High Performance MALDI-TOF Mass Spectrometry
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High Performance MALDI-TOF Mass Spectrometry Marvin Vestal and Kevin Hayden Virgin Instruments Corp. Sudbury, MA
description
High Performance MALDI-TOF Mass Spectrometry. Marvin Vestal and Kevin Hayden Virgin Instruments Corp. Sudbury, MA. Outline. Goals: Design and build MALD-TOF Analyzers Optimized for Selected Applications Approach: Modular construction using common elements - PowerPoint PPT Presentation
Transcript of High Performance MALDI-TOF Mass Spectrometry
High Performance MALDI-TOF Mass Spectrometry
Marvin Vestal and Kevin Hayden
Virgin Instruments Corp.
Sudbury, MA
Outline
• Goals: Design and build MALD-TOF Analyzers Optimized for Selected Applications• Approach:
– Modular construction using common elements– State-of-the-art technology, e.g. 5 khz laser– Focus on the needs of the application– Keep it small, simple, and reliable without sacrificing performance
• Analyzer geometries– Linear for broad mass range and sensitivity at high mass (to 500 kDa).– Reflector for high resolution, mass accuracy, and sensitivity at low mass
(0.1-5 kDa)– Reversed geometry reflector for both resolution and sensitivity in intermediate
mass range (1-50 kDa)
• Optimization Strategy• Results
Time-of-flight analyzer geometries
Ion Source Drift Tube Detector
ReflectorLinear
Reflecting
Reversed Geometry ReflectingReflectors
Reflector MS for MALDI mass fingerprinting peptides, metabolites, and other small molecules
• High sensitivity and dynamic range in mass range 0.1-5 kDa
• Highest possible resolving power and mass accuracy (1 ppm RMS)
• High throughput
• Low cost, stable, reliable, and small
• No operator expertise required
V
V1
0
d0 d1
Ds
Dv
Sample Plate
Extraction Electrode
Space focus
Velocityfocus
y=V/(V-V1)Ds= 2d0y3/2
Dv =Ds +(2d0y)2/(vnt)Wherevn =(2zV/m)1/2 and t is time lag
For single field source, V1=0, y=1Simultaneous 1st and 2nd order focusDv=6d0, vnt=d0
Wiley and McLarenRSI (1955) 26, 1150-1157Vestal and JuhaszJASMS (1998) 9, 892-911
V
d0
Sample plate
Extractionelectrode
0
V1
V2
d3
d40
d4
Drift tube
velocityfocus
Dm
2-stage reflector
Dm1 = 4d4w3/2 +4d3[w/(w-1)][1-w1/2]3Dm2 = 4d4w5/2 +4d3[w/(w-1)][1-w3/2]
d4 = d40(V-V1)/(V2-V1)
w=V/(V-V1)
Simultaneous 1st and 2nd order focus4d3/Dm=(w-3)/w4d4/Dm = w-3/2 + (4d3/Dm)/(w + w1/2)
D
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
Instrument Parameters for Reflecting Analyzer(single-field source and two-field mirror)
De=3200, d0=3, 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*=1.6 kDaDv=6d0 = 18 mm, vn (for m*)=0.03215 mm/nsecTime lag t=d0/vn = 93 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
0
10000
20000
30000
40000
50000
60000
0 500 1000 1500 2000 2500 3000 3500 4000
m/z (Da)
Reso
lvin
g Po
wer
Calculated resolving power for reflecting analyzer. v0=400m/s, x=0.01 mm for I and III and 800 m/s and 0.02 mm for II.t=1.5 nsec for I and II and 0.75 nsec for III,
I
III
II
Conditions for minimizing relative peak width, m/m
Initial position, x: Rs1 = 2K-1(x/De)
Initial velocity, v0: Rv1 =(4d0y/De)(v0/vn)[(1-(m/m*)1/2] Rv3 =2K3 (v0/vn)3 Rv = Rv1 + Rv3
Time error, t: Rt = 2t/t = 2tvn/De
Peak Width: R= [Rs12 + Rv
2 + Rt2 + RL
2 + RV2
]1/2
Optimum focus: dR/dK =0, K=2d0/Dv-Ds
Optimum velocity: dR/dvn=0, vn=C(V/m*)1/2
(can be optimized at m* or any other mass)
0
5
10
15
20
25
30
35
0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2
peak
wid
th (p
pm)
nominal optimum
R
Rs1
Rv3
K=2d0/(Dv-Ds)
Minimum peak width as function of focusing parameter K. Optimum value corresponds to dR/dK=0
0
5
10
15
20
25
30
0.8 1.2 1.6 2 2.4 2.8
V/m*
Peak
Wid
th (p
pm)
t=1.5
t=0.75
Rv3+
Rt
Minimum peak width as function of V/m*(indicated by arrows fort= 1.5 and 0.75 nsec). For m*= 1600 Da optimum voltages are2.4 and 3.4 kV, respectively.
