Miniaturized Time-of-Flight Mass Spectrometers with a Broad Mass Range
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1 Miniaturized Time-of-Flight Mass Spectrometers with a Broad Mass Range Robert J. Cotter Middle Atlantic Mass Spectrometry Laboratory Pharmacology and Molecular Sciences Johns Hopkins University School of Medicine Baltimore, MD (originally: Mass-Correlated Acceleration for the Analysis of Mixtures)
2 The analytical problem: polymer distributions cover a very broad mass range multiplex advantage: the TOF mass spectrometer has the ability to record the entire mass range from each ionization event without scanning focusing, or mass resolution, in a TOF is effected by the initial spatial and kinetic energy distributions of the ions space focusing can be achieved across the entire mass range with a single geometry and set of voltages kinetic energy focusing is achieved by pulsed extraction but is mass-dependent
3 The challenge: we would like to achieve broad mass range focusing on a miniaturized instrument (2 to 4 mass analyzer) maintaining mass resolution on a small instrument at any mass is itself a challenge
4 The solutions: fixed delay pulsed extraction on a 3 linear mass analyzer with approximately 2 nd order kinetic energy focusing at m/z 5, dynamic focusing using mass-correlated acceleration to compensate for different focusing parameters at different masses non-linear field mass spectrometers the examples used in this work will generally be mixtures of peptides and proteins
5 The time-of-flight mass spectrometer is the simplest of all mass analyzers: Ions formed in the ion source (s) appear at the detector with flight times through the drift region (D) proportional to the square root of their mass/charge: t = m 2eV 1/ 2 D What are the issues for miniaturization?
6 Generally, time-of-flight mass spectrometers have become relatively large instruments
7 with very high mass range %Int [c] Mass/Charge and resolution. %Int [c] Mass/Charge
8 The effects of initial energy and location time in ion source time in flight tube ( 2m) 1/ 2 [( ) ] 1/ 2 ( ) 1/ 2 1/ 2 2m D U + ees U + 2( U + ees) t = m + t 1/ 2 ee initial kinetic energy distribution turn-around time distribution of initial position in the source distribution in time of ion formation
9 Strategy for a miniaturized TOF MS using a fixed delay pulsed extraction Development of the 3-inch linear TOF mass spectrometer maintain highest possible resolution maintain high mass range design strategy
10 2 nd order space focusing occurs at a short distance Space distribution Space focus plane (a) E Single stage, first order space focusing s d = 2s (b) E E 1 Dual stage, first order space focusing s s 1 d (c) E E 1 Dual stage, second order space focusing s s 1 d Source, s Drift region, D
11 Miniaturization Strategy Delayed extraction results in both spatial and energy distributions But, the strategy is similar: do not shrink the ion source and provide high order focusing to a detector located at a relatively short distance. Sample plate Sample stage 3-inch flight tube Detector Laser in
12 A similar strategy is used for pulsed or delayed extraction focusing: During the delay, ions with different energies array themselves in different locations, though they still retain their energy distribution. before delay s s 1 d after delay High order focusing can be used to provide a short focal distance. before delay after delay s s 1 d
13 Miniaturized TOF Mass Spectrometer And, float the flight tube to prevent any post-acceleration Sample stage Grid 1 Grid 2 Laser Detector Pulsed extraction to space-focus plane Drift region (3 inches) 3.3 kv 25 MΩ 6.45 kv Floated -1.9 to -2.2 kv
14 Pulsed extraction on the Miniaturized TOF Mass Spectrometer Laser pulse 9.75 kv Source XY stage 6.45 kv Grid kv Grid 2, flight tube and detector, floated -2.2 kv delay extraction Time (ns)
