The Future of Time-of-flight Mass Spectrometry

Marvin Vestal

Applied Biosystems

Framingham, MA

A Brief History of Time (of flight)with apologies to Stephen Hawking

• 1946 W.E Stephens, Phys. Rev. 69,641– “Advances in electronics seem to make practical a type of mass

spectrometer in which microsecond pulses of ions are selected every millisecond from an ordinary low-voltage ion source. In travelling down the vacuum tube, ions of different M/e have different velocities and consequently separate into groups spread out in space. … This type of mass spectrometer should offer many advantages over present types. The response time should be limited only by the repetition rate (milliseconds)… Magnets and stabilization equipment would be eliminated. Resolution would not be limited by smallness of slits or alignment. Such a mass spectrometer should be well suited for composition control, rapid analysis, and portable use.”

Brief History of Time (of flight)

• 1948 Cameron & Eggers, Rev. Sci. Instr. 19, 605.– First working TOF

• 1953 Wiley & McLaren, Rev. Sci. Instr. 26, 1150– First practical TOF. Energy & Time Lag Focusing.

• 1959 Gohlke, Anal. Chem. 31, 535– GC-TOF

• 1963 Vestal & Wharhaftig, ASMS, 358.– Coincidence TOF, first ion counting TDC

• 1973 Mamyrin et al, Sov. Phys. JETP 37, 45.– Reflectron, higher resolution

• 1974 Macfarlane et al, Biochem. Biophys. Res. Comm. 60, 616– 252Cf Plasma desorption. Proteins Fly!!!!

Brief History of Time (of flight)

• 1988 Karas & Hillenkamp, Anal. Chem. 60, 2299.– MALDI - Really big proteins fly!!!

• 1991 Dodenov et al, 12th Int. MS Conf.– O-TOF - Electrospray works with TOF

• 1993 Kaufman, Spengler & Lutzenkirchen, RCM 7, 902.– Post-source dcay MALDI

• 1994 Brown & Lennon, Sunriver, p63.– Delayed extraction MALDI - Makes MALDI-TOF routine

• 1996 Morris et al, RCM 10, 889.– Q-TOF

• 2001– TOF-TOF

Today’s Instruments

• Ionization TechniquesTOF Analyzers

Electrospray o-TOFQq-o-TOF

MALDI linear TOFreflector TOFTOF-TOFTrap-TOF

Why TOF?

• Speed

• Efficiency (Sensitivity)

• Dynamic Range

• Resolving Power

• Mass Accuracy

• Mass Range

• Simplicity

Competitivein

All Respectswith

UnmatchedSpeed

Speed Matters!Particularly with MALDI

• More Samples - Higher Throughput

• More Measurements/Sample

• Better data - Precision of mass and intensity

• Increased dynamic range

MALDI-TOFyesterday, today, and tomorrow

• Applications– Better sample utilization (>100,000 shots/spot)– Interface with separations– Molecular scanner– Tissue Imaging

then now futureLaser Rate (hz) 2 200 10,000Acq. Time/Spect.(sec) 60 2 0.1Spectra/day 1000 40,000 1,000,000*

*If we can process and interpret the results

1 cm2 @100 micron resolution=10,000 pixels

599.0 1379.4 2159.8 2940.2 3720.6 4501.0

Mass (m/z)

2.6E+4

0

10

20

30

40

50

60

70

80

90

100

% Int

ensit

yT O F T O F R e fle c to r S p e c # 1 [B P = 1 3 9 4 .8 , 2 6 0 7 1 ]

1394.7

583

1265.6

307

1742.9

248

1299.6

482

666.01

75

1083.5

358

1757.8

953

2265.2

253

622.03

99

1361.7

411

1067.5

093

1787.7

473

2466.2

288

1428.7

198

871.02

70

2408.1

682

1507.7

241

989.50

97

2847.4

409

1584.8

077

2002.9

247

1248.6

605

675.98

28

1341.6

882

1891.0

026

736.44

76

2731.3

547

3001.5

198

2555.2

749

3423.7

747

4280.9

302

4135.8

774

4432.0

645

3830.9

902

4069.4

912

4218.2

739

4348.7

676

3010 3308 3606 3904 4202 4500

Mass (m/z)

