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MALDI TOF/TOF technology:
applications and workflows for a mass
spectrometry research lab
Volker Kruft
Manager Business Development EMEA, Proteomics
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Seminar Outline
• TOF/TOF family: technology overview
• Protein analysis challenges
• Intelligent protein identification
• Quantitative proteomics
• MSI: Mass Spectrometric Imaging
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Seminar Outline
• TOF/TOF family: technology overview
• Protein analysis challenges
• Intelligent protein identification
• Quantitative proteomics
• MSI: Mass Spectrometric Imaging
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
The AB SCIEX TOF/TOF Family
4800 MALDI
TOF/TOFTM
Analyzer
The AB SCIEX TOF/TOFTM
5800 System
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
4800 MALDI TOF/TOF
System
Laser
Camera
Sample stage and
sample plate
Reflector detector
Source 2 region
Mirror region
Linear detector
Deceleration Stack
QuanTIS™ Precursor Selector
OptiBeam™ on-axis laser
Source 1 Region
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
OptiBeam™ On-Axis Laser
● The angular distribution of the MALDI ion plume is directed back
towards the direction of the incident laser
● On-axis OptiBeam™ Laser configuration on the 4800 MALDI
TOF/TOF™ Analyzer and the TOF/TOF™ 5800 Systems provides
optimum MS and MS/MS sensitivity
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Off axis 30 On axis
y
x
Plume formation before acceleration (v0=300300m/s, = 45)
Trajectory after acceleration
Scale
enla
rged f
or
eff
ect
On-Axis vs. Off-Axis Ionization by MALDI
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Seminar Outline
• TOF/TOF family: technology overview
• Protein analysis challenges
• Intelligent protein identification
• Quantitative proteomics
• MSI: Mass Spectrometric Imaging
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Proteome 1 Proteome 2 Proteome 3 Proteome 4 Proteome 5 Proteome 6 Proteome 7
Proteomics: It´s all about time courses
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
mg/mL
LC/MS range:
~3.5 order of
magnitude
ng/mL
pg/mL
ug/mL
N.L. Anderson et al.; The human plasma proteome; MCP; 2002
The Dynamic Range Challenge: Plasma Proteomics
Deplete top 10 proteins
Where we need to be
*Prostrate specific antigen
Cancer marker
*
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential 12
34
5
67
9
8
10
Dynamic range in proteomics
Courtesy of B. Domon, ETH Zurich
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
The problem of
biological
variation unrelated
to disease
Although human genome > 99.9% identical …
… considerable differences in proteome
unrelated to disease biomarkers = “noise“
(courtesy of Karl Mechtler, IMP, Vienna)
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
The “Do´ers” in a Cell
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Importance of MS in the life sciences(as indexed in PubMedLine)
0
2000
4000
6000
8000
10000
12000
14000
16000
´82 ´84 ´86 ´88 ´90 ´92 ´94 ´96 ´98 ´00 ´02 ´04 '06 '08
MS total
MS protein
MS environmental
MS metabolism
MS quantitation
MS clinical
Last updated August 5, 2008
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Seminar Outline
• TOF/TOF family: technology overview
• Protein analysis challenges
• Intelligent protein identification
• Quantitative proteomics
• MSI: Mass Spectrometric Imaging
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
4800 MALDI TOF/TOF™ Analyzer
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Small proteins linear mode
1 6 0 0 6 0 8 0 1 0 5 6 0 1 5 0 4 0 1 9 5 2 0 2 4 0 0 0
M a s s (m /z )
1 3 2 .6
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0
% I
nte
ns
ity
4 7 0 0 L i n e a r S p e c # 1 M C = > S M 9 [ B P = 1 1 6 7 4 . 2 , 1 3 3 ]
Calibration Mix
