MALDI TOF/TOF technology: applications and workflows for a …€¦ · The angular distribution of the MALDI ion plume is directed back towards the direction of the incident laser

MALDI TOF/TOF technology:

applications and workflows for a mass

spectrometry research lab

Volker Kruft

Manager Business Development EMEA, Proteomics

[email protected]

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



Seminar Outline








The AB SCIEX TOF/TOF Family

4800 MALDI

TOF/TOFTM

Analyzer

The AB SCIEX TOF/TOFTM

5800 System



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



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



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



Seminar Outline








Proteome 1 Proteome 2 Proteome 3 Proteome 4 Proteome 5 Proteome 6 Proteome 7

Proteomics: It´s all about time courses



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

*


© 2009 Applied Biosystems - Confidential 12

34

5

67

9

8

10

Dynamic range in proteomics

Courtesy of B. Domon, ETH Zurich



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)

http://www.maniacworld.com/robert-wadlow-picture-1.htm



The “Do´ers” in a Cell



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



Seminar Outline








4800 MALDI TOF/TOF™ Analyzer



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



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




Other Unique Applications:

• Polymers

• Fatty Acids

• Carbohydrates



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



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



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



TOF/TOF: Recent Carbohydrate Publications



TOF/TOF: Growing list of publications



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


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”)



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”)



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?





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.


Greatest depth of coverage with intelligent LC

MALDI protein identification workflows.



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)



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)



Methods: Copy precursor list plus rentention time

Easy in Peak Explorer™



Method: Paste list into the exclusion list



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



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



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 !!


© 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



● 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



● 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



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).



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



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



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



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



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



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



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



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



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



● 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



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



Seminar Outline








• 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)



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

http://en.wikipedia.org/wiki/Image:Smiley.svg



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



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‟



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




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)



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!



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!



*VLVDTDYK*

MS/MS: reporter ions quantitate 4 individual samples!



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



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




MSMS

MSMS

MSMS

MSMS

114

115

116

117

MS

iTRAQ - Isobaric Tags for Rel. and Abs. Quantification

MSMS



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



Selected iTRAQ Applications(submitted or in press)



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



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



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



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



150 publications



Seminar Outline






4800 MALDI

TOF/TOF™ Analyzer

Slice Tissue Sections

Mount Sample

Spray Coat

with Matrix

Load Sample

Acquisition Software

Data Processing

oMALDI QSTAR® Elite



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



Tissue Imaging

• TissueView Software

• Applications:

- Extracted Ion Image (EII)

- Regions of Interest (ROI)





Mass Spectral Images : Whole Rat Body (head)

-TOF MS imaging

m/z+=616 Optical Image m/z+=798

Low High

Ion Intensity:



Overlaying Multiple Extracted Ion Images

Red m/z ~ 796

Green m/z ~ 635

Blue m/z ~ 867



TissueView™ Software

B

G

R

B

G

R

Overlay up to 3

cluster images:

Complementary nature of

different clusters apparent



● 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:



MALDI MSI covering a wide mass range

2510 Da 2028 Da

4618 Da

1540 Da

1909 Da optical image21.8 kDa

18 kDa



Whole rat body TOFMS image of compound Q (462 Da)

-some background interference observed



MSMS analysis eliminates

Background interference

Parent at 462 Da

MS/MS fragment

at 168 Da



Use of on-tissue trypsin digest to aid identification



Use of on-tissue trypsin digest to aid identification



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





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



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



Picture from www.fz-juelich.de/oea/Fotoarchiv.html

but beware: not every TOF/TOF is a TOF/TOF .....




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.



ThanksDanke

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Ačiu

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Dank

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ありがとう

Tack

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Спасибо

Tesekkurler

谢谢

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MulţumescKöszönöm

(

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Documents

MALDI TOF/TOF technology: applications and workflows for a …€¦ · The angular distribution of the MALDI ion plume is directed back towards the direction of the incident laser