752.9

0

10

20

30

40

50

60

70

80

90

100

% Intens

ity

T O F T O F R e fle c to r S p e c # 1 [B P = 1 3 9 4 .8 , 2 6 0 7 1 ]

3423.7747

4280.9302

4135.8774

4432.0645

4179.6128

3830.9902

4069.4912

4218.2739

4348.7676

4315.9785

4424.1982

4388.3730

50 Laser shots in 0.25 secon 125 femtomoles of trypic digest

1 11 21 31 41 51 61 71MTMITDSLAV VLQRRDWENP GVTQLNRLAA HPPFASWRNS EEARTDRPSQ QLRSLNGEWR FAWFPAPEAV PESWLECDLP

81 91 101 111 121 131 141 151EADTVVVPSN WQMHGYDAPI YTNVTYPITV NPPFVPTENP TGCYSLTFNV DESWLQEGQT RIIFDGVNSA FHLWCNGRWV

161 171 181 191 201 211 221 231GYGQDSRLPS EFDLSAFLRA GENRLAVMVL RWSDGSYLED QDMWRMSGIF RDVSLLHKPT TQISDFHVAT RFNDDFSRAV

241 251 261 271 281 291 301 311LEAEVQMCGE LRDYLRVTVS LWQGETQVAS GTAPFGGEII DERGGYADRV TLRLNVENPK LWSAEIPNLY RAVVELHTAD

321 331 341 351 361 371 381 391GTLIEAEACD VGFREVRIEN GLLLLNGKPL LIRGVNRHEH HPLHGQVMDE QTMVQDILLM KQNNFNAVRC SHYPNHPLWY

401 411 421 431 441 451 461 471TLCDRYGLYV VDEANIETHG MVPMNRLTDD PRWLPAMSER VTRMVQRDRN HPSVIIWSLG NESGHGANHD ALYRWIKSVD

481 491 501 511 521 531 541 551PSRPVQYEGG GADTTATDII CPMYARVDED QPFAVPKWS IKKWLSLPGE TRPLILCEYA HAMGNSLGGF AKYWQAFRQY

561 571 581 591 601 611 621 631PRLWGGFVWD WVDQSLIKYD ENGNPWSAYG GDFGDTPNDR QFCMNGLVFA DRTPHPALTE AKHQQQFFQF RLSGQTIEVT

641 651 661 671 681 691 701 711SEYLFRHSDN ELLHWMVALD GPKPLASGEVP LDVAPQGKQL IELPELPQPE SAGQLWLTVR VVQPNATAWS EAGHISAWQQ

721 731 741 751 761 771 781 791WRLAENLSVT LPAASHAIPH LTTSEMDFCI ELGNKRWQFN RQSGFLSQMW IGDKKQLLTP LRDQFTRAPL DNDIGVSEAT

801 811 821 831 841 851 861 871RIDPNAWVER WKAAGHYQAE AALLQCTADT LADAVLITTA HAWQHQGKTL FISRKTYRID GSGQMAITVD VEVASDTPPHP

881 891 901 911 921 931 941 951ARIGLNCQLA QVAERVNWLG LGPQENYPDR LTAACFDRWD LPLSDMYTPY VFPSENGLRC GTRELNYGPH QWRGDFQFNI

961 971 981 991 1001 1011 1021SRYSQQQLME TSHRHLLHAE EGTWLNIDGF HMGIGGDDSW SPSVSAEFQL SAGRYHYQLV WCQK

E Coli beta-galactosidase, MW 116,483.916/20 matched, MOWSE Score 3e14, 23% sequence coverage

599.0 1379.4 2159.8 2940.2 3720.6 4501.0

Mass (m/z)

2.5E+4

0

10

20

30

40

50

60

70

80

90

100

% Int

ensit

yT O F T O F R e fle c to r S p e c # 1 = > M C [B P = 1 3 9 4 .7 , 2 5 3 9 8 ]