Resolution Thioredoxin: 11300
Resolution Myoglobin: 11101
16
74
.7
57
34
.6
16
95
2.4
11
72
8.9
11
88
6.9
58
39
.1
17
17
1.9
84
76
.0
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
9.0 2473.8 4938.6 7403.4 9868.2 12333.0
Mass (m/z )
396.6
0
10
20
30
40
50
60
70
80
90
100
% In
te
ns
ity
4700 MS/MS Precursor 11674 Spec #1=>NF1.0[BP = 4914.6, 397]
4912.8267
4895.8833
6487.6792
9448.3438
6916.33404670.0229
9431.21486470.800310540.32236899.73054868.2427
98.0329 8863.8330 10638.94926357.93994878.9834
30.0763 9476.35944785.0996 6584.0532 8343.54393898.371652.4390
MSMS MALDI Spectrum of Intact Thioredoxin
m/z 11574
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
4800 MALDI TOF/TOF™ Analyzer
Other Unique Applications:
• Polymers
• Fatty Acids
• Carbohydrates
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
4800 TOF/TOF: Polymer Fragmentation
90 302 514 726 938 1150
Mass (m/z)
270.7
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
1049.5
4
1093.5
8
1005.5
4
1111.6
2
961.5
1
142.0
5
1067.5
5
303.1
4
681.3
6
725.4
2
1023.5
5
935.4
8
769.4
1
391.2
0
637.3
1
917.3
9
979.5
2
785.3
7
287.1
6
375.2
2
873.4
6
829.4
2
349.2
2
259.1
3
845.3
7
751.8
3
1007.6
5
891.4
6
653.3
5
593.3
6
1039.4
7
813.4
4
549.2
8
703.2
8
1083.6
6
O
HO - [CH CH O]2 2 w
[OCH CH ] - OH2 2 x
[OCH CH ] - OH2 2 y
[OCH CH ] - OH2 2 z
MS/MS of sorbitan polyethoxylate (Tween component) MNa+ 1243.72
no CID
90 302 514 726 938 1150
Mass (m/z)
706.9
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
1049.5
6
725.4
0
681.3
8
637.3
6
961.5
1
142.0
6
1005.5
2
769.4
2
1065.5
4
977.5
1
1093.5
9
1021.5
1
593.3
4
785.4
1
1109.5
9
813.4
4
917.4
7
933.4
6
156.0
7
873.4
5
186.0
9
829.4
3
549.3
2
741.4
0
669.3
8
305.1
9
1023.5
3
889.4
5
347.1
9
697.3
7
261.1
7
1067.5
5
391.2
4
505.3
0
857.4
6
713.3
8
581.3
3
1111.5
9
112.0
4
435.2
6
979.4
8
801.4
2
639.3
6
287.1
9
623.3
5
919.4
9
331.2
0
479.2
9
565.3
2
155.0
7
419.2
7
771.4
3
537.3
0
963.5
2
375.2
4
217.1
3
243.1
4
461.2
7
171.0
7
935.4
8
875.4
6
891.4
7
743.4
4
128.0
4
519.3
1
200.1
1
401.2
4
271.1
6
357.2
3
597.3
3
817.4
2
1033.5
5
715.3
1
1061.0
1
675.3
6
1080.6
1
1003.5
2
851.4
8
985.5
6
1131.6
9
with CID
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
TOF/TOF: MS/MS of a Triglycerid
10 199 388 577 766 955
Mass (m/z)
3.1E+4
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsit
y
22.9
9
625.5
2
603.5
4
697.5
5
907.8
6
417.3
4
475.3
4
909.9
7
753.6
0
531.3
9
808.6
5
711.5
6
83.1
0
403.3
2
345.3
1
599.4
5
489.3
5
641.5
0
684.5
3
911.8
2
559.4
2
517.3
9
265.2
8
111.1
2
57.0
8
135.1
3
41.2
1
459.3
7
305.2
6
835.7
2
767.6
2
195.0
9
431.3
5
73.4
2
793.6
4
377.2
3
615.4
7
415.3
2
588.4
5
93.0
8
751.0
0
395.3
4
734.0
0
543.3
9
864.8
7
220.0
0
323.1
9
237.1
4
361.3
1
657.4
9
153.1
4
283.2
4
171.0
9
339.3
1
- C17H33COOH50 230 410 590 770 950
Mass (m /z)
1219.9
0
10
20
30
40
50
60
70
80
90
100
% Inte
nsity
S t i t c h e d P S D = > S M 2 5 [ B P = 5 8 4 . 8 , 1 2 2 0 ]
PSD
CID-MSMS
cleavage of carbon chain:
-loss of (CnH2n+2)
-loss of (CnH2n)
-loss of (CnH2n-2)
808 (even)
indicates C=C
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
124.0 421.6 719.2 1016.8 1314.4 1612.0
Mass (m/z )
1527.3
0
10
20
30
40
50
60
70
80
90
100
% In
te
ns
ity
TOF TOF MS/MS Precursor 1582 Spec #1= >NF1.0[BP = 1579.3, 1527]
1579.5009
1171.8664832.9398 1283.6907476.1597
960.4986 1445.6344745.2733
510.0605 1173.8828 1422.16791285.6490815.0296273.2654 687.5010414.2672 930.5787170.0208 1046.2710591.7584
-OH
M+Na+
Mass (m/z)
MSMS of (Man)7(GlcNAc)2(D3):
Mix of Glycosidic Cleavages and Cross Ring Cleavages
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
TOF/TOF: Recent Carbohydrate Publications
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
TOF/TOF: Growing list of publications
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Direct Spotting onto
MALDI target
MS Analysis and
