers

Suitable for Direct Unsuitable for Direct

Analysis by MALDI MS Analysis by MALDI MS

Ammonium bicarbonate Phosphate buffers

Ammonium acetate Sulfate buffers

Bis-Tris Tris (> 100 mM)

Tris (≤ 100 mM) Hepes (>100 mM)

Hepes (< 100 mM)

 Sample Clean up
For 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 Source Field free drift tube Ion detector

ttotal = tsource + ttof

 r r r
F = m a = qE
Time-of-flight in Source Time-of-flight in tube (field-free)
(constant field acceleration)
Kinetic energy: Ekin = qU = ½ mV2
(U, accelerating
Force and acceleration: a = Eq/m potential)
(a, acceleration; E, electric field d d tof
in source; q, charge; m, mass) Drift time : V = t tof or t tof =
tof V
Velocity and time: a = dV/dt
(V, velocity; t, time) d tof m
t tof = = d tof
V 2 qU
V =⌠ Eq/m = Vo + (Eq/m)t
⌡
~0 Total time-of-flight
V - Vo
t = (m/q) ttot = ts + ttof
E
~0
Position: d =⌠ V dt
⌡ 2 ds m m
ttot = + d tof
(d, position
in source)
d = do + Vot + ½(Eq/m)t2 qE 2qU

~0 ~0
t =a m q +b
2 ds m
ts = where a and b are defined by the physical dimensions
qE of the instrument and the operating parameters.
 Time-of-Flight Mass Spectrometry (TOF-MS)
Linear TOF :
Ionizing Probe (start)
Ion detector (MCP)
M3 M2 M1
Ion
signals

+/- U M2

M1

M3

t1 t2 t3 Time or M
Start

t = a M + b
MALDI: Data acquisition Transient Recorder

Ion detector (MCP)

+ Ion
+
signals

0 200 400 600 800 1000 1200

Time [100 ns]
Data transfer to PC, Software
Calibration

4000 6000 8000 10000

Mass [m/z]
MALDI DE-STR TOF MS from Applied Biosystems
Time-of-Flight Mass Spectrometers, General Organization
Attenuator
TC1 Prism
Laser

Inlet Detectors BA2

BA1
Sample Plate Guide Wire

Grids
Ion Selector Reflector
Camera

Valves
Turbo Pumps
TC2
Fore Pump
 Instrument Panel
Indicates that the
laser is powered on Mayor problem! High voltage is on

Indicates the Computer is

laser is firing Pumps are controlling the
operating instrument (software
normal is ready)
Pressure Select Gauge
Gauges
Press to Switch
on / off Gauge

Gauge Measures Expected Pressure

BA1 Pressure in main source chamber Less than 5 x 10 –7
BA2 Pressure in mirror chamber Less than 5 x10 -8

TC2 Pressure in sample loading chamber Less than 10 –2 during

operation
Higher when loading or
ejecting sample plate.
TC1,TC3, Not used displays E03 (indicates
TC4 gauge not connected)
[torr]
 Isotopic Distribution
100 100
90 C1 90
C10
80 80
70 70
60 60
50 50
40 40
30 30
20 20
10 10
0 0
12 13 120 121 122

100 100
90 90
80 C100 80
C1000
70 70
60 60
50 50
40 40
30 30
20 20
10 10
0 0
1,200 1,202 1,204 1,206 12,000 12,010 12,020 12,030
 1673.9 Average Mass
Neurotensin
100
90
80
70
60
50

Res. 1’000 40
30
20
10

1,672 1,674 1,676 1,678 1,680 1,682

pQLYENKPRRPYIL
MW 1672.9 1672.9 Monoisotopic Mass
[M+H]+
100
90
80 [M+H+1]+
70
60
[M+H+2]+
Res. 10’000 50
40
30
[M+H+3]+
20
10 [M+H+4]+
0
1,672 1,674 1,676 1,678 1,680 1,682
 Constant field extraction TOF MS

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

25 kV

+ +
+ +
Sample
+εE
E1

∆t
LD, MALDI
25 kV

+ +
+ +
Sample
E1

∆d ∆t
+εE, + ∆d
MALDI ion plume

DHB matrix and Substance P

[M+H]+ ions densities measured
1 µs after laser impact.