1394.7

321

1742.8

959

1265.6

179

1083.5

219

1299.6

221

1757.8

625

1361.7

229

666.01

21

2265.1

990

1428.6

871

1067.4

916

1787.7

350

1507.6

984158

4.7861

2466.2

036240

8.1423

622.04

23

989.49

22

2847.4

292

871.01

29

2002.8

771

1341.6

591141

4.7480

1252.6

586

750.37

35681

.9842

1050.4

740

1890.9

662

1501.7

145157

7.8330

3001.4

573

1133.5

934

1770.7

158

2730.3

289

2554.2

290248

2.1875

2137.9

861222

1.1733

3423.7

664

3292.6

782

3679.9

226

4304.8

716

3775.9

045

4185.6

772

3010 3308 3606 3904 4202 4500

Mass (m/z)

309.4

0

10

20

30

40

50

60

70

80

90

100

% Intens

ity

T O F T O F R e fle c to r S p e c # 1 = > M C [B P = 1 3 9 4 .7 , 2 5 3 9 8 ]

3036.43

68

3423.76

64

3292.67

82

3679.92

26

3441.81

96347

1.8052

4304.87

16

3775.90

45

4185.67

72

1000 Laser shots(5 seconds)

2900 4320 5740 7160 8580 10000

Mass (m/z)

5.7E+4

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

T O F T O F R e fle c to r S p e c # 1 = > M C [B P = 3 0 0 2 .4 , 5 6 5 9 4 ]

3001

.417

3423

.727

3036

.399

3292

.614

4324

.016

3678

.874

3827

.872

3961

.899

4449

.117

4113

.046

5432

.598

5045

.541

5187

.636

3100 3380 3660 3940 4220 4500

Mass (m/z)

2.3E+4

0

10

20

30

40

50

60

70

80

90

100

% Intens

ity

T O F T O F R e fle c to r S p e c # 1 = > M C [B P = 3 0 0 2 .4 , 5 6 5 9 4 ]

3423.727

3292.614

3112.389

4324.016

3678.874

3471.659

3439.688

3379.607

3774.868

3523.741

3712.820

3827.872

3961.899

4449.117

4282.009

4113.046

4374.002

9000 9100 9200 9300 9400 9500

Mass (m/z)

5.7

0

10

20

30

40

50

60

70

80

90

100

% Intensity

T O F T O F R e fle c to r S p e c # 1 = > S M 3 5 [B P = 3 4 2 5 .1 , 6 8 6 ]

9177

9194

10,000 laser shots, increased gain Low masses suppressed (50 seconds)

4311.0 4316.2 4321.4 4326.6 4331.8 4337.0

Mass (m/z)

7071.6

0

10

20

30

40

50

60

70

80

90

100

% Intens

ity

T O F T O F R e fle c to r S p e c # 1 = > M C [B P = 3 0 0 2 .4 , 5 6 5 9 4 ]

4324.016

4318.938

563-600

975-1014

389.0 911.4 1433.8 1956.2 2478.6 3001.0

Mass (m/z)

5.8E+4

0

10

20

30

40

50

60

70

80

90

100

% Int

ensit

y

T O F T O F R e fle c to r S p e c # 1 [B P = 4 4 1 .0 , 5 7 6 7 5 ]

441.01

73433

.0129

413.26

56

568.13

48

454.99

46

1394.7

312

1265.6

132

666.01

53

1083.5

148

524.14

56

1742.9

187

750.36

58

989.48

15

622.05

23

871.02

54

1507.7

096

397.02

86

2266.2

732

1446.7

974

1787.7

615

1584.8

086

707.36

06

2409.2

188

945.44

99

2467.3

071252

3.3069

2848.5

593

801.49

88

2003.9

152

1624.8

276

1139.5

703

1341.6

725

1891.0

288194

9.9652

2731.4

375

2139.0

732

450 560 670 780 890 1000

Mass (m/z)

1.0E+4

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ityT O F T O F R e fle c to r S p e c # 1 [B P = 4 4 1 .0 , 5 7 6 7 5 ]

568.

