Purdue-UAB Botanicals Center for Age-Related Disease

Principles of MALDI-TOF
mass spectrometry
Stephen Barnes, Ph.D.

Purdue-UAB Botanical Center Workshop 2002

Mass Spectrometry Methods in Botanicals Research
 Purdue-UAB Botanicals Center for Age-Related Disease

Shining the light on samples

• A focused laser beam, either in the UV or infra-

red ranges, can “evaporate” compounds from the
solid phase

• The resulting ions are injected into a tube (1 - 2 m

in length), accelerated and allowed to drift
towards a detector. Their time-of-flight is
proportional to their (MW)1/2
 Matrix-Assisted Laser
Desorption Ionization (MALDI)
Short laser
pulse
Flight tube and drift region to
measure the time-of-flight (TOF)

detector
Accelerating pulse
 MALDI-TOF ionization
N2 laser pulse (337 nm)

[M+H]+
[M-H]-
matrix ions

Protein/peptide/nucleotide/saccharide
deposited on crystal surface
 How a TOF instrument works

Linear flight tube (1.0 - 1.5 m)

Oscilloscope
Source to time ion
arrival (1 µsec)
Accelerating
pulse

Oscilloscope
to time ion Reflectron
arrival Effective flight tube (3.0 m)
 Protein analysis 2002
destain

Eppendorf
tube Speed-Vac

Incubate trypsin
Water
Bath
37oC
overnight 1:20

1048.72

2284.25
MALDI plate

1122.791165.80

1937.22
15000
Counts

684.11

2137.21
1705.16
1461.00
1277.83

2384.27
10000

789.57

2647.50
1838.26
1994.24
Desalting 5000
Ziptip
0
1000 1500 2000 2500 3000
Mass (m/z)
 Purdue-UAB Botanicals Center for Age-Related Disease

Applications of time-of-flight

• Whole proteins - modified antibodies

• Peptide mixtures following trypsinolysis
of spots or bands from 2D-gels
• Polyphenols
• Bile acids and many other biologically
interesting molecules
Cytochrome C Modified by HNE
MALDI-TOF Mass Spectrum
-

100
+
12521.80 PROTEIN + ONE 4HNE
[M+H]
12365.22
12680.12 PROTEIN + TWO 4HNE
CONTROL
% Intensity

12837.86 PROTEIN +THREE 4HNE

2+
[M+2H]
6260.97 +
[2M+H]
25207.30

1000 8800 16600 24400 32200 40000

Mass (m/z)
 Trypsin Digest of porin-P1;
Voltage-dependent anion-selective channel
100 1181.59

90

80

70
% Intensity

60

50

40
1461.75
1443.76
1950.89
30
1583.79 2368.13
1016.55 2164.05
20
1034.54 1687.98
10 1184.60 2185.02
1756.86
2813.32
0
900 1520.6 2141.2 2761.8 3382.4 4003.0

Mass (m/z)
 MALDI-TOF Spectrum for Daidzein

100 255.71 [M+H]+

Daidzein
80
% Intensity

60

277.71 Daidzein + Na+

40

293.66
256.71
20
224.69 358.78
122.57 192.73

0
0
100 180 260 340 420 500

Mass (m/z)
 MALDI-TOF of mouse bile
TC [M-H]-
514.946
100 TDC [M-H]-
498.897

80
CONHCH2CH2 SO3 -

HO OH
60 H
Intensity

40

TC [M-H+Na]-
20
536.920

0
400 448 496 544 592 640
Mass (m/z)

Matrix: α-cyano-4-hydroxycinnamic acid

N2 laser 337 nm, 1:100 fold dilution of bile, 1 µl spotted
LC-MS analysis of mouse bile

Tauro-triOH

Total ion current

Tauro-diOH

Mouse bile does not contain ANY

glycine conjugates of bile acids, only
taurine conjugates. Several isomers are
present.
Connecting CE and LC to
MALDI analysis

CE nanoLC
analysis analysis

Creates 20 mm wide tracks that Parallel capture of effluents of 8

can be scanned by MALDI laser nanoLC separations on Mylar - can be
for MS analysis scanned simultaneously by fast laser
 Pros/Cons of laying down LC or
EC separations on matrix plate

• Allows off-line analysis both in real time and then

in a retrospective mode
• MALDI-TOF analysis is very fast
• Can do TOF-TOF MS-MS analysis
• BUT what happens chemically on the acidic
environment on the surface of the plate during
storage
• Also, can the laser beam cause chemical
changes?