New MALDI-TOF MS with very high resolving power and mass accuracy Presented at ASMS June 3,2008 Marvin Vestal and Kevin Hayden Virgin Instruments Corp

New MALDI-TOF MS with very high resolving power and mass accuracy

Presented at ASMSJune 3,2008

Marvin Vestal and Kevin Hayden Virgin Instruments Corp.

Sudbury, MA

Objectives

• Determine optimum design parameters for a high-performance bench-top MALDI-TOF– Resolving power and mass accuracy– Sensitivity and dynamic range– High throughput– Efficient sample utilization– Simple and reliable– Low cost

• Build prototype instrument and compare experimental results with theoretical predictions

Common Features of Virgin MALDI TOF

• Simplified sample loading with super microplate format (102x108 mm)

• Benchtop cabinet – 1.75m high x 0.5 m wide x 0.7 m deep– 100 kg max weight

• 5 kHz laser, up to 100 spectra/sec• Fully automated, no operator expertise

required– No joystick, no camera

• 24/7 operation with minimal preventative maintenance

V

d0

Sample plate

Extractionelectrode

0

V1

V2

d3

d40

d4

Drift tube

velocityfocus

Dm

2-stage reflector

D

Potential diagram for high-resolution TOF with single-field source and two-field reflector. The field-free distance D is the distance from the source to the mirror plus the distance from the mirror to the detector (not shown).

Instrument Parameters for Reflecting Analyzer(single-field source and two-field mirror)

De=3200, d0=6, D=2264, d3=114.2, d40=127.7 all in mm

V=8.56, V1=6.31, V2=9.23 all in kVt=1.5 nsec (0.5 nsec bins, 1 nsec single ion pulse width) Focus mass m*= 3 kDaDv=6d0 = 36 mm, vn (for m*)=0.0235 mm/nsecTime lag t=d0/vn = 250 nsecK=2d0/(Dv-Ds)=0.5

Initial Conditions for MALDI (typical)v0=400 m/sec, x=0.01 mm

Trajectory error and voltage error assumed to be small

References• M. L. Vestal and P. Juhasz, “Resolution and Mass Accuracy in

Matrix-Assisted Laser Desorption Time-of-Flight Mass Spectrometry”, J. Am. Soc. Mass Spectrom. 9, 892-911 (1998).

• M. L. Vestal and K. Hayden, “High-performance MALDI-TOF mass spectrometry for proteomics”, Int. J. Mass Spectrometry 268, 83-92 (2007).

• M. L. Vestal and K. Hayden, “High-performance MALDI-TOF Mass Spectrometry”, paper TOFam08:15 presented at 55th ASMS Conference on Mass Spectrometry and Allied Topics, Indianapolis, June 3-7, 2007.

• S. J. Hattan and M. L. Vestal, “Novel 3-D sample plate using monolithic capture media in collimated-hole structures for interfacing high capacity separations with MALDI-TOF”, poster TBP-060 presented at 56th ASMS Conference on Mass Spectrometry and Allied Topics, Indianapolis, June 3, 2008.

•

Contributions to relative peak width, m/m, with 1st and2nd order velocity focusingInitial position, x: Rs1 = 2[(Dv-Ds)/2d0y](x/De)

Initial velocity, v0: Rv1 =(4d0y/De)(v0/vn)[(1-(m/m*)1/2] Rv2 =2[2d0y/(Dv-Ds)]2 (v0/vn)2=0 Rv3 =2[2d0y/(Dv-Ds)]3 (v0/vn)3 Rv = Rv1 + Rv3

Time error, t: Rt = 2t/t = 2tvn/De

Trajectory error, L: RL = 2L/De

Voltage error, V: RV = V/V

Resolving power:R-1 = [Rs1

2 + Rv2 + Rt

2 + RL2 + RV

2 ]-1/2

Neglected in initialcalculation

0

20000

40000

60000

80000

100000

120000

0 1 2 3 4 5 6

0 0.5 1 1.5 2

m/z (kDa) V=9 kV

m/z (kDa) V=3 kV

4 3

2 1

ResolvingPower

4 GS/s 2 GS/s

2 GS/s

4 GS/s

Calculated resolving power as function of m/z, curve 1 corresponds to experimental instrument