1348

454.

9946

666.

0153

524.

1456

550.

1259

569.

1384

506.

1363

660.

0019

480.

1578

750.

3658

622.

0523

871.

0254

455.

9986

644.

0301

522.

1321

681.

9896

886.

9975

900.

3804

553.

3895

855.

0515

566.

2953

962.

4616

707.

3606

945.

4499

604.

0451

736.

4346

801.

4988

823.

5007

840.

5246

918.

9470

401.5 402.3 403.1 403.9 404.7 405.5

Mass (m/z)

3633.3

0

10

20

30

40

50

60

70

80

90

100

% Intensit

y

T O F T O F R e fle c to r S p e c # 1 [B P = 4 4 1 .0 , 5 7 6 7 5 ]

402.0796

402.0004

403.0881

403.2230

484.0 486.8 489.6 492.4 495.2 498.0

Mass (m/z)

1105.9

0

10

20

30

40

50

60

70

80

90

100

% Intens

ity

T O F T O F R e fle c to r S p e c # 1 [B P = 4 4 1 .0 , 5 7 6 7 5 ]

495.278

0

488.318

8

10,000 laser shots Focused at low mass (50 seconds)

335-337EVR

290-293VTLR

?

Summary of Database Searching Results1000 Laser Shots, 20 ppm window

m/z No. No. Match Seq.Cov. Mean Error Data Tol. S/N Sub. Match % % ppm ppm

1000-3000 100 18 14 78 19 1.65 9.0 30 29 23 79 36 1.56 8.3 10 49 33 67 45 1.07 8.9 3 196 52 27 53 0.60 15.03000-10000

10 26 9 35 31 -2.18 10.8400-1000

30 7 5 71 3 -7.58 13.9 10 18 11 61 5 -6.02 9.8 3 156 24 15 12 -5.01 15.4

Total 3 378 85 23 96

0

2

4

6

8

10

12

-20 -18 -16 -14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 14 16 18

Numberof

PeptidesMatched

Error (ppm)

M/z 1000-3000, 1000 laser shotsPeaks detected down to S/N =352/196 matched

1 11 21 31 41 51 61 71MTMITDSLAV VLQRRDWENP GVTQLNRLAA HPPFASWRNS EEARTDRPSQ QLRSLNGEWR FAWFPAPEAV PESWLECDLP

81 91 101 111 121 131 141 151EADTVVVPSN WQMHGYDAPI YTNVTYPITV NPPFVPTENP TGCYSLTFNV DESWLQEGQT RIIFDGVNSA FHLWCNGRWV

161 171 181 191 201 211 221 231GYGQDSRLPS EFDLSAFLRA GENRLAVMVL RWSDGSYLED QDMWRMSGIF RDVSLLHKPT TQISDFHVAT RFNDDFSRAV

241 251 261 271 281 291 301 311LEAEVQMCGE LRDYLRVTVS LWQGETQVAS GTAPFGGEII DERGGYADRV TLRLNVENPK LWSAEIPNLY RAVVELHTAD

321 331 341 351 361 371 381 391GTLIEAEACD VGFREVRIENIEN GLLLLNGKPL LIRGVNRHEH HPLHGQVMDE QTMVQDILLM KQNNFNAVRC SHYPNHPLWY