Quantitation
TOF/TOF MALDI
System
GPS Explorer for
Data Analysis
MS/MS Analysis and
Quantitation (iTRAQTM)
The Power of LC-MALDI
Tempo™ LC MALDI
oMALDI QSTAR
Elite
© 2008 Applied Biosystems - Confidential
ESI threshold
150 990 1830 2670 3510 4350m/z
0
10
20
30
40
50
60
70
80
90
100
% In
ten
sit
y
y11 y12
y10
y9
y8
y7y6
y5
y4
y2
y14
y13
y15
y16 y17y18y19
y20
y21y22
y23
y24y25
y26y27
y28 y29y30y31
y32y33b33
b35 b36
MH+
LAADEDDDDDDEEDDDEDDDDDDFDDEEAEEKAPVKK
This fraction is used
for MS/MS in MALDI
XIC for m/z 4246.5
Selecting the precursor ion at the
optimum elution time affords the
highest quality MS/MS spectra
LC/MALDI result dependent experiment:
Optimized selection of precursors for MS/MS
2100 2140 2180 2220 2260 2300Mass (m/z)
2370.7
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
2170.2
19
2159.9
21
2117.9
83
2115.9
73
2288.0
18
2226.1
23
2186.0
53
2221.1
02
2113.9
18
2274.1
09
2242.1
08
2111.9
37
2292.0
28
2124.9
49
2282.0
73
2153.9
65
2164.9
71
2262.0
80
2254.9
81
2165.9
09
2285.1
21
2212.9
68
2265.0
16
2272.0
54
719.0 1268.2 1817.4 2366.6 2915.8 3465.0
Mass (m/z)
5.3E+4
0
10
20
30
40
50
60
70
80
90
100
% In
ten
sit
y
876.4
28
1326.7
60
1133.6
54
1728.7
25
1273.6
51
900.5
34
1529.7
42
1015.5
41
830.3
99
960.5
61
1126.5
69
1215.6
35
1514.7
19
2371.2
96
1286.6
67
751.2
92
1690.8
32
1169.6
29
1063.5
88
984.5
00
908.4
68
1464.7
11
820.4
96
2170.2
19
872.5
63
1632.8
22
3081.4
53
1121.5
47
2812.3
51
1811.8
40
1350.6
28
1774.8
66
2416.2
05
1229.6
49
1393.6
56
2115.9
70
1021.5
20
2288.0
18
1536.7
11
1594.8
22
2226.1
24
2472.3
44
1933.8
17
3353.5
50
20 sec LC fraction of serum
# of monoisotopic peaks: 302 (>S/N 3)
Range of intensities: 0.1-100.0%
Selected for MS/MS: 43 most abundant
MALDI MS/MS: Dynamic Range(“digging deeper”)
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
160.0 645.8 1131.6 1617.4 2103.2 2589.0
Mass (m/z)
1392.7
0
10
20
30
40
50
60
70
80
90
100%
In
ten
sity
716.1
7
504.1
1
1803.5
2
1642.5
0
175.0
6
2518.6
1
928.2
0
1343.4
0
213.0
2
1414.5
4
1562.4
6
290.0
8
405.1
3
1598.5
7
2308.6
6
672.1
7
2459.4
7
1664.5
3
1495.6
0
1157.3
9
2014.5
7
1056.3
4
1740.4
3
849.2
7
1632.5
7
1528.5
6
1784.5
6
814.2
2
2343.7
4
563.1
5
1307.3
8
1240.4
4
602.1
4
1199.2
1
496.1
3
1956.7
0
748.2
6
444.1
1
329.1
0
2113.6
1
2389.7
4
2201.5
7
362.0
7
9.0 191.4 373.8 556.2 738.6 921.0
Mass (m/z)
9.3E+3
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
876.4
1
120.1
1
509.3
2
422.2
8
294.2
1
136.1
1
583.3
1
729.4
1
365.2
5
672.3
9
555.3
2
420.2
4
702.3
8
147.1
4
512.2
7
436.2
3
565.3
0
408.2
5
368.2
1
308.1
7
130.1
2
193.1
4
290.1
7
221.1
5
276.1
8
205.1
3
481.2
8
73.4
1
712.3
8
30.1
4
347.1
9
455.2
6
877.9
4
251.1
6
538.8
9
113.5
7
191.1
6
685.9
4
177.1
3
394.2
3
379.2
0
335.2
2
748.3
8
292.1
8
233.1
4
806.5
5
273.2
0
494.2
9
145.1
2
57.3
5
125.9
2
91.1
0
217.1
3
44.0
7
615.3
3
160.1
4
507.3
7
410.3
3
470.2
4
438.2
5
526.2
3
628.3
2
670.3
3
836.0 859.2 882.4 905.6 928.8 952.0
Mass (m/z)
5.3E+4
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
876.4
28
900.5
34
869.4
39
938.4
80
898.4
04
908.4
69
927.4
95
872.5
64
940.4
66
933.2
65
883.4
21
914.3
65
929.4
74
845.4
36
836.3
83
891.4
16
893.4
64
922.4
70
920.4
63
948.4
83
852.3
73
916.5
11
855.4
03
864.3
89
925.4
34
53,000 counts
2451.0 2476.4 2501.8 2527.2 2552.6 2578.0Mass (m/z)
630.5
0
10
20
30
40
50
60
70
80
90
100
% I
nte
nsi
ty
2472.3
44
2518.2
08
2548.2
29
380 counts
Expanded regions MS and MS/MS of well B10
Corresponding MS/MS of Precursor Ions
(Y)FGYSGAFK from transferrin MH 876(P)LPPTSAHGNVAEGETKPDPDVTER
from beta-1B-glycoprotein MH 2517
MALDI MS/MS: Dynamic Range(“digging deeper”)
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Martinsried Contest
• Tryptic digest of 10,000 cells
(human kidney cell line, HEK 293)
• Dried pellet (about 2mg each) in triplicate
(per instrument)
How many proteins can you identify?
