Purdue-UAB Botanicals Center for Age-Related Disease Principles of MALDI-TOF . mass spectrometry. Stephen Barnes, Ph.D. Purdue-UAB Botanical Center Wo...
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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 infrared 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)
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 [M+H]
+
% Intensity
12365.22 CONTROL
[M+2H]
12521.80
PROTEIN + ONE 4HNE
12680.12
PROTEIN + TWO 4HNE
12837.86
PROTEIN +THREE 4HNE
2+ + [2M+H] 25207.30
6260.97
1000
8800
16600
24400
Mass (m/z)
32200
40000
Trypsin Digest of porin-P1; Voltage-dependent anion-selective channel 100
1181.59
90
80
% Intensity
70
60
50
1461.75 1443.76
40
30
1583.79
1016.55 20
10
0
900
1950.89 2368.13 2164.05
1687.98
1034.54 1184.60 1520.6
1756.86
2185.02 2813.32 2141.2
2761.8
Mass (m/z)
3382.4
4003.0
MALDI-TOF Spectrum for Daidzein 255.71 [M+H]+
100
Daidzein % Intensity
80
60 277.71
Daidzein + Na+
40
256.71 20
224.69
358.78
192.73
122.57 0 100
293.66
0
180
260
340
Mass (m/z)
420
500
MALDI-TOF of mouse bile TC [M-H]514.946 TDC [M-H]498.897
100
80 CONHCH2CH2 SO3 -
Intensity
60
HO
H
OH
40
TC [M-H+Na]536.920
20
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 analysis
nanoLC analysis
Creates 20 mm wide tracks that can be scanned by MALDI laser for MS analysis
Parallel capture of effluents of 8 nanoLC separations on Mylar - can be 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?