15 Mass spectrum of ACTH on the miniature linear TOF mass spectrometer shows peak width of only 2 ns! 22 ACTH 7-38, Da 2.47 ns (FWHM) ACTH 1-39, Da 2.ns (FWHM) 215 R = 87 at m/z 366 R = 12 at m/z uS 4.4uS 4.6uS 4.8uS Desc: NewFile Device: Lecroy 945/24/2 Date Thu Sep 25 11:13: e+9s/S mas,Unc 1 of 1 Pulsed extraction is optimized for mass 4542
16 High mass range is retained on a miniature TOF mass spectrometer Relative Intensity Ubiquitin, 8566 Da 5.8 ns (FWHM) R = 685 at m/z uS 7.6uS 8uS 8.4uS Cytochrome C, Da FWHM=7.75 ns sinapinic acid, analyte 2 pm C fragment of tetanus toxin: 52 kda uS 14uS 15uS 16uS 17uS Bovine serum albumin : 66 kda ,43 9uS 9.2uS 9.4uS 9.6uS 9.8uS 13uS 14uS 15uS 16uS 17uS 18uS
17 Interleukin-8 and interleukin-2 on the 3-inch miniaturized TOF mass spectrometer Cytokines (IL-8, IL-2, TNF-α, etc.) and other biomarkers Clusters and multiplycharged ions span a wide mass range MH MH M 2 H + M 5 H M 4 H + M 3 H uS 1uS 15uS MH MH MH M 3 H M 2 H uS 1uS 15uS M 4 H
18 Analysis of Bacillus Spores on the 3-inch TOF s s 1 detector XY sample stage 3-inch flight tube
19 Bacillus Spore Identification via Proteolytic Peptide Mapping with a Miniaturized MALDI TOF Mass Spectrometer Figure 2. MALDI spectra of the tryptic digests generated in situ from (a) Bacillus subtilis 168, (b) Bacillus anthracis Sterne, (c) Bacillus cereus T, (d) Bacillus thuringiensis subs. Kurstaki HD-1, and (e) Bacillus globigii spores, and analyzed with the miniaturized TOF mass spectrometer. Peaks that were matched to peptides in the SASP database are numbered Peaks that occur in more than one spectrum carry the same number. Relative Intensity (arb. units) (a) 3 (d) 15 (e) (b) (c) m/z
20 Mass-correlated acceleration (MCA) Ions distributed in space are focused at a point dependent upon E, s, E 1 and s 1 E E 1 s s 1 d Using delayed extraction techniques, ions of different mass desorbed from a surface are distributed differently in space: E E 1 s s 1 d
21 Mass-correlated acceleration (MCA) In the mass-correlated acceleration (MCA) technique, the field in the second ion extraction region or accelerating region changes as each mass enters the region. E 1 E 2 delay E 2 /E 1 changes Time Using mass-correlated acceleration ions across the mass range are brought into simultaneous focus: E E 1 s s 1 d
22 Schematic diagram of the 1 meter MCA Pulse instrument generator Iris Filter (neutral) Mirror Nitrogen laser TTL In Optosync Out Pulse generator In Out Out Out To correction pulse generator TTL To HV switch Lens Grid 1 Grid 2 Einzel ion lens Anode Detector Oscilloscope Probe tip MCP Drift Region C4 R1 R2 R3 Reflectron HV switch R4 C1 C2 C3 Power supply Correction pulse generator Power supply Power supply
23 Comparison of pulsed extraction and mass-correlated acceleration on a linear TOF mass spectrometer uS 17.3uS 17.35uS 17.4uS uS 35.45uS 35.5uS 35.55uS uS 38.68uS 38.7uS 38.72uS 38.74uS S 1uS 2uS 3uS uS 17.15uS 17.2uS 17.25uS uS 35.45uS 35.5uS 35.55uS uS 38.68uS 38.7uS 38.72uS S 1uS 2uS 3uS Figure 1. Averaged mass spectra of a mixture of 11 peptides obtained with normal pulsed (delayed) extraction (top) or with mass-correlated acceleration (bottom) in a linear TOF. Insets are shown for methionine enkephalin-arg-gly- Leu, 9 Da; biocytin-β-endorphin, 3819 Da; ACTH 1-39, 4541Da.
24 Comparison of pulsed extraction and mass-correlated acceleration on a reflectron TOF mass spectrometer uS 33.65uS 33.7uS 33.75uS 49.45uS 49.5uS 49.55uS 49.6uS 67.5uS 67.55uS 67.6uS 67.65uS uS 9.8uS 9.85uS 9.9uS S 2uS 4uS 6uS 8uS uS 33.55uS 33.6uS 33.65uS 49.4uS 49.45uS 49.5uS 49.55uS 67.45uS 67.5uS 67.55uS 67.6uS uS 9.75uS 9.8uS 9.85uS S 2uS 4uS 6uS 8uS Figure 2. Averaged mass spectra of a mix of 9 peptides obtained with normal pulsed (delayed) extraction (top) or with mass-correlated acceleration (bottom) in a reflectron TOF. Insets are shown for bradykinin, fragment 1-7, 758 Da; neurotensin, 1674 Da; somatostatin 28, 315 Da; insulin, 5734 Da.