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
High Performance Instruments for Robust
Protein Identification
QSTAR® Elite LC/MS/MS System
High quality protein identification results in
the fastest time with Smart Exit and up to 7
MS/MS per second.
4800 MALDI TOF/TOF™ Analyzer
Greatest depth of coverage with intelligent LC
MALDI protein identification workflows.
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Maximizing number of protein identifications when
sample is limited: (I) 4800
● In-depth protein id in complex samples with a wide dynamic range:
– Tryptic digest of 10,000 cells
○ (human kidney cell line, HEK 293)
● LC-MALDI with the 4800
– Separation frozen on target plate
– Complete control in precursor selection without separation time constraints
● Dynamic Exclusion of precursors:
– Run 1of 2: 50% of 1 sample○ Prot ID MALDI MS and MS/MS
– Run 2 of 2: 50% of 1 sample○ Prot ID MALDI MS and MS/MS (dynamic exclusion of all precursors from run 1 by mass
and retention time)
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Maximizing number of protein identifications when
sample is limited: (II) QSTAR Elite
● In-depth protein id in complex samples with a wide dynamic range:
– Tryptic digest of 10,000 cells
○ (human kidney cell line, HEK 293)
● Optimized IDA runs:
– Dynamic background substraction
– Smart CE and Smart Exit
● Dynamic Exclusion of precursors:
– Run 1of 2: 50% of 1 sample○ Prot ID LC/MS/MS
– Run 2 of 2: 50% of 1 sample○ Prot ID LC/MS/MS (dynamic exclusion of all precursors from run 1 by mass and retention
time)
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Methods: Copy precursor list plus rentention time
Easy in Peak Explorer™
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Method: Paste list into the exclusion list
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Results: Heat map of the 1st LC run
100 200 300 400 500 600 700 800 900 1000Fraction Number
700
900
1100
1300
1500
1700
1900
2100
2300
2500
2700
2900
3100
3300
3500
Ma
ss
(m
/z
)
Heat Map - Demo\Martinsried\LC-02 2h Martinsried 2005 Run #7 Trace: 1
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Results: XIC of 1264
Signal/Rauschen 1264.64 +/- 0.2 Da
0
200
400
600
800
1000
1200
1400
1600
1800
200 250 300 350 400 450 500 550 600
Fraktionsnummer
Sig
na
l/N
ois
e
1264.52
not identified
1264.634
LWDLTTGTTTR
guanine binding protein
1264.620
RAPFDLFENR
heat shock protein 1264.660
DFLLQQTMLR
glyoxalase I
1264.674
AFITNIPFDVK
ribonucleoprotein
XIC
Fraction Number
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Results: # of MSMS and # of proteins
● 1st LC: 4276 MSMS ⇒ 820 proteins
(5.2 MSMS = 1 protein)
● 2nd LC: 2563 MSMS ⇒ 430 new proteins
(6.0 MSMS = 1 new protein)
Get as many good MSMS as possible !!
