Advantages of Ion Mobility QTOF for Characterization of Large Molecules

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1 Advantages of Ion Mobility QTOF for Characterization of Large Molecules Add a New Dimension to your Research Capability with Agilent s New Drift Ion Mobility QTOF System Ning Tang, Ph.D. Application Scientist Anne Blackwell, Ph.D. Application Scientist 1

2 IM-QTOF Instrument Overview System sensitivity optimized using electrodynamic ion funnels to focus and transmit ions Ion Mobility resolution optimized while maintaining QTOF performance (mass resolution and accuracy) Ion Fragmentation can be selected using standard QTOF collision cell (CID) Bandwidth of QTOF data acquisition and processing channel was increased by 10 fold to match the ion mobility data rates 2

3 Ion Mobility System Design Ionization source: Ion generation (ESI, AJS, Nano ESI, ChipCube, APCI etc.) Front ion funnel: Efficient ion collection, desolvation and excess gas removal Trap funnel: Ion accumulation and introducing ion packets into drift cell Drift cell: Uniform low field ion mobility allows direct determination of accurate CCS (Ω) Rear funnel: Efficient ion refocusing and introduction into mass analyzer 3

4 Basic Operational Principle of Ion Mobility For Conventional DC Uniform Field IMS V H Ion Mobility Cell V L Analyte Ions Detector Gating Optics Electric Field Stacked ring ion guide gives linear field v = K E e E P T Ω 4

5 Resolution Is Important! Chromatographic Ion Mobility Mass ~seconds ~60 milli-seconds ~ 100 m seconds 5

6 Ion Mobility Resolution IMS Resolution R = t d t d = LEQ 16kTln

7 Ion Mobility Resolution - Continued Resolution = 84!

8 It s All About Separation Chromatography Ion Mobility Mass ~seconds ~60 milli-seconds ~ 100 m seconds 8

9 It s All About Separation Chromatography Ion Mobility Mass Peak Capacity = IM Resolution x Mass Resolution x Orthogonality Peak Capacity = 60 x 40,000 x 14% = 336,600 ~ 8-fold increase Dwivedi P, Schultz AJ, Hill HH, Metabolic profiling of human blood by high-resolution ion mobility mass spectrometry (IM-MS). Int J Mass Spectrom 298:

10 Separation of Isobaric Pesticides Theoretical Plot Aldicarb-sulfone (C 7 H 14 N 2 O 4 S) [M+Na] + = Acetamiprid (C 10 H 11 ClN 4 ) [M+Na] + = D mass is 0.2 mda requires ~ 2,000,000 resolution! IMS Drift Separation 4 x x Acetamiprid Aldicarb-sulfone Drift Time (ms) x10 4 +IMS DriftSpec (m/z: ) (rt: min) Aldicarbsulfone_A 5.5 * * Drift Time (ms) 10

11 Resolving Structural Sugar Isomers C 18 H 32 O 16 Raffinose Melezitose Resolving two isobaric tri-saccharides 11

12 Mobility Drift Time (ms) Crude Bacterial Extract Mass (Da) Mass (Da) siamycin II Ion Mobility Drift Time (ms) 12

13 IMS-MS for Proteomics: Transmembrane Spanning Peptides Drift Time (ms) vs. m/z Drift Time (ms) vs. m/z Characteristics: Large hydrophobic peptides X-ray: 7-helical domains Confirmation impacts function of membrane Membrane proteins are drug targets Working Hypothesis: α-helical membrane spanning domains stay intact, and differ in drift-time from denatured peptides Drift Time (ms) vs. m/z 13

14 IMS-MS for Proteomics: Transmembrane Spanning Peptides A B Drift Time (ms) vs. m/z Drift Time (ms) vs. m/z C D Drift Time (ms) vs. m/z Drift Time (ms) vs. m/z Figure Seq. Loc. Chg. State Helix A 1-40 [M+3H] 3+ A B 1-82 [M+6H] 6+ A-B C [M+5H] 5+ C-D-E D [M+4H] 4+ F-G 14

