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)
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
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
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
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?
Generally, time-of-flight mass spectrometers have become relatively large instruments
with very high mass range %Int. 1 9 574.61 5735.64 5777.65 588.66 5779.65 8 7 6 5 4 5693.9 572.36 5738.63 5766.85 572.31 577.65 5732.56 585.64 5782.72 5811.73 3 5748.58 5797.77 2 1 568 57 572 574 576 578 6 58 582 1[c] Mass/Charge 5 2 1 and resolution. %Int. 1 9 8 7 5814.4 585.64 5812.7 587.67 581.66 5811.73 589.68 588.66 4 3 1[c] 585 586 587 588 589 581 5811 5812 5813 5814 5815 Mass/Charge
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
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
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
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
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
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
Pulsed extraction on the Miniaturized TOF Mass Spectrometer Laser pulse 9.75 kv Source XY stage 6.45 kv Grid 1 6.45 kv Grid 2, flight tube and detector, floated -2.2 kv delay extraction Time (ns)
Mass spectrum of ACTH on the miniature linear TOF mass spectrometer shows peak width of only 2 ns! 22 ACTH 7-38, 366.2 Da 2.47 ns (FWHM) ACTH 1-39, 4542.1 Da 2.ns (FWHM) 215 R = 87 at m/z 366 R = 12 at m/z 4542 21 25 4.2uS 4.4uS 4.6uS 4.8uS Desc: NewFile Device: Lecroy 945/24/2 Date Thu Sep 25 11:13:55 193 1pts @ 2e+9s/S 495-182.mas,Unc 1 of 1 Pulsed extraction is optimized for mass 4542
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 8566 7.2uS 7.6uS 8uS 8.4uS Cytochrome C, 12328 Da FWHM=7.75 ns sinapinic acid, analyte 2 pm C fragment of tetanus toxin: 52 kda 2598 13uS 14uS 15uS 16uS 17uS Bovine serum albumin : 66 kda 51819 66,43 9uS 9.2uS 9.4uS 9.6uS 9.8uS 13uS 14uS 15uS 16uS 17uS 18uS
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 25 2 15 1 5 144 15 1 5 144 MH +2 2 419 MH + 838 M 2 H + M 5 H + 1675 418 246 593 M 4 H + M 3 H + 3341 256 5uS 1uS 15uS 396 191 45294 MH +3 3 517 MH +2 2 775 MH + 1548 M 3 H + 4619 287 133 M 2 H + 384 5uS 1uS 15uS 396 191 45294 M 4 H + 6142
Analysis of Bacillus Spores on the 3-inch TOF s s 1 detector XY sample stage 3-inch flight tube
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 1-39. Peaks that occur in more than one spectrum carry the same number. Relative Intensity (arb. units) 5 4 3 2 1 2 15 1 5 1 8 6 4 2 25 2 15 1 5 1 5 (a) 3 (d) 15 (e) 11 36 13 1 16 2 3 17 32 2 24 4 25 26 2 21 18 19 24 27 11 12 14 17 22 23 13 15 16 (b) (c) 11 17 16 13 31 32 2 24 37 25 26 25 26 27 1 15 2 25 3 27 m/z 28 28 5 28 6 33 8 7 29 9 3 38 39 1 34 35
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
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
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
Comparison of pulsed extraction and mass-correlated acceleration on a linear TOF mass spectrometer 24 165 17 165 18 22 16 155 16 155 17 16 2 15 17.25uS 17.3uS 17.35uS 17.4uS 15 35.4uS 35.45uS 35.5uS 35.55uS 15 38.66uS 38.68uS 38.7uS 38.72uS 38.74uS 18 16 S 1uS 2uS 3uS 18 175 18 24 175 17 175 22 17 165 16 165 16 17 165 16 2 155 17.1uS 17.15uS 17.2uS 17.25uS 155 35.4uS 35.45uS 35.5uS 35.55uS 155 38.66uS 38.68uS 38.7uS 38.72uS 18 16 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.
Comparison of pulsed extraction and mass-correlated acceleration on a reflectron TOF mass spectrometer 65 36 6 355 355 355 4 55 5 35 35 35 45 4 345 345 345 38 35 33.6uS 33.65uS 33.7uS 33.75uS 49.45uS 49.5uS 49.55uS 49.6uS 67.5uS 67.55uS 67.6uS 67.65uS 34 9.75uS 9.8uS 9.85uS 9.9uS 36 34 S 2uS 4uS 6uS 8uS 4 36 352 38 39 35 37 38 355 348 36 37 35 346 4 35 34 36 35 34 345 344 342 33 33 34 34 38 33.5uS 33.55uS 33.6uS 33.65uS 49.4uS 49.45uS 49.5uS 49.55uS 67.45uS 67.5uS 67.55uS 67.6uS 67.65 9.7uS 9.75uS 9.8uS 9.85uS 36 34 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.