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential679.0 1265.6 1852.2 2438.8 3025.4 3612.0
M ass (m/z )
3322.4
0
10
20
30
40
50
60
70
80
90
100
% I
nte
ns
ity
4 7 0 0 Re fle c tor Spe c # 1 M C[BP = 1 2 6 4 .6 , 3 3 2 2 ]
MS - Fraktion 500
1264.6
36
1650.7
10
1786.9
33
1081.5
64
2225.1
17
1314.7
28
835.4
42
2040.9
76
1971.0
27
1393.6
87
1450.7
25
2283.0
10
2439.1
61
2154.0
78
1776.8
36
2239.1
34
969.5
03
1590.7
66
1815.9
20
1516.8
08
1318.6
72
1259.6
85
1157.6
50
1869.9
33
1955.0
09
1667.8
03
1710.8
77
2087.0
50
782.4
53
1996.0
22
1915.9
50
922.4
76
1029.5
39
1219.6
57
2046.0
21
1399.6
80
2317.0
46
2142.0
65
1620.8
11
1359.6
52
1110.6
15
2705.1
90
1764.8
79
2211.1
21
1453.7
39
713.4
13
1563.7
88
877.0
34
1521.7
60
2257.0
11
2455.1
49
1289.6
86
2512.1
86
1176.6
30
984.4
69
1071.5
92
2621.2
02
2382.1
81
1823.8
93
2966.4
60
2748.3
54
820.4
30
3457.4
39
2812.2
83
776.4
60
2578.2
65
3375.6
07
2921.3
97
3129.3
63
3039.5
30
3495.6
26
2881.4
05
Peak Selection in LC-MALDIMS – fraction 500 (= 81.3 min.)
= fr. 500
= fr. 499
= fr. 498
= fr. 501
= fr. 502
= fr. 497
MS/MS performed in
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
● Reverse database searching
was used to analyze the false
positive rate in protein detection
from ProteinPilot Software for
each workflow.
● Both ROC curves show good
discrimination, specifically both
curves have a sharp transition
from a nearly vertical segment
– (correct high scoring
peptides) to a nearly
horizontal segment (incorrect
low scoring peptides).
Independent False Positive Rate
Assessment by Decoy Searching
QSTAR® Elite System
4800 TOF/TOF Analyzer
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
● Proteins identified at the 5%
aggregate false positive rate
● Determined from the ROC plot
of the ProteinPilot™ Software
results
Proteins Identified by ProteinPilot™ Software Venn Diagram Comparison of Proteins Found
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Comparison of Acquisition Techniques
● Proteins identified at the 5% aggregate false positive rate
● Total analysis time - time required to acquire and process the
MS/MS data
● Processing time was determined by running both datasets on a
single processor computer (Dell Latitude D810 laptop with 2.0 GB
RAM and 1.86 GHz Pentium M processor).
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Assessing the Complementarity of ESI and MALDI
● Two dominant „soft‟ ionization strategies
● LC MALDI has been more flexible due to decoupling of
acquisition and LC, however, the technique has been
speed limited.
● Previous work has suggested a complementarities in
information
● Here, we wanted to assess the complementarities vs.
random acquisition variation of ESI vs MALDI
● Sample is “universal performance standard“ E.coli total
protein tryptic digest
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
LC MALDI AcquisitionAB SCIEX TOF/TOF™ 5800 System
● Monolithic CapRod column (100 µm ID x 15cm, Merck)
– 90 minute gradient - 2µL/min (0.1% TFA)
● Tempo™ LC MALDI Spotting System
– 720 spots were collected per LC run, 8sec/spot
– Online mixing of α-cyano-4-hydroxycinnamic acid matrix (Agilent).
● TOF/TOF™ 5800 System
– 25 most intense precursors per spot
– Laser frequency of 1000Hz
– DynamicExit™ Algorithm
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
LC MALDI Workflow
975.0 1525.2 2075.4 2625.6 3175.8 3726.0
Mass (m/z)
2381.291843.07
2424.33
2465.20
1282.67 2302.36
1150.671662.83
1456.83 1976.151016.64
1644.952328.251278.65
2702.46
1948.10
2134.111738.942447.24
2913.51 3237.61 3502.76
Protein Pilot™
Software for
Data Analysis
Interpretation
331.6 654.2 976.8 1299.4 1622.0
Mass (m/z)
985.66114.14
116.14
857.59
951.64551.3370.12
72.12983.69387.2439.02 486.31 1245.77142.12 679.40 957.68585.35 1391.861149.78829.60359.24228.1958.12 1263.79458.32 549.36 934.65 1048.77 1415.80136.11 664.54
113 116 119 122
Mass (m/z)
116114 117115113 121
120.10(A14747.87)
112.10(A9043.70)
118 119
Direct Spotting onto
MALDI target
-2kV pulse-2kV pulse0kV
250msec
Nano-LC
AB SCIEX TOF/TOFTM
5800 System
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
ESI Orbitrap LC MS Acquisition
● Reverse phase nanoLC was performed using the Agilent 1100 LC system
● Magic AQ C18 column (75 µm x 100cm column, self packed) at 300 nL/min
● Same 90 min gradient
● Acquisition settings used according to vendor specifications
● *.mgf file was generated using ReAdw4Mascot2.exe, a NIST derivative of the ISB tool, ReAdW for processing the Orbitrap data with ProteinPilot™ Software Version 3.0
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Comparing Sample Loading at the Protein LevelOnline ESI vs LC MALDI Analysis
● Similar protein ID rates achieved at 20ng
● More proteins found by LC MALDI at 100 and 500 ng
– Acquisition time was 2.5x longer
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Comparing Sample Loading at the Peptide LevelOnline ESI vs LC MALDI Analysis
● Similar peptide ID rates achieved at 20ng
● More peptides found by LC MALDI at 100 and 500 ng
– Acquisition time was 2.5x longer
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Protein / Peptide Intersection500 ng Loading Level – Comparison of Single Best Replicates
● ESI with MALDI acquisitions appear complementary and suggest greater
depth of coverage from combination than a signal technique alone.