15 LC Ion Mobility QTOF Protein Isoform Analysis Cry34AB Drift Time (ms) vs. m/z

16 Resolving Isoforms of IgG2 16

17 Resolving Isoforms of IgG Drift Time (ms) vs. m/z x IMS Drift Spectrum ( ms) Counts vs. Drift Time (ms) Publication Paul Schnier, Synapt: 3uM direct infusion in 160 mm NH 4 Ac Drift Time (ms) vs. m/z 17

18 Why Ion Mobility? Separation! Protein Digest, Erin Baker, PNNL 18

19 Lipid Analysis: Mixture of L-α-phosphotidylethanolamine (PE) Lipids Ion Mobility Drift Time (ms) PE 92:N PE 90:N +1 lipids PE 19:N PE 48:N PE 46:N PE 44:N PE 40:N PE 38:N PE 37:N PE 36:N PE 34:N PE 33:N PE 29:N PE 63:N PE 61:N PE 59:N +2 lipids +3 lipids +4 lipids PE 38:8 PE 38:6 PE 38:4 PE 38:2 PE 38:0 PE 38:7 PE 38:5 PE 38:3 PE 38:1 PE 37:1 19

20 Mobility Drift Time (ms) Why Ion Mobility? Specificity! Integrated Mass Spectrum: Mobility-Filtered Mass Spectrum: Mass (Da) Mass (Da) Crude bacterial extract, John McLean, Vanderbilt Mass (Da) 20

21 single pulse 1 ms trapping single pulse 4 ms trapping multi-pulse 4x1 ms trapping single pulse 8 ms trap time multi-pulse 8x1 ms trapping Area Response Signal intensity (A.U.) Why Ion Mobility? Sensitivity! Linear Dynamic Range y = 59060x R² = E+06 5E+06 4E+06 3E+06 2E+06 1E Sample Amount (pg) 0E+00 Sensitivity: ~ 50 fg of Reserpine Dynamic range: ~ 3-4 orders Integrated signal intensity for tetrakis decyl ammonium bromide ion versus ion trapping time for single pulse and multi-pulse experiments. These data indicate that for the same amount of trapping time, multiplexing experiments result in at least 10X higher signal intensity possibly due to less space charge effects and detector saturation issues. 21

22 Why Ion Mobility? Selectivity! Data browser can be used to isolate a group of glycans from matrix Isolation of RNAseB native Glycans, Cathy Costello, Boston University 22

23 Why Ion Mobility? Selectivity! RNaseB native glycans Simplified view of RNaseB native glycan Glycan obscured by matrix is identified after cleaning up background chemical noise using ion mobility 23

24 Ion Mobility of Polymeric Ink Dispersants 24

25 Determining Cross Sectional Areas Charge state of the analyte ion Charge on an electron Electric field Ω = (18π)1/2 16 ze [ ] 1 (kbt) 1/2 2 t E d m i m B L 760 P T N Boltzmann constant Reduced mass of the ion and neutral Number density of the drift gas K 0 = 273 K T P 760 Torr L 2 Vt d Molecular size K 0 = Reduced Ion Mobility T = Temperature P = Pressure L = Drift length V = Voltage Drop across drift region t d = Drift time X-ray crystallography Ion mobility (using Helium) 25

26 Standard Procedure for Calculating CCS (Auto Charting, Curve Fitting and Calculating the CCS) Time segments with different drift fields 26

27 Collision Cross Section Benchmark --- Vanderbilt University Mobility Drift Time (ms) TAA-5 N-(CH 2 CH 2 CH 2 CH 2 CH 3 ) 4 Tetraalkylammonium salts (TAA) Proposed as an ideal ion mobility standard Wide CCS range (TAA-4 to TAA-18; 100 to 400 Å 2 ) TAA salts do not form clusters Literature CCS values exist N 2 drift gas TAA-8 TAA-7 TAA-6 TAA-5 TAA-4 TAA-10 TAA-12 TAA-16 TAA ions +2 ions +3 ions Mass-to-Charge (m/z)