3.2 m

6.4 m

\\Domainserver\public\Shared Spectra\minitof\MiniTof_AutoSave-2007_05_22_16_21\MiniTof_AutoSave-000000001-0105-2007_05_22_16_21_56.427-(3687.025,8.098)-@31077,14847.dataXML

496.2443

508.2488

545.3450

565.2775

572.2940

573.3050

592.1041

658.3223

686.3764

689.3639

712.3753

733.4203

746.3996

927.4999

928.4904

929.5121

984.5116

985.5207

1121.4562

1149.4662

1163.6183

1284.7146

1306.6880

1479.7937

1480.8139

1536.8329

1537.8164

1567.7525

1881.9388

60.0000 80.0000 100.0000 120.0000

0.20

0.40

0.60

\\Domainserver\public\Shared Spectra\minitof\MiniTof_AutoSave-2007_05_22_16_21\MiniTof_AutoSave-000000001-0105-2007_05_22_16_21_56.427-(3687.025,8.098)-@31077,14847.dataXML

1479.7937

102.4000 102.5000 102.6000

0.10

0.20

0.30\\Domainserver\public\Shared Spectra\minitof\MiniTof_AutoSave-2007_05_22_16_21\MiniTof_AutoSave-000000001-0105-2007_05_22_16_21_56.427-(3687.025,8.098)-@31077,14847.dataXML

689.3639

69.9000 70.0000

0.10

0.20

0.30

0.40

11,60013,000BSA digest

Resolution is 40% ofcalculated

Spectrum from original version

10000

11000

12000

13000

14000

15000

16000

17000

400 800 1200 1600 2000

m/z (Da)

Res

olvi

ng P

ower

Comparison of calculated resolving power with experimental resultsWith m/m=70 ppm due to mass independent errors. Measured 5 channel plate ETP MagnetTOFTM (DM167)Original version before trajectory error correction

calculated

Changes to correct mass independent errors

• Add voltage regulator at input to mirror HV supplies (removes low frequency noise)

• Correct trajectory error due to ion deflector

• Use faster detector (0.5 ns) – ETP DM167 in place of 5 m dual channel plate

• Use faster digitizer – 0.5 ns bins>0.25 ns bins

11

2

+ _

Dd

mirror

detector

Ion source

d

2

2 2

2

Dd

mirror

detector

Ion source

d

Ray diagrams indicating source of trajectory error

1 = 2

+ -

1

2

1+2

1+2

2

Dd

Ion source

detector

mirror

1 = 2

Ray diagram showing general correction to trajectory error due to deflection

Low mass gate

Laser beam

4.60

0.40 0.40

0.80

0.80

Laser mirrorIon mirror

detector

Ray diagram of final version employed in the corrected version of theexperimental analyzer

\\domainserver\public\Shared Spectra\stevenew\MiniTof_AutoSave-2008_05_15_16_30\MiniTof_AutoSave-000007219-0360-2008_05_15_16_31_49.921-100 femto.mzml#0

1252.1202R:24363

1296.6884R:40099

1320.6501R:40718

1372.5907R:35361

1570.6801R:44096

1672.9220R:41688

1726.8405R:41310 2448.2654

R:6590

2465.1966R:40825

100.00000 110.00000 120.00000 130.00000 140.00000

0.00

0.05

0.10

Microseconds

Volts


1672.9220

R:41688

1673.9260

R:41714

1674.9288

R:39459

118.70000000 118.80000000

0.00

0.01

0.02

MicrosecondsVolts


2465.1966

R:40825

2466.1987

R:35961

2467.1994

R:35759

2468.2007

R:41383

2469.2030

R:44410

144.10000000 144.20000000

0.00

0.01

0.02

0.03

0.04

Microseconds

Volts


1296.6884

R:40099

1297.6908

R:41195

1298.6958

R:38673

1299.7013

R:38491

104.6000000

0.00

0.05

0.10

Microseconds

Volts

100 fm peptide standards

Example of spectrum obtained with corrected analyzer

\\domainserver\public\Shared Spectra\stevenew\MiniTof_AutoSave-2008_04_09_18_06\MiniTof_AutoSave-000005332-0093-2008_04_09_18_07_00.631-.mzml#35