● But how similar are replicates from the same instrument?
234 466 78(30.1%) (59.9%)
(10.0%)
5800 system700 proteins
Orbitrap544 proteins
3302 1408 851
(59.4%) (25.3%)(15.3%)
5800 System4710 peptides
Orbitrap2259 peptides
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Peptide Level Intersection of Replicates
● Average intersection at the peptide
level between 500 ng replicates:
– For 5800 system: 64% (Sample 1-3)
– For Orbitrap: 61% (Samples 4-6)
– Between instruments: 25% (red
boxes)
● This clearly shows that
intra-instrument replicates
are more similar than
inter-instrument replicates
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Protein Level Intersection of Replicates
● Average intersection at the protein
level between 500 ng replicates:
– For 5800 system: 78% (Sample 1-3)
– For Orbitrap: 74% (Samples 4-6)
– Between instruments: 60% (red
boxes)
● Even at the protein level,
differences are apparent
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
● Average Unused
ProtScores for all proteins
were aligned from each
instrument (500 ng
replicates).
● Points above the diagonal
mean those proteins have
more peptides and
therefore get better Protein
scores with 5800 system.
● The „clumps‟ at the lowest
limits are proteins not
found at all by one
instrument or the other.
Comparison of Protein Identification QualityProteinPilot™ Software Results from Orbitrap and 5800 System
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Reproducibility of LC MALDI Acquisition
● LC MALDI is a unique acquisition strategy as the MS is decoupled from the chromatographic separation, enabling as much or as little time to be taken during MS and MS/MS acquisition
● Intelligent precursor selection (using MS peak apex selection) and intelligent MS/MS acquisition (DynamicExit™ Algorithm) enable the most information to be obtained from each sample spot in an LC MALDI run
55
5572
61369%
29
44
22
Rep1 741
Rep2 719Rep3 751
~ 5% global protein level used for Venn
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Seminar Outline
• TOF/TOF family: technology overview
• Protein analysis challenges
• Intelligent protein identification
• Quantitative proteomics
• MSI: Mass Spectrometric Imaging
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
• Profiling approaches (not MS-based):
- gel-based, e.g. DIGE
- SELDI
- chromatography
• Non-tagging approaches (MS-based):
- peptide count (“protein abundance index”)
- ion-current-based (XIC)
• Tagging/labeling (MS-based):
- metabolic stable isotope labeling (SILAC, 15N)
- enzymatic stable isotope labeling
(18O incorporation during digestion)
- chemical stable isotope labeling (cICAT, iTRAQ, ICPL, mCAT)
Experimental Approaches to Biomarker Discovery(Relative and Absolute Quantitation)
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Advantages of Stable Isotope Labeling
• simultaneous quant and ID in a single run
• flexibility in fractionation strategy (not limited to 1D LC)
• high protein coverage
• simple and easy to use
• reproducibility of sample prep across samples
• multiplexing possible: multiple sample comparisons in a single run,
reduction in analysis time
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Characteristics of a good “tagging” chemistry
• induces mass shift by stable isotopes (not deuterium)
• low molecular weight
• reporters in a quiet region in MS/MS
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
0
40000000
80000000
120000000
160000000
0 200 400 600 800 1000 1200 1400 1600 1800 2000
m/z
Su
mm
ed
Io
n In
ten
sit
y
Summed Ion Intensity
(~75,000 Spectra)
Reporter Group Placement:Selection of „Quiet Region‟
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Characteristics of a good “tagging” chemistry
• induces mass shift by stable isotopes (not deuterium)
• low molecular weight
• reporters in a quiet region in MS/MS
• simple and easy to use
• multiplexing
• isobaric
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Isobaric Tag
Total mass = 145
Reporter Group mass
114 –117 (Retains Charge)
Balance Group
Mass 31-28 (Neutral loss)
Amine specific peptide
reactive group (NHS)
N
N
O
O
N
O
O
iTRAQ Workflow
MS
Mix-NH
114 31-NH
115 30-NH
116 29-NH
117 28 MS/MS b
y
b
yb
yb
y
+
+
+
+
-PRG114 31
-PRG115 30
-PRG116 29
-PRG117 28
S1
S2
S3
S4
Pa
ralle
l D
en
atu
re &
Dig
est
114
115
116
117
(Ross et. al, MCP 3,
1154-1169, 2004)
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
iTRAQ Workflow
MS
Mix-NH
114 31-NH
115 30-NH
116 29-NH
117 28 MS/MS b
y
b
yb
yb
y
+
+
+
+
-PRG114 31
-PRG115 30
-PRG116 29
-PRG117 28
S1
S2
S3
S4
Pa
ralle
l D
en
atu
re &
Dig
est
114
115
116
117
1347.0 1349.6 1352.2 1354.8 1357.4 1360.0
Mass (m/z)
1352.84MS: intact labelled peptides are
identical, signal intensities are
additive!