Bleu: DHB
Red: Substance P (MW 1347.7

V. Bokelmann, B. Spengler, R. Kaufmann,

Eur. Mass Spectrom. 1, 81-93 (1995)

B. Spengler
J. Mass Spectrom., 32, 1019-1036 (1997)
 Delayed Extraction in MALDI TOF MS
25 t=0, start
Ubias=25 kV
t=Text
Upulse<25 kV
+

+
0
[M+H]+

E2
E1
Sample

velocity
distribution
(300<v<1000 m/s)
M/∆M = f(Ubias, Upulse, Text., m)

Q 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
[M+H]+ M/∆M = 300 [M+H]+ M/∆M = 1100

[M+SA+H]+

[M+SA+H]+

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

100

20
40
60
80

0
2000
2865.4, [M+2H]2+ Ins.

5734.6
[M+H]+ Insulin

6181.4, [M+2H]2+ Cyt.C

7000
8476.8, [M+2H]2+ Myo.

11996.8, [M+2H]2+ Tryp.

12000
12361.1
[M+H]+ Cytochrome C

Mass (m/z)
16952.5
17000
[M+H]+ Myoglobin
22000
MALDI TOF MS of Protein Mixture

23992.8
[M+H]+ Trypsinogen
27000
 MALDI TOF MS of Protein Mixture, Ubias= 25 keV
[M+H]+ M/∆M = 800 [M+H]+ M/∆M = 220

delay = 150+ε ns
Upulse = 23625 V
[M+SA+H]+

5550 5650 5750 5850 5950 6050 23100 23540 23980 24420 24860 25300
[M+H]+ [M+H]+
M/∆M = 350 M/∆M = 500

delay = 250+ε ns
Upulse = 22750 V
[M+SA+H]+

[M+SA+H]+
[M+2SA+H]+

5550 5650 5750 5850 5950 6050 23100 23540 23980 24420 24860 25300
Mass (m/z)
Delayed extraction in linear MALDI TOF MS

Usample = 10 kV
Text = 1050 ns

Sub P, MW 1347
Melittin, MW 2848
Insulin, MW 5734
2Ins., MW 11467
 Reflex Time-of-Flight Mass Spectrometry
Detector
+ Reflectron
Ion source +
E
+
+U +
E+ε
+U1, (U1>U)
Q Increase in resolution
Q Tandem mass spectrometry

Single stage Two-stage Curved-field

+U1 + U1 +U1 +U1

 Reflex MALDI TOF Mass Spectrometer
Laser Nitrogen Laser
Optics (337 nm)

TOF
Analyzer

Microchannel
Detector

MALDI
’
Target

Ion Mirror
Ion
Grid

(1)
 Reflex MALDI TOF Mass Spectrometer
Laser Nitrogen Laser
Optics (337 nm)

TOF
Analyzer

Microchannel
Detector

MALDI
’
Target

Ion Mirror
Ion
Grid

(2)
Time-of-Flight variation f(energy variation)

Two-stage electrostatic reflectron

(-600 eV)

(+600 eV) (20 keV)

U, Nominal energy
∆U, Energy variations acquired during the desorption process
T, Nominal time-of-flight
∆ T, Time-of-flight variations
 MALDI TOF MS of Neurotensin, MW 1671.92
DE Linear Mode DE Reflex Mode

+U +U +U1
1672.92

1672.92
M/∆M = 4000 M/∆M = 12700

∆M = 1 u
∆M = 1 u
Counts

1665 1670 1675 1680 1670 1675 1680 1685

Mass (m/z)
 MALDI TOF MS of Bovine Insulin, MW 5733.5
DE Linear Mode DE Reflex Mode

+U +U +U1

C254 H377 N65 O75 S6 M/∆M ≥ 1000 M/∆M ≥ 10,000

Q Calculated
(FWHM 4000)
∆M = 1 u
Q Measured

(FWHM)

5725 5730 5735 5740 5745 5725 5730 5735 5740 5745
Mass (m/z)
Delayed extraction in reflex MALDI TOF MS

+U +U1

Sub P, MW 1182

Laser irradiance less

critical under DE
conditions
 Ion detection: Microchannel plate detector
-2 kV Single Ion counting, n < 1
R2 R3 R2 R4
(PDMS, SIMS…)
Time to Digital Converter
(20 keV) e- + Histogram PC card
~106-8 e-
+
1 < Nb ions < 10
TDC + charge coder,
Anode or Multi Anode Detector
MCP
e-
+
Nb ions > 10, (n1000)
(LD, MALDI…)
Transient Recorder
Gain / MCP ~ 103-4
 Electronic emission from a CsI coated surface

γe

γe ~ M0.6 f(V)(1-4)