25 Mass-correlated acceleration (MCA) On a 1 meter TOF instrument MCA provides isotopicallyresolved peaks across the mass range: (a) Normal pulsed extraction (b) Mass-correlated acceleration Relative Intensity (a) Bradykinin Dynorphin A Bovine insulin (b) Bradykinin Dynorphin A Bovine insulin
26 Mass-correlated acceleration (MCA) on a miniaturized instrument On a 4-inch TOF instrument: the ion source is the same size Probe tip (sample) UV laser G1 G2 TOF 4. inches ExtractionMCA region region 4 inches MCP detector
27 Mass-correlated acceleration (MCA) on a miniaturized instrument On a 4-inch TOF instrument: unit mass resolution is not observed, but mass resolution is maintained across the mass range (a) Normal pulsed extraction (b) Mass-correlated acceleration Relative Intensity (a) bradykinin 2-9 (94. Da) 1.6 ns 2.4uS 2.45uS 2.5uS 2.55uS 2.6uS (c) dynorphina ( Da) 8.8 ns 3.5uS 3.55uS 3.6uS 3.65uS 3.7uS (f) Bovine insulin (5733.5Da) 4.4 ns 5.45uS 5.5uS 5.55uS 5.6uS (a) (b) time (microseconds) (a) bradykinin 2-9 (94. Da) 3.5 ns 1.95uS 2uS 2.5uS 2.1uS 2.15uS (c) dynorphin A ( Da) 3.1 ns 3.1uS 3.15uS 3.2uS 3.25uS (f) Bovine insulin (5733.5Da) 4. ns 5.5uS 5.1uS 5.15uS 5.2uS
28 Lysozyme (14,313.14) tryptic digest Peptide Sequence PE MCA* WWCNDGR (937.2 Da) 15.6 ns 3.7 ns GTDVQAWIR ( Da) 14.7 ns 3.6 ns FESNFNTQATNR ( Da) 12.1 ns 3.4 ns IVSDGNGMNAWVAWR ( Da) 11.4 ns 3.7 ns NTDGSTDYGILQINSR ( Da) 11.7 ns 2.7 ns KIVSDGNGMNAWVAWR ( Da) 11.7 ns 3.5 ns FESNFNTQATNRNTDGSTDYGILQINSRR ( Da) 6.8 ns 3.4 ns Parameters set to focus on M+H + =
29 BSA (66, Da) tryptic digest: comparison of peak widths using pulsed extraction and mass correlated acceleration. Peptide Sequence PE MCA* YLYEIAR ( Da) 11.4 ns 3.2 ns LVNELTEFAK ( Da) 11. ns 4.2 ns HPEYAVSVLLR ( Da) 11.6 ns 3.1 ns HLVDEPQNLIK ( Da) 8. ns 2.8 ns RHPEYAVSVLLR ( Da) 9.2 ns 3.1 ns LGEYGFQNALIVR( Da) 1.6 ns 3.2 ns DAFLGSFLYEYSR ( Da) 1.8 ns 2.6 ns KVPQVSTPTLVEVSR ( Da) 9.4 ns 3.1 ns MPCTEDYLSLILNR ( Da) 1.4 ns 3.4 ns RPCFSALTPDETYVPK ( Da) 1.3 ns 3. ns DDSPDLPKLKPDPNTLCDEFKADEK ( Da) 8.6 ns 4.5 ns Parameters set to focus on M+H + =
30 Non-linear geometries Single-stage reflectron: first order focusing of energy ev Quadratic reflectron: t is independent of kinetic energy Endcap reflectron: is nearly quadratic 1/ 2 m t = eV t = π 2 m ea [ L + L + d ] 1/ 2
31 Endcap reflectron TOF 4.5 diameter/3. depth Mass Technologies, Inc. Cylinder (grounded) Multichannel plate detector (-2.3 kv, pulsed) Laser beam (IR or UV) Reflecting potential (up to ±28 kv) Ions Ion collector (to data acquisition) Sample probe (pulsed up to ±25 kv with the delay of.2-1 µsec) Extraction electrode (grounded) Ion lens (up to ±7.5 kv)
32 Mass spectra from the endcap reflectron TOF NI UV MALDI mass spectrum of bovine insulin (MW=5,733 Da) NI IR MALDI mass spectrum of cytochrome C (MW=12,327 Da) 25 (M-H) (M+H) (M+2H) (2M-H) (M+3H) (2M+H) NI IR MALDI mass spectrum of bovine insulin (MW=5,733 Da) UV MALDI mass spectrum of bovine serum albumin (MW=66,429 Da) 2 (M+2H) (M+H) (M+3H) (2M+H) BA+++++ BA+++ BA++++ BA++ BA
33 Turning the endcap around: a TOF with a single non-linear field region Electrode 3 V V Electrode 4 JHU technology DM477: Non-Linear Time-of-Flight Mass Spectrometer 18 kv Beam Collimating Field Electrode 1 Sample Probe Ion Trajectories Electrode 1 Sample Probe 15 kv 1 kv 5 kv 2 kv Detector 1 kv 15 kv 1 kv 5 kv Iso-potential Lines 2 kv 1 kv Figure 4: Cross-sectional and 3-dimensional topographical views of the physical geometry and non-linear electrical field distribution for the first embodiment.