0
10000
20000
30000
40000
50000
60000
70000
80000
0 500 1000 1500 2000 2500 3000 3500 4000
m/z (Da)
Reso
lvin
g Po
wer
Optimized resolving power for reflecting analyzer, v0=400m/s, x=0.01 mm. t=1.5 nsec for I and II and 0.75 nsec for III and IV. Red lines nominal, black at optimum for focus mass.
I
III
IV
II
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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
8000
9000
10000
11000
12000
13000
14000
15000
500 1000 1500 2000
m/z (da)
resolv
ing p
ow
er
Experimental resolving power for reflecting analyzer on BSA digest
Contributions to relative peak width, m/m
Initial position, x: Rs1 = [(Dv-Ds)/De](x/d0y)
Initial velocity, v0: Rv1 =(4d0y/De)(v0/vn)[(1-(m/m*)1/2] Rv2 =2[2d0y/(Dv-Ds)]2 (v0/vn)2
Rv3 =2[2d0y/(Dv-Ds)]3 (v0/vn)3
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
Not small!!!
Calculated resolving power for reflecting analyzer with V/V =70 ppm
8000
9000
10000
11000
12000
13000
14000
15000
400 600 800 1000 1200 1400 1600 1800 2000
m/z (Da)
Reso
lvin
g Po
wer
I
II
III
10000
11000
12000
13000
14000
15000
400 600 800 1000 1200 1400 1600 1800 2000
m/z (Da)
Res
olv
ing
Po
wer
Comparison of calculated resolving power with experimental resultsWith V/V=70 ppm
calculated
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 V/V=70 ppm. Measured 5 channel plate ETP MagnetTOFTM
Calculatedt=0.75 nsec
Calculatedt=0.75 nsec
Contributions to relative peak width, m/m, with 1st and2nd order velocity focusing (size matters)Initial 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
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
0
4
8
12
16
0.4 0.5 0.6 0.7 0.8 0.9 1
peak
wid
th (p
pm) 10.4
6.2
De=3.2 m
De=6.4 m
Rv3
K=2d0/(Dv-Ds)
Minimum peak width as function of focusing parameter K for effectiveLengths of 3.2 and 6.4 m. Optimum values correspond to dR/dK=0,And is proportional to De
0.84 for these initial conditions.
Calculated maximum resolving power as function of effective lengthneglecting voltage error.
0
20000
40000
60000
80000
100000
120000
0 1 2 3 4 5 6 7
Effective Length (m)
Max
. Res
olvi
ng P
ower
t=0.75 nsec
t=1.5 nsec
Current instrument
0
200000
400000
600000
800000
1000000
1200000
1400000
0 20 40 60 80 100
Effective Length
Max
. Res
olvi
ng P
ower
t=1.5 nsec
t=0.75 nsec
(m)
Calculated maximum resolving power as function of effective lengthneglecting voltage error.
Conclusions
• Resolving power of prototype reflecting analyzer is limited by voltage error or trajectory error
• If the total contribution of these two mass independent errors can be reduced to <10 ppm, then maximum resolving power of 50,000 is feasible with current prototype.
• Maximum resolving power is proportional to De0.84
• 1,000,000 resolving power is theoretically feasible with an effective length of 100 m (physical length about 40 m)
• But!!! • Errors independent of length must be less than 0.5 ppm
(5 millivolts at 10 kilovolts)
The Virgins