401 411 421 431 441 451 461 471TLCDRYGLYV VDEANIETHG MVPMNRLTDD PRWLPAMSER VTRMVQRDRN HPSVIIWSLG NESGHGANHD ALYRWIKSVD

481 491 501 511 521 531 541 551PSRPVQYEGG GADTTATDII CPMYARVDED QPFAVPKWS IKKWLSLPGE TRPLILCEYA HAMGNSLGGF AKYWQAFRQY

561 571 581 591 601 611 621 631PRLWGGFVWD WVDQSLIKYD ENGNPWSAYG GDFGDTPNDR QFCMNGLVFA DRTPHPALTE AKHQQQFFQF RLSGQTIEVT

641 651 661 671 681 691 701 711SEYLFRHSDN ELLHWMVALD GPKPLASGEVP LDVAPQGKQL IELPELPQPE SAGQLWLTVR VVQPNATAWS EAGHISAWQQ

721 731 741 751 761 771 781 791WRLAENLSVT LPAASHAIPH LTTSEMDFCI ELGNKRWQFN RQSGFLSQMW IGDKKQLLTP LRDQFTRAPL DNDIGVSEAT

801 811 821 831 841 851 861 871RIDPNAWVER WKAAGHYQAE AALLQCTADT LADAVLITTA HAWQHQGKTL FISRKTYRID GSGQMAITVD VEVASDTPPHP

881 891 901 911 921 931 941 951ARIGLNCQLA QVAERVNWLG LGPQENYPDR LTAACFDRWD LPLSDMYTPY VFPSENGLRC GTRELNYGPH QWRGDFQFNI

961 971 981 991 1001 1011 1021SRYSQQQLME TSHRHLLHAE EGTWLNIDGF HMGIGGDDSW SPSVSAEFQL SAGRYHYQLV WCQK

E Coli beta-galactosidase, MW 116,483.950 shots 1000 shots high mass low mass

Missing Peptides MH+

811-812 WK 333.

338-353 IENGLLLLNGKPLLIR 1776.111

390-405 CSHYPNHPLWYTLCDR 2004.885

450-474 NHPSVIIWSLGNESGHGANHDALYR 2744.329

448-474 DRNHPSVIIWSLGNESGHGANHDALYR 3015.457

1730 1796 1862 1928 1994 2060

Mass (m/z)

1.9E+4

0

10

20

30

40

50

60

70

80

90

100

% Inte

nsity

T O F T O F R e fle c to r S p e c # 1 [B P = 1 3 9 4 .8 , 2 5 3 9 8 ]

1742.9

320

1757.8

989

1778.1

279178

7.7719

2002.9

165

1984.9

833

1891.0

045

1949.9

800

1770.7

523

1740.8

965

1762.8

960

1804.7

662

1923.0

077

2016.9

587

1939.9

860

1914.9

846

2025.9

783

1770 1776 1782 1788 1794 1800

Mass (m/z)

7607.5

0

10

20

30

40

50

60

70

80

90

100% In

tensity

T O F T O F R e fle c to r S p e c # 1 [B P = 1 3 9 4 .8 , 2 5 3 9 8 ]

1778.12

79

1787.77

19

1777.13

27

1770.75

23

1773.86

25

1776

2700 2766 2832 2898 2964 3030

Mass (m/z)

6119.6

0

10

20

30

40

50

60

70

80

90

100

% Intensit

y

T O F T O F R e fle c to r S p e c # 1 [B P = 1 3 9 4 .8 , 2 5 3 9 8 ]

2847.4634

2866.4224

3001.4880

2730.3652

2883.4368

2985.1558

2804.4163

2849.0925

257-283

358-381

9.0 380.8 752.6 1124.4 1496.2 1868.0

Mass (m/z)

2850.7

0

10

20

30

40

50

60

70

80

90

100

% Inte

nsity

T O F T O F M S /M S P re c u rs o r 1 7 7 7 S p e c # 1 [B P = 8 6 .0 , 2 8 5 1 ]

I,L,a1(+1) y7(+1)

y1(+1)

P

170.00

R

y13(+1)y8(+1)211.10 y5(+1)492.19 1759.82b4(+1) 735.30227.13136.02 324.18 1535.93K b5(+1) 1762.951399.62y8 - 17(+1)y6(+1)641.34 y10(+1)424.17

1398.0 1435.6 1473.2 1510.8 1548.4 1586.0

Mass (m/z)

489.1

0

10

20

30

40

50

60

70

80

90

100

% Intensit

y

T O F T O F M S /M S P re c u rs o r 1 7 7 7 S p e c # 1 [B P = 8 6 .0 , 2 8 5 1 ]

y13(+1)