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
MS
Mix-NH
114 31-NH
115 30-NH
116 29-NH
117 28 MS/MS b
y
b
yb
yb
y
+
+
+
+
-PRG114 31
-PRG115 30
-PRG116 29
-PRG117 28
S1
S2
S3
S4
Pa
ralle
l D
en
atu
re &
Dig
est
114
115
116
117
MS/MS: y- and b-ions are additvefrom 4 individual samples!
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
*VLVDTDYK*
MS/MS: reporter ions quantitate 4 individual samples!
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
The iTRAQ workflow
From: Ross PL et al. Ross PL et al. MCP 3(12), 1154ff (2004) and Lenčo J, personal communication
NO
O
O
O
N
NCH
3
NO
O
O
O
X3C
NO
O
O
OX
X
X
X
X
NH2- reactive group: NHS
Balance:31-28
Reporter:114-117
iTRAQ - Isobaric Tags for Rel. and Abs. Quantification
4 isobaric tags:145 Da each
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
The iTRAQ workflow
ON
ONCH
3
Peptid1
ON
ONCH
3
Peptid4
ON
ONCH
3
Peptid3
ON
ONCH
3
Peptid2
Fragment s m/z 114.1
Fragment s m/z 115.1
Fragment s m/z 116.1
Fragment s m/z 117.1
MSMS
MSMS
MSMS
MSMS
114
115
116
117
MS
iTRAQ - Isobaric Tags for Rel. and Abs. Quantification
MSMS
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Selected iTRAQ Applications
• Discovery and Identification of Protein Markers in
Endometrial Carcinoma
• Study of Deregulated Signaling Pathways in Cancer
(c-kit/KDR kinase dual inhibitor)
• Discovery of Putative Biomarkers for Anaplastic
Oligodendrogliomas in Human Brain
• Biomarker Discovery in Human Cerebral Spinal Fluid of Patients
with Various Neurodegenerative Diseases
• Studies on the Composition of Synapsis in Mouse Brain
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Selected iTRAQ Applications(submitted or in press)
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Selected iTRAQ Applications
• Quantitation of Amino-Phospholipids in Tissue
• Quantitation of Non-Enzymatic Glycosylation of Milk Products
“Current iTRAQ applications are limited
by our lack of imagination rather than the
iTRAQ technology“
(Phil Andrews)
• Quantitation of Natural Peptidomes
• Localization of Proteins in the Plant Golgi
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
iTRAQ™ Reagents 8-plex Chemistry
N+
N
N+
N
13CH2
15N+
N
13CH2
13CH2
13CH215N+
N
13CH2
13CH2
13CH215N+
15N
13CH3
13CH2
13CH215N+
15N
13CH2
13CH3
13CH2
13CH215N+
N
13CH2
13CH215N+
13CH2
13CH2
15N
13CH2
13CH3
113 114 115 116
117 118 119 121
Isobaric Tag: Total mass = 305
Reporter Group
Peptide Reactive Group
(PRG)
N
N
O
O
N
O
O
Balance Group
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
9.0 403.4 797.8 1192.2 1586.6 1981.0
Mass (m/z)
1.1E+4
0
10
20
30
40
50
60
70
80
90
100
% In
ten
sit
y
y9(+1)
y6(+1)
116.1147
y13(+1)
b2(+1) y7(+1)y1(+1)
b3(+1)
b6(+1)y2(+1) 1039.4237y8(+1)y4(+1) b4(+1) 1268.5028 b14(+1) y15(+1)V b5(+1) y14(+1)E R 344.1307188.1420 795.3037 a8(+1) 1423.7990565.6216 1163.4664
MS/MS
Expected Ratio:
1:1:1:1:1:1:1:1
Glu-Fib peptide 8plex
1864.8 1871.6 1878.4 1885.2
Mass (m/z)
1874.8842
1873.8741
MS
113 116 119 122
Mass (m/z)
116114 117115113 121
120.10(A14747.87)
112.10(A9043.70)
118 119
114 117 120 123
Mass (m/z)
116 121117
118115 120.08
113 119
123.03(A199.04)111.04(A162.86)
114
Reporter ions
Expected Ratio:
1:2:2:5:5:2:1:5
A. B.
RF
R
F
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Seminar Outline – but no time ……. ;-)))
Examples of iTRAQ applications
1) organellar proteomics, protein location
2) signaling pathways, c-Kit/KDR inhibitor
3) cell cycle proteomics, Caulobacter cresentus
4) ocular melanoma, protein quant in vitreous fluid
5) Fragile X, protein quant in hippocampal
synapses
6) Alzheimer, protein quant in CSF
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
150 publications
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Seminar Outline
• TOF/TOF family: technology overview
• Protein analysis challenges
• Intelligent protein identification
• Quantitative proteomics
• MSI: Mass Spectrometric Imaging
4800 MALDI
TOF/TOF™ Analyzer
Slice Tissue Sections
Mount Sample
Spray Coat
with Matrix
Load Sample
Acquisition Software
Data Processing
oMALDI QSTAR® Elite
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Total Solution MS Imaging Package
● MALDI Imaging Starter kit for the 4800 System
from Laser Biolabs
● Leap™ Sprayer from Leap Technologies
● MSI Imaging data acquisition tool
● TissueView™ 1.0 Software
● MarkerView™ Software for statistical analysis
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Tissue Imaging
• TissueView Software
• Applications:
- Extracted Ion Image (EII)
- Regions of Interest (ROI)
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Mass Spectral Images : Whole Rat Body (head)
-TOF MS imaging
m/z+=616 Optical Image m/z+=798
Low High
Ion Intensity:
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Overlaying Multiple Extracted Ion Images
Red m/z ~ 796
Green m/z ~ 635
Blue m/z ~ 867
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
TissueView™ Software
B
G
R
B
G
R
Overlay up to 3
cluster images:
Complementary nature of
different clusters apparent
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
● Calculate statistics of region
of interest, providing the
average intensity of the mass
imaged in the region of
interest
● Export images as .tiff or .jpeg
images, or simply copy and
paste into another program
TissueView™ Software:
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
MALDI MSI covering a wide mass range
2510 Da 2028 Da
4618 Da
1540 Da
1909 Da optical image21.8 kDa
18 kDa
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Whole rat body TOFMS image of compound Q (462 Da)
-some background interference observed
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
MSMS analysis eliminates
Background interference
Parent at 462 Da
MS/MS fragment
at 168 Da
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Use of on-tissue trypsin digest to aid identification
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Use of on-tissue trypsin digest to aid identification
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
High Resolution MSI: Oversampling
● Perceived limitation of MSI for spatial resolution is laser
spot size
● Jurchen et. al recently described „oversampling‟ technique
that increases spatial resolution without requiring changes
to MALDI laser
● Using this technique, resolution can be increased beyond
the laser spot size
50 µm
50 µm
Laser
Spot “Burnt-Out
Spot”
25 µm
~500 msShift
Plate
Burnt-Out
Spot
Fresh
Sample
(25 µm width)
Sample
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Outlook: Advanced data processing (research grade)
(spectrum normalization, exclusion of margin spectra, periodic noise removing/FFT
filtering, prior to clustering and visualization)
Normalization: peptida ca. 7 kDa
before after
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Conclusions
• protein research/proteomics is an interesting field
to be in
• MALDI TOF/TOF technology offers the most
versatile applications
• in protein ID MALDI and ESI are complementary
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Picture from www.fz-juelich.de/oea/Fotoarchiv.html
but beware: not every TOF/TOF is a TOF/TOF .....
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
Trademarks & Licensing
iTRAQ, MIDAS and TEMPO are trademarks and QTRAP, QSTAR and
MALDI TOF/TOF are registered trademarks of Applied Biosystems/MDS
Analytical Technologies, a joint venture between Applera Corporation
and MDS Inc. Applied Biosystems is a registered trademark of Applera
Corporation or its subsidiaries in the U.S. and/or certain other countries.
All other trademarks are the property of their respective owners.
For Research Use Only. Not for use in diagnostic procedures.
© 2008 Applera Corporation and MDS Inc. All rights reserved.
Information subject to change without notice.
Praha, March12, 2009
© 2009 Applied Biosystems - Confidential
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