733.4155

R:34222

835.0562

R:24141

918.9307

R:29707

927.4924

R:28012

1034.4701

R:35004

1121.4735

R:34571

1167.4780

R:36461

1283.7135

R:37578

1305.7020

R:34480

1419.6942

R:29470

1479.7953

R:35719

1511.8443

R:39338

1633.8930

R:18207

1639.9416

R:42237

1749.6712

R:37912

1880.9256

R:51427

2465.1983

R:57252

80.00000 100.00000 120.00000 140.00000

0.00

0.05

0.10

Microseconds

Volts


246

5.1

983

R:5

7252 24

66.2

026

R:4

5350

246

7.2

050

R:3

924

1

2468

.212

1R:5

8763

2469

.213

8R

:49685

144.05069677 144.10069677 144.15069677

0.00

0.01

0.02

0.03

Microseconds

Vo

lts


188

0.92

56R:5

142

7

1881

.928

4R:5

2904

188

2.92

59

R:4

839

3

125.85000000 125.90000000 125.95000000

0.000

0.005

0.010

Microseconds

Vo

lts


1639

.941

6R:42237

1640

.942

0R:453

25

1641

.943

3R:33861

1642.94

15R:420

72

117.50805559 117.60805559

0.00

0.02

0.04

0.06

Microseconds

Volts

Example of spectrum obtained with corrected analyzer

BSA digest

0

10000

20000

30000

40000

50000

60000

70000

500 1000 1500 2000 2500 3000

m/z

Reso

lvin

g Po

wer 1

2

Comparison of experimental results with theoretical calculations. Bars aremeasured resolving power with standard deviation of 5 replicates. Curve 1 corresponds to digitizer with 0.5 ns bins and curve 2 to 0.25 ns bins.

4 GS/s

2 GS/s

Chromatogram

N/A

0 200 400 600

0

1

2

3

Spot Position

RMS (PPM

)

RMS error for 10 peaks in spectrum of tryptic digest of BSA for all 675spots on a 102x108 mm sample plate with automatic 2-point internal Calibration (D0 and D1) with D3 and D4 fixed.

Calibration Equation: m1/2=D0 +D1t[1+D2t+D3t2]Determine coefficients by least square fit to multiplePeaks covering broad mass range

-2

0

2

4

800 1000 1200 1400 1600 1800 2000

mass (Da)

mas

s er

ror

(mD

a)

Average=-0.1 mDa

RMS =0.99 ppm

Serial Dilution Experiment

Peptide Standards

Equimolar mixture diluted in 4-hydroxy--cyanocinnamic acid from 1 picomole/L to 1 attomole/L

Automated acquisition and calibration averaging 2000 laser shots/spectrum at 1 kHz

No data processing

MH+ No.Angiotensin Fragment 1-7 899.47 1 des-Pro2 Bradykinin 963.51 2Angiotensin II Acetate 1046.54 3Angiotensin I 1296.69 4Neurotensin 1672.92 5ACTH (18-39) 2465.20 6Glu-1 Fibrinopeptide B 1570.68 7(<10% pure)


