FOURIER TRANSFORM MASS SPECTROMETRY

FT-ICR Theory Ion Cyclotron Motion Inward directed Lorentz force causes ions to move in circular orbits about the magnetic field axis Alan G. Marshall, Christopher L. Hendrickson, and George S. Jackson Encyclopedia of Analytical Chemistry, R.A. Meyers (Ed.), John Wiley & Sons Ltd, Chichester, 2000, pp. 11694 11728

FT-ICR Theory Ion Cyclotron Motion Ion cyclotron motion. Ions rotate in a plane perpendicular to the direction of a spatially uniform magnetic field, Note that positive and negative ions orbit in opposite senses.

FT-ICR Theory Ion Cyclotron Motion X Y ω c = qb m Z

Once we make an ion, we move it into the center of the Magnet. Then, we trap it before it can escape. Electrostatic Barrier ION + From Primer 1998 Marshall.

Once we make an ion, we move it into the center of the Magnet. Then, we trap it before it can escape. Electrostatic Barrier ION + Ion sees barrier and is turned back From Primer 1998 Marshall.

Once we make an ion, we move it into the center of the Magnet. Then, we trap it before it can escape. Electrostatic Barrier ION + Gate shut before the ion escapes From Primer 1998 Marshall.

Once we make an ion, we move it into the center of the Magnet. Then, we trap it before it can escape. Ion is now trapped in the magnet. ION + From Primer 1998 Marshall.

FT-ICR Theory - Ion Trapping X Z T T Magnetic Field (B) Y E Axial Position

FT-ICR Theory Combined Ion Motion v m = magnetron motion v c = cyclotron motion v t = trapping oscillations Alan G. Marshall, Christopher L. Hendrickson, and George S. Jackson Encyclopedia of Analytical Chemistry, R.A. Meyers (Ed.), John Wiley & Sons Ltd, Chichester, 2000, pp. 11694 11728

FT-ICR Theory - Excitation 0.05 0.04 0.03 0.02 0.01 Image 0-0.01-0.02-0.03-0.04-0.05 0 100 200 300 400 500 600 700 800 Time (ms)

FT-ICR Theory - Excitation X Z or B o Y Amplitude Excitation Electrodes Time

FT-ICR Theory Excitation Alan G. Marshall, Christopher L. Hendrickson, and George S. Jackson Encyclopedia of Analytical Chemistry, R.A. Meyers (Ed.), John Wiley & Sons Ltd, Chichester, 2000, pp. 11694 11728

SWIFT Excitation Single Notch SWIFT Event (MS/MS) Data Count Affects Resolution (Limited to < 512K) Freq Cutoff Bandwidth 100% Power Start Frequency End Frequency 0% Frequency

SWIFT Excitation 100% Power 0% Frequency IFT

FT-ICR Theory - Excitation X Z or B o Y Amplitude Time Excitation Electrodes

SWIFT Excitation 10+ 11+ 9+ 8+ 12+ 778.5 779.0 779.5 m/z 780.0 780.5 781 11+ On -the -fly SWIFT isolation of a single Isotope of Bovine Ubiquitin M+4 Isotope 778.5 779.0 779.5 m/z 780.0 780.5 781 700 800 900 1000 1100 m/z 1200 1300 1400 1500

FT-ICR Theory - Detection X Z or B o Y

Multiplex Detection in FT-ICR 0.05 Time Domain Transient Differential Amplifier Image Current 0.04 0.03 0.02 0.01 0-0.01-0.02-0.03-0.04-0.05 0 100 200 300 400 500 600 700 800 Time (ms)

Multiplex Detection in FT-ICR 0.05 0.04 Time Domain Transient Differential Amplifier Image Current 0.03 0.02 0.01 0-0.01-0.02-0.03-0.04-0.05 0 100 200 300 400 500 600 700 800 Time (ms)

Signal Apodization in FT MS

Fourier Transforms in FT-ICR Differential Amplifier Image Current 0.05 0.04 0.03 0.02 0.01 0-0.01-0.02-0.03-0.04-0.05 0 Time Domain Transient 100 200 300 400 500 600 700 800 Time (ms) Fourier Transform Frequency Spectrum 0 50 100 150 200 Frequency (khz) 250 300

Mass Calibration in FT-ICR Frequency Spectrum Mass Spectrum m z A = + BV t f f 2 0 50 100 150 200 250 Frequency (khz) 300 500 600 700 800 900 10001100120013001400 m/z

Space Charge and Resolution in FT-ICR T T LOW ION DENSITY FT 0 200 400 600 Detection Time (msec) 800 952.0 952.5 953.0 m/z 953.5 FT T T 0 200 400 600 Detection Time (msec) 800 HIGH ION DENSITY 952.0 952.5 953.0 m/z 953.5