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) 34 338 336 334 332 33 36 355 35 345 34 335 37 36 35 34 Bradykinin 1-7 754.637 756.948 759.263 761.581 Dynorphin A 2143.72 2147.62 2151.51 2155.41 Bovine insulin 5727.42 5733.78 574.15 5746.52 (b) Bradykinin 1-7 6 55 5 45 4 35 755.776 758.87 76.4 762.718 Dynorphin A 55 5 45 4 35 2143.81 2147.7 2151.6 2155.49 Bovine insulin 4 39 38 37 36 35 34 5729.26 5735.62 5741.98
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
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 (2147.5 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 (2147.5 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
Lysozyme (14,313.14) tryptic digest Peptide Sequence PE MCA* WWCNDGR (937.2 Da) 15.6 ns 3.7 ns GTDVQAWIR (146.17 Da) 14.7 ns 3.6 ns FESNFNTQATNR (1429.488 Da) 12.1 ns 3.4 ns IVSDGNGMNAWVAWR (1676.89 Da) 11.4 ns 3.7 ns NTDGSTDYGILQINSR (1754.85 Da) 11.7 ns 2.7 ns KIVSDGNGMNAWVAWR (185.64 Da) 11.7 ns 3.5 ns FESNFNTQATNRNTDGSTDYGILQINSRR (3165.316 Da) 6.8 ns 3.4 ns Parameters set to focus on M+H + = 5734.5
BSA (66,432.96 Da) tryptic digest: comparison of peak widths using pulsed extraction and mass correlated acceleration. Peptide Sequence PE MCA* YLYEIAR (928.75 Da) 11.4 ns 3.2 ns LVNELTEFAK (1164.343 Da) 11. ns 4.2 ns HPEYAVSVLLR (1284.5 Da) 11.6 ns 3.1 ns HLVDEPQNLIK (136.54 Da) 8. ns 2.8 ns RHPEYAVSVLLR (144.688 Da) 9.2 ns 3.1 ns LGEYGFQNALIVR(148.76 Da) 1.6 ns 3.2 ns DAFLGSFLYEYSR (1568.725 Da) 1.8 ns 2.6 ns KVPQVSTPTLVEVSR (164.92 Da) 9.4 ns 3.1 ns MPCTEDYLSLILNR (1668.963 Da) 1.4 ns 3.4 ns RPCFSALTPDETYVPK (1825.89 Da) 1.3 ns 3. ns DDSPDLPKLKPDPNTLCDEFKADEK (2832.18 Da) 8.6 ns 4.5 ns Parameters set to focus on M+H + = 5734.5
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 = 1 2 4 2eV t = π 2 m ea [ L + L + d ] 1/ 2
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)
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)- 5784.63 14 (M+H)+ 12328.5 2 12 15 1 (M+2H)++ 6164.82 1 2416.97 3474.61 (2M-H)- 11416.5 8 (M+3H)+++ 419.81 (2M+H)+ 24644.6 5 6 2 4 6 8 1 12 14 NI IR MALDI mass spectrum of bovine insulin (MW=5,733 Da) 5 1 15 2 25 3 35 UV MALDI mass spectrum of bovine serum albumin (MW=66,429 Da) 2 (M+2H)++ 2867.89 (M+H)+ 5734 16 14 15 12 1 5 (M+3H)+++ 1912 (2M+H)+ 1146 1 8 6 BA+++++ BA+++ BA++++ BA++ BA+ 2 4 6 8 1 12 1 2 3 4 5 6 7
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.
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
The platform for the non-linear TOF has an adjustable aspect ratio
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
Results for 2.25 with delayed extraction 1 8 6 * 95 1349 2467 4542 5735 * 8566 Compound Bradykinin Substance P Neurotensin ADTH 18-39 m/z (M+H + ) 95 1349 1675 2467 Insulin Chain B 3497 ADTH 1-39 4542 Bovine Insulin 5735 Ubiquitin 8566 Cytochrome C 12328 1674 3497 4 2 12329 time (usec) m/z 2uS 177 4uS 1445 6uS 3935 8uS 7645 1uS 12575 12uS 18725 14uS 261
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 1 8 6 4 2 3 25 2 15 1 5 3 25 2 15 1 5 15 1 5 2 15 1 5 25 2 15 1 5 2 15 1 5 8 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 35 3 25 2 15 1 5 16 14 12 1 8 6 4 2 3 25 2 15 1 5 35 3 25 2 15 1 5 15 1 5 15 1 5 15 1 5 5 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)
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
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
The MAMS Laboratory