835.0620

MI:45455.9

R:28942 963.5155

MI:93147.7

R:29910

990.6680

MI:14042.1

R:29454

1296.6873

MI:264973.9

R:36130

1350.6004

MI:19705.4

R:37075

1672.9151

MI:113961.5

R:38539

2465.1994

MI:334018.2

R:28231

2520.0991

MI:5112.6

R:38235

80.00000 100.00000 120.00000 140.00000

0.0

0.1

0.2

0.3

Microseconds

Volts

100 fm

1 fm

Examples of spectra from serial dilution of peptide standards

12

3

5

4 6


1161

.936

1121

6.38

00

1225

.154

6127

9.07

98129

6.68

53135

0.5945

157

0.67

74

167

2.9175

1726.84

40

246

5.19

892520

.098

9

100.00000 120.00000 140.00000

0.000

0.005

0.010

0.015

Microseconds

Vo

lts


4.6914

1046

.173

510

46.651

9

1048

.156

0

93.85000000 94.00000000

0.000

0.002

0.004

0.006

0.008

Microseconds

Vo

lts


963.6704

90.1000000

0.000

0.002

0.004

0.006

0.008

Microseconds

Volts

1 fm4

5 6

3

2

7

7

\\domainserver\public\Shared Spectra\stevenew\MiniTof_AutoSave-2008_05_15_15_56\MiniTof_AutoSave-000006194-0254-2008_05_15_15_58_01.268-100 atto.mzml#0

1198.6940

1225.1357

1267.1141

1296.6783

1328.7196

1434.9402

1488.8633

1672.9175

2465.2013

100.00000 120.00000 140.00000

0.000

0.002

0.004

0.006

0.008

Microseconds

Volts

100 am


1198.6968

1203.5876

1225.1479

1252.6085

1296.6884

1330.2040

1402.7768

1433.9517

1487.8683

1541.8048

1596.8532

1673.9235

1727.8435

2465.1974

100.00000 120.00000 140.00000

0.000

0.005

0.010

Microseconds

Volts

10 am

4 5

6


1250.1104

1297.6997

1301.0323

1351.9429

1456.1489

1534.9803

1613.8237

1672.9035

2465.2050

110.00000 120.00000 130.00000 140.00000

0.000

0.002

0.004

0.006

Microseconds

Volts


1292.02

38

129

6.69

32129

8.04

19

130

1.032

3

1304

.022

5

104.4000000 104.8000000

0.000

0.002

0.004

0.006

Microseconds

Volts


1672.9035

1673.9124

118.7000000 118.9000000

0.0000

0.0005

0.0010

MicrosecondsV

olts


246

5.20

50246

6.17

94

144.1000000 144.3000000

0.0000

0.0002

0.0004

0.0006

Microseconds

Volts

4

65

1 attomole

Threshold 100 ions/peak

5 6

\\Domainserver\public\Shared Spectra\stevenew\MiniTof_AutoSave-2008_05_15_15_24\MiniTof_AutoSave-000005105-0172-2008_05_15_15_27_41.156-matrix.mzml#0

38.951

58.051 84.065

122.071 146.050

147.043

155.143

174.049

178.123

190.042

191.044

200.228

231.069

240.224

266.158

271.268

272.272

301.306

311.304

335.104

336.107

379.093

379.196

380.098

381.102

430.284

433.028

434.025

493.023

574.219

592.106

624.066

674.005

676.006

823.137

835.097

836.097

918.984

994.177

1078.064

1225.198

20.00000 40.00000 60.00000 80.00000 100.00000

0

1

Microseconds

Volts


126

7.16

113

00.090

1302

.089

1332

.141

1402

.199

1463

.193

1534.056

1617

.211

1686.518

175

5.79

0

1808

.014

1844

.757

1937.681

2052.64

0

110.00000 120.00000 130.00000

0.000

0.002

0.004

0.006

0.008

Microseconds

Vo

lts

Matrix blank, 2V digitizer range, major matrix peaks off-scale

threshold 100 ions/peak


1275

.132

127

9.13

1128

0.133

1291

.089

129

5.12

2

130

0.090

1302.089

1305

.077

1307

.952

104.0000000 105.0000000

0.000

0.005

0.010

MicrosecondsV

olts

4

Test Mixture for Determining Dynamic Range

Peptide Conc. (fmole/L) MH+ No.Angiotensin Fragment 1-7 10,000 899.47 1 Angiotensin II Acetate 1,000 1046.54 3des-Pro2 Bradykinin 100 963.51 2 Angiotensin I 2 1296.69 4Neurotensin 1 1672.92 5Glu-1 Fibrinopeptide B (synthetic) 0.2 1570.67 7ACTH (18-39) 0.01 2465.20 6BSA digest 0.1-1 1479.79 B