34 An extraction field that is both non-linear in space and time Ion Source Exit A quadratic field would provide infinite order space focusing at the end of the source region (i.e. there would be no drift region). When space and velocity are correlated as in MALDI, delayed extraction will provide energy focusing, but still be mass dependent. Therefore, need a field that is nonlinear in time as well as in space. Potential (V) Potential (V) Non-linear Field Gradient S Ion Source Exit Non-linear Field Gradient Dynamic Field S
35 The platform for the non-linear TOF has an adjustable aspect ratio
36 Infinite order space focusing at the end of the single region requires a quadratic field 2.9 inches 2.25 inches 3 inches The optimal aspect ratio approximates this field 1.5 inches 4 inches
37 Results for 2.25 with delayed extraction * * 8566 Compound Bradykinin Substance P Neurotensin ADTH m/z (M+H + ) Insulin Chain B 3497 ADTH Bovine Insulin 5735 Ubiquitin 8566 Cytochrome C time (usec) m/z 2uS 177 4uS uS uS uS uS uS 261
38 Energy focusing by delayed extraction still shows mass dependence Next stage will be to develop time dependence of the non-linear field 2.25-inch Configuration Intensity Intensity Intensity Intensity Intensity Intensity Intensity µ sec delay 2uS 4uS 6uS 8uS 1uS 1 µ sec delay 2uS 4uS 6uS 8uS 1uS 12 µ sec delay 2uS 4uS 6uS 8uS 1uS Time ( µ sec) 14 µ sec delay 2uS 4uS 6uS 8uS 1uS Time ( µ sec) 16 µ sec delay 2uS 4uS 6uS 8uS 1uS Time ( µ sec) 18 µ sec delay 2uS 4uS 6uS 8uS 1uS Time ( µ sec) 2 µ sec delay 3-inch Configuration Intensity Intensity Intensity Intensity Intensity Intensity Intensity µ sec delay 2uS 4uS 6uS 8uS 1uS 12uS 14uS Time ( µ sec) 8 µ sec delay 2uS 4uS 6uS 8uS 1uS 12uS 14uS Time ( µ sec) 1 µ sec delay 2uS 4uS 6uS 8uS 1uS 12uS 14uS Time ( µ sec) 12 µ sec delay 2uS 4uS 6uS 8uS 1uS 12uS 14uS Time ( µ sec) 16 µ sec delay 2uS 4uS 6uS 8uS 1uS 12uS 14uS Time ( µ sec) 2 µ sec delay 2uS 4uS 6uS 8uS 1uS 12uS 14uS Time ( µ sec) 3 µ sec delay 2uS 4uS 6uS 8uS 1uS Time ( µ sec) 2uS 4uS 6uS 8uS 1uS 12uS 14uS Time ( µ sec)
39 Such an instrument can also be configured as a reflectron or tandem (TOF/TOF) MS 18 kv Beam Collimating Field Electrode 3 V Electrode 4 V Electrode 5 V Electrode 6 19 k V JHU technology DM477: Non-Linear Time-of-Flight Mass Spectrometer Ion Trajectories Electrode 1 Sample Probe Detector 15 kv 1 kv 5 kv 2 kv 1 kv 1 kv 2 kv 5 kv 1 kv 15 kv Iso-potential Lines Figure 6: Cross-sectional view of the physical geometry and non-linear electric field distribution for the second embodiment
40 Acknowledgements: 3-inch instrument Slava Kovtoun, Mari Prieto, Robert English Mass correlated instrument Slava Kovtoun, Robert English Endcap reflectron mass spectrometer Timothy Cornish, Vladimir Doroshenko (MassTech) Non-linear instrument Ben Gardner
41 The MAMS Laboratory
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