Effect of Magnetic Field Strength Resolving Power Highest Non-Coalesced Mass Scan Speed (LC/MS) Axialization Efficiency 3 4.7 7 9.4 11.5 15 25 Number of Ions Trapped Ion Upper Mass Limit 2D-FT Resolving Power Ion Trapping Time Ion Energy 3 4.7 7 9.4 11.5 0 5 10 15 20 25 Field Strength (Tesla) 15 25

FT-ICR Experiment - Event Sequences - Use a single mass analyzer but separate the mass analysis and ion isolation events in time - Can perform many successive stages of MS (MS n ) Event Sequence Ion Transfer / Ion Trapping Parent Ion Fragmentation Ionization Parent Ion Isolation Daughter Ion Detection

Ultra-high Resolving Power Peak Capacity = (m/z) max - (m/z) min Dm 50% Dm 50% (m/z) min m/z (m/z) max

Separation Method Maximum # of Components Maximum Peak Capacity Theoretical Plates HP-TLC 6 25 1,000 Isocratic LC 12 100 15,000 Gradient LC 17 200 60,000 HPLC 37 1,000 1,500,000 CE 37 1,000 1,500,000 Open Tubular GC 37 1,000 1,500,000 ESI FT-ICR MS 525 200,000 60,000,000,000 m/dm 50% > 200,000 200 < m/z < 1,000 m average +/- 0.25 Da Skip Prior Chemical Separation and Identify Components by MS!

17,000+ Compositionally Distinct Components Resolved by High Resolution 9.4 Tesla Electrospray FT-ICR MS Negative Ion ESI Mass Spectrum 6,118 resolved components Positive Ion ESI Mass Spectrum 11,127 resolved components 0 ~ -900-800 -700-600 -500-400 -300-200 200 m/z 300 400 500 600 700 800 900 )Ryan P. Rodgers, Alan G. Marshall, Petroleomics: Advanced Characterization of Petroleum-Derived Materials by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICRMS, Asphaltenes, Heavy Oils, and Petroleomics 2007, pp 63-93

)Ryan P. Rodgers, Alan G. Marshall, Petroleomics: Advanced Characterization of Petroleum-Derived Materials by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICRMS, Asphaltenes, Heavy Oils, and Petroleomics 2007, pp 63-93

Isotopic Fine Structure Resolving power M DM 50% = 8,000,000 S. D.-H. Shi, C. L. Hendrickson, and A. G. Marshall Proc. Natl. Acad. Sci. USA, 1998, 95, 11532 11537.

Unit Mass Baseline Resolution for an Intact 148 kda Therapeutic Monoclonal Antibody by FT-ICR Mass Spectrometry Anal Chem. 2011 November 15; 83(22): 8391 8395. doi:10.1021/ac202429c.

Unit Mass Baseline Resolution for an Intact 148 kda Therapeutic Monoclonal Antibody by FT-ICR Mass Spectrometry Valeja et al. Page 10 NIH-PA Author Manuscript NIH-PA Author Manuscript FigureAnal 3. Chem. 2011 November 15; 83(22): 8391 8395. doi:10.1021/ac202429c. Top: Positive ESI 9.4 T magnitude-mode FT-ICR mass spectrum for quadrupole-selected IgG1k showing 57+ charge state molecular ions along with various adducts, based on an

Single-scan electrospray FT-ICR mass spectrum of the isolated 48+ charge state of bovine serum albumin Bovine Serum Albumin 66,433 Da [M+48H] 48+ m = 2,000,000 m 50% 1384 1385 1386 m/z Figure 3. J. Am. Soc. Mass Spectrom. (2015) 26:1626Y1632 Single-scan electrospray FT-ICR mass spectrum of

Principle of Trapping in the Orbitrap The Orbitrap is an ion trap but there are no RF or magnet fields! Moving ions are trapped around an electrode - Electrostatic attraction is compensated by centrifugal force arising from the initial tangential velocity Potential barriers created by end-electrodes confine the ions axially One can control the frequencies of oscillations (especially the axial ones) by shaping the electrodes appropriately Thus we arrive at Orbital traps Kingdon (1923)

Orbitrap - Electrostatic Field Based Mass Analyser k U ( r, z) = 2 { 2 2 2 z - r / 2 + R ln( r / )} r m R m z φ Korsunskii M.I., Basakutsa V.A. Sov. Physics-Tech. Phys. 1958; 3: 1396. Knight R.D. Appl.Phys.Lett. 1981, 38: 221. Gall L.N.,Golikov Y.K.,Aleksandrov M.L.,Pechalina Y.E.,Holin N.A. SU Pat. 1247973, 1986.

