purdue-uab botanicals center for age-related disease
TRANSCRIPT
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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
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Shining the light on samplesShining the light on samples
Purdue-UAB Botanicals Center for Age-Related Disease
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
Purdue-UAB Botanicals Center for Age-Related Disease
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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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
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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?