Purdue-UAB Botanicals Center for Age-Related Disease

8/14/2019 Purdue-UAB Botanicals Center for Age-Related Disease

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Purdue-UAB Botanicals Center for Age-Related Disease

Principles of MALDIPrinciples of MALDI--TOFTOFmass spectrometrymass spectrometry

Stephen Barnes, Ph.D.

Purdue-UAB Botanical Center Workshop 2002

Mass Spectrometry Methods in Botanicals Research


2/14

Shining the light on samplesShining 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 drifttowards a detector. Theirtime-of-flightis

proportional to their (MW)1/2


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Matrix-Assisted Laser

Desorption Ionization (MALDI)

Accelerating pulse

detector

Short laserpulse

Flight tube and drift region to

measure the time-of-flight (TOF)


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MALDI-TOF ionization

N2 laser pulse (337 nm)

[M+H]+

[M-H]-[M-H]-

matrix ions

Protein/peptide/nucleotide/saccharidedeposited on crystal surface


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How a TOF instrument works

Linear flight tube (1.0 - 1.5 m)Oscilloscopeto time ionarrival (1 sec)

Source

Acceleratingpulse

Oscilloscopeto time ionarrival

Reflectron

Effective flight tube (3.0 m)


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Protein analysis 2002

Water

Bath

37oC

destain

Speed-Vac

trypsinIncubate

overnight

Eppendorf

tube

1:20

MALDI plate

Desalting

Ziptip

684

.11

789.5

7

1048.7

2

1122.7

91

165.8

0

1277.8

3

1461.0

0

1705.1

6

1838.2

6

193

7.2

2

1994.2

4 2137.21

2284.2

5

2384.2

7

2647.5

0

00

5000

10000

15000

Coun

ts

1000 1500 2000 2500 3000

Mass (m/z)


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Applications of timeApplications of time--ofof--flightflight


Whole proteins - modified antibodies

Peptide mixtures following trypsinolysis

of spots or bands from 2D-gels

Polyphenols

Bile acids and many other biologicallyinteresting molecules


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-

CytochromeCytochrome C Modified by HNEC Modified by HNEMALDIMALDI--TOF Mass SpectrumTOF Mass Spectrum

Mass (m/z)

1000 8800 16600 24400 32200 40000

10012521.80

12680.12

12837.86

6260.97

25207.30

12365.22

[M+2H]2+

[2M+H]+

[M+H] +

CONTROL

PROTEIN + ONE 4HNE

PROTEIN + TWO 4HNE

PROTEIN +THREE 4HNE

%I

ntensity


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Trypsin Digest ofTrypsin Digest ofporinporin--P1;P1;

VoltageVoltage--dependent aniondependent anion--selective channelselective channel

0

10

20

30

40

50

60

70

80

90

100

%I

ntensity

1181.59

1461.75

1950.89

2368.131583.79

1443.76

1016.55 2164.05

1687.981034.54

2185.021184.60 1756.862813.32

900 1520.6 2141.2 2761.8 3382.4 4003.0

Mass (m/z)


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11/14

MALDIMALDI--TOF of mouse bileTOF of mouse bile

TDC [M-H]-

498.897

TC [M-H+Na]-

536.920

Mass (m/z)

544 592496448400

Intensity

20

40

60

80

100

0640

CONHCH2CH2SO3-

OHH

HO

TC [M-H]-

514.946

Matrix: -cyano-4-hydroxycinnamic acidN2 laser 337 nm, 1:100 fold dilution of bile, 1 l spotted


12/14

LC-MS analysis of mouse bile

Tauro-triOH

Tauro-diOH

Total ion current

Mouse bile does not contain ANY

glycine conjugates of bile acids, only

taurine conjugates. Several isomers arepresent.


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Connecting CE and LC toConnecting CE and LC to

MALDI analysisMALDI analysis

CE

analysis

nanoLCanalysis

Parallel capture of effluents of 8nanoLC separations on Mylar - can be

scanned simultaneously by fast laser

Creates 20 mm wide tracks thatcan be scanned by MALDI laser

for MS analysis


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Pros/Cons of laying down LC orPros/Cons of laying down LC or

EC separations on matrix plateEC separations on matrix plate

Allows off-line analysis both in real time and thenin 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 duringstorage

Also, can the laser beam cause chemical

changes?

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Purdue-UAB Botanicals Center for Age-Related Disease