Ion Motion in Orbitrap Only an axial frequency does not depend on initial energy, angle, and position of ions, so it can be used for mass analysis The axial oscillation frequency follows the formula w = k m / z

Ions of Different m/z in Orbitrap Large ion capacity - stacking the rings Fourier transform needed to obtain individual frequencies of ions of different m/z

HOW DOES IT WORK? The Orbitrap mass analyzer consists essentially of three electrodes as shown in Figure 1. The cut-outs represent both How Big Is the Orbitrap? E sub eﬃ to cha use em ion reso ad dev this hig key deﬁ the ma T sem cen as a ma

Getting Ions into the Orbitrap The ideal Kingdon field has been known since 1950 s, but not used in MS. Why? There is a catch how to get ions into it? Ions coming from the outside into a static electric field will zoom past, like a comet from the outer space flies through a solar system The catch: The field must not be static when ions come in! A potential barrier stopping the ions before they reach an electrode can be created by lowering the central electrode voltage while ions are still entering Thus we arrive at the principle of Electrodynamic Squeezing A.A. Makarov, Anal. Chem. 2000, 72: 1156-1162. A.A. Makarov, US Pat. 5,886,346, 1999. A.A. Makarov et al., US Pat. 6,872,938, 2005.

Curved Linear Trap (C-trap) for Fast Injection Gate Deflector Push Trap Pull Lenses Orbitrap Ions are stored and cooled in the RF-only C-trap After trapping the RF is ramped down and DC voltages are applied to the rods, creating a field across the trap that ejects along lines converging to the pole of curvature (which coincides with the orbitrap entrance). As ions enter the orbitrap, they are picked up and squeezed by its electric field As the result, ions stay concentrated (within 1 mm 3 ) only for a very short time, so space charge effects do not have time to develop Now we can interface the orbitrap to whatever we want! A.A. Makarov et al., US Pat. 6,872,938, 2005. A. Kholomeev et al., WO05/124821, 2005.

Comparison of Resolving Power as a function of mass for Orbitrap and ICR Dependence of resolving power on m/z for the following analyzers (all data are shown for a 0.76 s scan): (i) standard trap (magnitude mode, 3.5 kv on central electrode), (ii) compact high-field trap (eft, 3.5 kv on central electrode), (iii) FTICR (magnitude mode, 15 T), (iv) FTICR (absorption mode, 15 T). Anal. Chem. 2013, 85, 5288 5296

Hybrid Orbitrap XL

Hybrid Orbitrap Elite

Hybrid Orbitrap Fusion

Highly Parallel Data Acquisition

Parallel Detection in Orbitrap and Linear Ion Trap ControlB3a #4869 RT: 41.56 AV: 1 NL: 7.39E6 T: FTMS + p NSI Full ms [465.00-1600.00] ControlB3a #4870 RT: 41.57 AV: 1 NL: 7.16E3 T: ITMS + c NSI d Full ms2 598.99@cid30.00 [150.00-1810.00] 437.9462 100 95 90 85 80 75 542.7487 70 65 RT: 41.57 MS/MS of m/z 598.6 Scan # 4870 100 95 90 85 80 75 70 65 60 55 600.9776 558.7548 532.2505 804.3450 RT: 41.56 High resolution Full scan # 4869 60 50 55 45 50 40 45 35 40 35 30 25 20 15 10 5 0 590.2733 983.4816 776.4982 623.5060 301.2447 1084.6279 1171.8290 200 400 600 800 1000 1200 1400 1600 1800 m/z 30 25 20 15 10 5 0 649.9460 699.3472 897.9816 716.0311 956.8159 849.8573 974.9185 1116.5020 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 m/z ControlB3a #4871 RT: 41.58 AV: 1 NL: 4.17E3 T: ITMS + c NSI d Full ms2 547.32@cid30.00 [140.00-1655.00] 535.5252 100 95 Relative Abundance 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 234.2242 330.2767 262.1056 200 400 600 800 1000 1200 1400 1600 Relative Abundance 490.3550 450.8616 361.2963 575.8568 690.1100 747.4839 m/z ControlB3a #4872 RT: 41.58 AV: 1 NL: 3.27E3 T: ITMS + c NSI d Full ms2 777.39@cid30.00 [200.00-790.00] 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 RT: 41.58 MS/MS of m/z 547.3 Scan # 4871 900.6165 1022.6853 1088.7388 RT: 41.58 MS/MS of m/z 777.4 Scan # 4872 400.3238 354.2529 683.1174 10 371.1810 309.1429 5 547.4052 512.5754 469.5364 252.0748 0 200 250 300 350 400 450 500 550 600 650 700 750 m/z 592.5975 654.3235 701.4880 729.5197 ControlB3a #4873 RT: 41.59 AV: 1 NL: 1.54E3 T: ITMS + c NSI d Full ms2 974.92@cid30.00 [255.00-1960.00] 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 767.4117 436.2499 393.1896 539.2245 856.3868 965.7724 1092.6033 400 600 800 1000 1200 1400 1600 1800 Relative Abundance m/z 1223.7373 1294.7877 1409.7291 654.2495 757.5266 1801.9797 1513.5245 RT: 41.59 MS/MS of m/z 974.9 Scan # 4873 1674.7556 RT: 41.60 MS/MS of m/z 1116.5 Scan # 4874 ControlB3a #4873 RT: 41.59 AV: 1 NL: 1.54E3 T: ITMS + c NSI d Full ms2 974.92@cid30.00 [255.00-1960.00] 100 95 90 85 80 75 70 65 60 55 50 45 40 35 539.2245 856.3868 1092.6033 1409.7291 Total cycle is 2.4 seconds 1 High resolution scan 5 ion trap MS/MS in parallel 30 25 20 965.7724 1223.7373 1294.7877 15 10 5 0 654.2495 757.5266 1801.9797 1513.5245 436.2499 1674.7556 393.1896 400 600 800 1000 1200 1400 1600 1800 m/z