Mass (Da)
 Protein extract from mouse anterior prostate

2000 7600 13200 18800 24400 30000

Intensity

27000 41600 56200 70800 85400 100000

80000 164000 248000 332000 416000 500000

Mass (m/z)
 Signal Optimization

To Optimize… Adjust…
Signal intensity Laser intensity
Resolution Delay Time
Guide Wire Voltage%
Grid Voltage%
Signal-to-Noise Ratio Accelerating Voltage
Guide Wire Voltage%
Shots/Spectrum
Low Mass Gate
Instrument Control

1. Select method
2. Select data path
3. File name
4. Choose sample spot
5. Enter comment
Instrument Control

1. Select method
2. Select data path
3. File name
4. Choose sample spot
5. Enter comment
Instrument Parameters
 Instrument Parameters
Experiment Bin size Vertical scale

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

Proteins – linear mode

5 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

 Substance P in HCCA
40000
[M+H]+ [M+H]+
[Mox+H]+ +18 u
30000 [M+Na]+ +22 u
[M+K]+ +38 u
Counts

20000

[Mox+H]+ [M+Na]+
10000 [M+K]+

0
1340 1350 1360 1370 1380 1390 1400
Mass (m/z)
 Bovine Insulin in Sinapinic Acid
+206

[M+H]+
10000
[M+Matrix+H]+
50000
8000

+224
6000
40000
4000
Counts

30000 2000
[M+2H]2+

0
5500 5600 5700 5800 5900 6000 6100 6200
20000

[2M+H]+

[3M+H]+
10000

0
2000 4000 6000 8000 10000 12000 14000 16000 18000
Mass (m/z)
 Mixture of 3 Proteins in SA
8000
Ins+ Bovine Insulin, MW 5733.5
Cytochrome C, MW 12360.1
Trypsinogen, MW 23981
6000
Counts

Cyt.C+
4000

2Cyt.C+
Cyt.C2+
Ins2+ Trg2+ Trg+
2000

0
5000 10000 15000 20000 25000
Mass (m/z)
 Mixture of Trypsin and Trypsinogen
40000
Trg+ Trp, MW 23293
Trp+ Trg, MW 23981
30000
Counts

20000
Trp2+
Trg2+ Trp+, Trg+
[Trp+Trg]+
10000

0
10000 20000 30000 40000 50000
Mass (m/z)
 Mixture of Trypsin and Trypsinogen
40000
Trp, MW 23293
Trg+ Trg, MW 23981
Trp+
30000 [M+H]+

[M+Matrix+H]+
Counts

20000

[M+2Matrix+H]+
10000 [M+3Matrix+H]+

0
21000 22000 23000 24000 25000 26000 27000
Mass (m/z)
 MALDI TOF MS of Glucose 6 Phosphate Dehydrogenase
100 57432, [M+H]+
[M+H]+

57630, [M+SA+H]+
80
57827,
[M+2SA+H]+

60
% Intensity

[M+2H]2+
40

56500 56940 57380 57820 58260

20

0
10000 28000 46000 64000 82000 100000
Mass (m/z)
 Influence of the Choice of Matrix

Sinapinic Ac.

2+

[2M+H]+
Relative intensities

3+

[M+H]+
4+
α-Cyano-4OH-cinnamic Ac
5+
6+
2M3+
2M5+

20000 40000 60000 80000 100000 120000

Mass (m/z)
 Cox2 in Sinapinic Acid (Glycoprotein)

BSA+ Cox2+,
73 070 +/- 200 Da
20000

15000
Counts

68200

80500
10000

5000

0
50000 60000 70000 80000 90000 100000
Mass (m/z)
 IgG in Sinapinic Acid
4000

[M+H]+
Igg, 1.5 pmole on target
3000 [M+2H]2+
Counts

2000

[M+3H]3+
1000 [2M+H]+

0
50000 100000 150000 200000 250000 300000
Mass (m/z)
MALDI-TOF Mass Spectrometry
Performances
Q Biological materials : Peptides, Proteins, Polysaccharides,
Polynucleotides...
Q Mass accuracy : 1000 ≤ Mass ≤ 30,000 Da ⇒ ≤ 10-4
Mass > 30,000 Da ⇒ ≥ 10-4
Q Sample amounts : Mass analysis ⇒ a few fentomoles to
a few picomoles.
Structure analysis ⇒ high femtomole range

Advantages Disadvantages
Q Rapid, easy sample preparation, Q No on-line coupling
Q Large mass scan possibilities
(Routine, up to 150,000 Da),
Q Mixture analysis,
Q Tolerance towards impurities
(buffers, salts...).
 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