927.491283.711639.94, etc

\\domainserver\public\Shared Spectra\dynamic range)\MiniTof_AutoSave-2008_05_20_16_27\MiniTof_AutoSave-000011321-0301-2008_05_20_16_39_54.947-Run1.mzml#0

784

.045

R:1815

855.44

5R:174

0989

9.470

R:5880

953

.380

R:1992

1

1046

.550

R:153

26

1053

.570

R:183

48

120

0.642

R:2667

9

1296.685

R:281

25

1479.795

R:307

04

1672

.918

R:30996

80.80873 100.80873 120.80873 140.80873

0

1

2

3

4

5

Microseconds

Volts


1283.711

R:29998

1296.685

R:28125

1335.670

R:31408

1479.795

R:30704

1570.668

R:24565

1672.918

R:30996

1726.841

R:28323

110.00000 120.00000

0.00

0.01

0.02

Microseconds

Volts

Normal laser


1437.8174

1466.8455

1479.7985

1487.8878

1511.8489

1537.8066

1570.6850

1639.9341

1672.9120

1694.8790

1726.8187

1749.6452

1880.8425

1907.8395

110.000000 115.000000 120.000000 125.000000

0.00

0.01

0.02

Microseconds

Volts

Higher laser


927.482

9

931.48

60

88.4000000 88.60000000.00

0.01

0.02

Microseconds

Volts

1 3

2

4

57BB

B

7B

5

BB B B

B

B

\\domainserver\public\Shared Spectra\petides 100fm 5khz\MiniTof_AutoSave-2008_05_22_12_03\MiniTof_AutoSave-000024199-1877-2008_05_22_12_07_45.906-100 femto.mzml#2

899.4705

R:25276

963.5139

R:23761

1046.5399

R:26128

1296.6853

R:31271

1350.6021

R:33420 1672.9175

R:33033

2465.1989

R:31470

100.00000 120.00000 140.00000

0.00

0.02

0.04

0.06

Microseconds

Volts

\\domainserver\public\Shared Spectra\petides 100fm 200hz\MiniTof_AutoSave-2008_05_22_12_14\MiniTof_AutoSave-000025518-1897-2008_05_22_12_16_58.543-100 femto.mzml#2

899.4686

R:23721

963.5112

R:25789

1046.5394

R:27410

1296.6853

R:32090

1350.5962

R:30570

1672.9175

R:35179

2465.1989

R:32447

100.00000 120.00000 140.00000

0.00

0.02

0.04

0.06

Microseconds

Volts

200 Hz

5 kHz

100 fm/L peptide standards

No detectable effect of laser rate on spectrum quality.

• Present status

– Resolving power > 30,000 for peptides

– Mass error < 2 ppm RMS over entire sample plate

– Detection limit ~1 attomole/L

– Dynamic range ~105

– Performance independent of laser rate (to 5 kHz)

• Future goals

– Resolving power >100,000

– Mass error < 1 ppm RMS

– 10x improvement in detection limit and dyanamic range

– Isotopic resolution of proteins to 30,000 Da

– Multiplexed MS-MS with >4000 resolving power for precursor selection and very high sensitivity and throughput

– Instruments designed for specific applications, e.g. isotope ratios at ppt level

limited by chemical noise

Conclusion and Future

SteveGabelerEE

Joe FitzpatrickDesign Eng.

KevinHaydenIon OpticsME

RogerVoyerMachinist

MattGabeler-LeeSW

GeorgeMillsSW

JoeValentineEE

MarkDahlEE

SteveHattanAnalyticalChemist

ChrisVestalThe Boss

MarvinVestalHead Virgin

Documents

New MALDI-TOF MS with very high resolving power and mass accuracy Presented at ASMS June 3,2008 Marvin Vestal and Kevin Hayden Virgin Instruments Corp