The One Hour Yeast Proteome* S Alexander S. Hebert **, Alicia L. Richards **, Derek J. Bailey, Arne Ulbrich, Emma E. Coughlin, Michael S. Westphall, and Joshua J. Coon We describe the comprehensive analysis of the yeast proteome in just over one hour of optimized analysis. We achieve this expedited proteome characterization with improved sample preparation, chromatographic separations, and by using a new Orbitrap hybrid mass spectrometer equipped with a mass filter, a collision cell, a highfield Orbitrap analyzer, and, finally, a dual cell linear ion trap analyzer (Q-OT-qIT, Orbitrap Fusion). This system offers high MS 2 acquisition speed of 20 Hz and detects up to 19 peptide sequences within a single second of operation. Over a 1.3 h chromatographic method, the Q-OTqIT hybrid collected an average of 13,447 MS 1 and 80,460 MS 2 scans (per run) to produce 43,400 (x ) peptide spectral matches and 34,255 (x ) peptides with unique amino acid sequences (1% false discovery rate (FDR)). On average, each one hour analysis achieved detection of 3,977 proteins (1% FDR). We conclude that further improvements in mass spectrometer scan rate could render comprehensive analysis of the human proteome within a few hours. Molecular & Cellular Proteomics 13: 10.1074/ mcp.m113.034769, 339 347, 2014. pression levels of each yeast gene using either GFP or TAP tags (9). This seminal work established that 4500 proteins are expressed during log-phase yeast growth. Subsequent mass spectrometry-based studies have confirmed this early estimate (9 12). With this knowledge, we hereby define comprehensive proteome analysis as an experiment that detects 90% of the expressed proteome ( 4000 proteins for yeast). Note others have used the term nearly complete for this purpose; we posit that comprehensive has identical meaning (i.e. including many, most, or all things) (13). Initial MS-based proteomic analyses of yeast, each identifying up to a few hundred proteins, were conducted using a variety of separation and MS technologies (14 16). Yates and co-workers reported the first large-scale yeast proteome study in 2001 with the identification of 1483 proteins following 68 h of mass spectral analysis, i.e. 0.4 proteins were identified per minute (17). Their method two dimensional chromatography coupled with tandem mass spectrometry has provided a template for large-scale protein analysis for the past decade (18 20). By incorporating an offline first dimension of separation with more extensive fractionation (80 versus 15) Gygi et al. expanded on this work in 2003 (21). That January 1, 2014 Molecular & Cellular Proteomics, 13, 339-347.

FIG. 2. Overview of Q-OT-qIT scan cycle. At a retention time of 57.88 min scan #59,211, an MS 1, was acquired and presented several spectral features for MS 2 analysis. Triangles indicate the 22 precursors that were selected for subsequent MS 2 sampling all of which were acquired within 1 s of scan #59,211. 19 of these 22 MS 2 spectra were subsequently mapped to sequence. TABLE I Summary of identification results for the quintuplicate one hour yeast proteome experiments using the Q-OT-qIT mass spectrometer. Note SGD stems from the Saccharomyces Genome Database (www.yeastgenome.org) Experiment PSMs Peptides Proteins SGD verified SGD un-characterized SGD dubious 1 43,423 34,535 4002 3630 337 8 2 43,622 34,495 3966 3608 331 7 3 42,339 33,450 3959 3595 334 3 4 43,326 34,347 3968 3602 337 8 5 43,343 34,449 3991 3623 341 4 Total 216,256 47,624 4395 3976 381 16 January 1, 2014 Molecular & Cellular Proteomics, 13, 339-347.

Rate of protein identifications as a function of mass spectrometer scan rate for selected large-scale yeast proteome analyses over the past decade. Each data point is annotated with the year, corresponding author, type of MS system used, and reference number. January 1, 2014 Molecular & Cellular Proteomics, 13, 339-347.