1421.90

1419.94

1535.93

1422.87 y14(+1)1399.62

1423.85 y14 - 17(+1) 1537.801402.82

IENGLLLLNGKPLLIR Sequence expectedIENGLLLLDGKPLLIR Major component, labeledIEDGLLLLDGKPLLIR Minor component

9 428 847 1266 1685 2104

Mass (m/z)

453.6

0

10

20

30

40

50

60

70

80

90

100

% Int

ensit

y

A D D (T O F T O F M S /M S P re c u rs o r 2 0 0 2 .5 S p e c # 1 [B P = 2 0 0 3 .7 , 3 0 7 ] , < < 2 0 0 2 b g a lg a s o n 3 > > T O F T O F M S /M S P re c u rs o r 2 0 0 2 .5 S p e c #

H

W

P

y1(+1)Y

LHP(+1)

PNH(+1),NHP(+1)130.0005

1828.5574LCD(+1) - 28

44.0 90.8 137.6 184.4 231.2 278.0

Mass (m/z)

453.6

0

10

20

30

40

50

60

70

80

90

100% In

tensity

A D D (T O F T O F M S /M S P re c u rs o r 2 0 0 2 .5 S p e c # 1 [B P = 2 0 0 3 .7 , 3 0 7 ] , < < 2 0 0 2 b g a lg a s o n 3 > > T O F T O F M S /M S P re c u rs o r 2 0 0 2 .5 S p e c #

H

W

P

y1(+1)YL 112.02 HP(+1)PL(+1) - 28 PL(+1)

N 130.00101.01 PN(+1)195.04

1763.0 1824.2 1885.4 1946.6 2007.8 2069.0

Mass (m/z)

326.9

0

10

20

30

40

50

60

70

80

90

100

% Intensity

A D D (T O F T O F M S /M S P re c u rs o r 2 0 0 2 .5 S p e c # 1 = > N F 0 .7 [B P = 1 1 0 .0 , 4 4 8 ]

2002.74

2006.71

1987.51

1984.911996.691828.56S

CSHYPNHPLWYTLCDR

b15-2

10 590 1170 1750 2330 2910

Mass (m/z)

580.2

0

10

20

30

40

50

60

70

80

90

100

% Int

ensit

y

T O F T O F M S /M S P re c u rs o r 3 0 0 1 .5 S p e c # 1 = > N F 0 .7 [B P = 3 0 0 2 .2 , 3 3 8 2 ]

y12(+1)

b15(+1)

2481

H

304

y13(+1)y4(+1) b23 - 17(+1)15902818y1(+1)P 2617y13 - 17(+1) y17(+1) 24742230 y25(+1)1280 2367y6(+1) 2499I,L 1851 y18(+1) 21311724 2696y11(+1) 1611284 2362a4(+1) 1378 2234

1013 1527374 829 1218a4 - 17(+1) 1422934 1105

DRNHPSVIIWSLGNDSGHGANHDALYR

b23

y24

b21

EMH

+ = 3001.44

b14

Summary of Results

• 99.8 % of Sequence Covered (1022 of 1024)

• AA at position 462 is D not E

• Intact disulfide bond at 390-403

• Deamidation of NG at positions 340 and 346

– Discussed by N. E.Robinson PNAS 2002, 99,5283

• Dynamic range>1000 and resolving power>10,000 required to obtain good coverage

• Results required < 5 min. acquisition time on 125 fmoles loaded (51,000 laser shots @200 hz)

What will the future bring

• Laser rates to 10 khz will be routine within two years

• A major challenge is developing automated data acquisition, processing, and interpretation systems that can operate continuously at rates in the range of 10-100 finished

spectra/sec. • Applications to protein and peptide samples distributed on

surfaces become practical– 10,000 discrete spots/sample plate

– Multi-channel LC interfaced to single MALDI-TOF

– Molecular scanner for 1- and 2-D gels

– Direct tissue imaging