28 Tetraalkylammonium Salts --- CCS Values Compared to Literature Analyte Measured Cross-Section [Å 2 ] TAA ± 0.5% TAA ± 0.6% TAA ± 0.3% TAA ± 0.2% TAA ± 0.1% TAA ± 0.1% TAA ± 0.2% TAA ± 0.2% TAA ± 0.3% Literature Cross-Section [Å 2 ] ± 0.3% ± 0.1% ± 0.3% ± 0.2% ± 0.4% Relative Standard Deviation [%] High experimental precision (< 0.5% relative deviation) Agreement with literature (most < 0.5% deviation) 28

29 Cross Section Calculation of Ubiquitin Charge States m/z 612.8, [M+14H] 14+ m/z 659.8, [M+13H] 13+ Automated collision cross section calculation without the use of calibration curves approximate region for compact structures approximate region for elongated structures m/z 714.7, [M+12H] 12+ m/z 779.6, [M+11H] 11+ m/z 857.5, [M+10H] 10+ m/z 952.6, [M+9H] 9+ m/z , [M+8H] 8+ m/z , [M+7H] 7+ m/z , [M+6H] 6+ m/z , [M+5H] (Å) Reference: Koeniger and Clemmer J Am Soc Mass Spectrom 2007, 18,

30 Cross Section Calculation of Ubiquitin Charge States Ion Charge State CCS experimental (Å 2 ) [M+5H] [M+6H] , 1658 CCS literature (Å 2 ) [M+7H] , [M+8H] [M+9H] [M+10H] , [M+11H] , [M+12H] , [M+13H] , [M+14H] , 2726 Automated collision cross section calculation without the use of calibration curves Reference: Bush et al., Anal Chem. 2010, 82,

31 Carbohydrates -- Great complexity by linkage Source: Blixt et al., PNAS, 2004 Current dominant strategies: MS n or Library searches 31

32 Carbohydrates IM-MS Ion Mobility Drift Time (ms) Lacto-N-difucohexaose I 60 Mixture of Lacto-N-difucohexaose I & II Fuc Fuc Gal 50 GlcNAc Gal Glc Lacto-N-difucohexaose 40 II Mass (Da) 30 Fuc Fuc Gal GlcNAc Gal Glc Lacto-N difucohexaose I Drift Time (ms) 10 Lacto-Ndifucohexaose II Mass (Da) Drift Time (ms) 32

33 IM Drift Spectrum on Cytochrome C at various RFs: S1: Native S2-S5: Denatured S3 Waters Synapt G2 IM data RF 90V RF 110V RF 130V RF 150V RF 180V S1? S2 S4 S5 Preserve protein native structures better! Due to the much lower ions heating. 33

34 IMS-MS for Proteomics: Transmembrane Spanning Peptides of HeLa digest All Ions Fragmentation Low dt High dt Low dt Drift Time (ms) vs. m/z Drift Time (ms) vs. m/z High dt Low dt 34

35 A Word About Instrument Design LC Drift IMS MS and MS/MS High Resolution Accurate Mass Feature Agilent Waters Drift Mobility Advantage Mobility Resolution Highest (can be > 80) 80cm drift tube (L) higher voltage (E) No RF fields, Uniform low DC field Generally around 30 25cm drift TriWave, Multi-section device RF fields Over 2X the IM resolution of T-wave Sensitivity High efficiency ion funnels - trapping and rear Step wave lens Pressure barrier between Q and TriWave 10X to 50X better than T-wave Collision Cross Section (CCS) measurement (Ω) Direct determination of Ω Low electric field and constant drift tube pressure Ω cannot be directly determined from drift time. Need calibration tables. 1-2% precision Much better than Synapt (5-10%) Molecular structures Lower RF fields, less ion heating. Higher RF fields, tendency for higher fragmentation and ion heating Lower RF allows preservation of molecular structures LC Q IMS MS High Resolution Accurate Mass 35

36 Summary Next generation of IM Q-TOF Technology Added dimension of separation based on size, charge and molecular conformation Resolve and characterize the complex samples -- Increased peak capacity Direct determination collision cross sections Preservation of molecular structures 36

37 Acknowledgments Ruwan Kurulugama Alex Mordehai Mark Werlich Chris Klein Tom Knotts Ed Darland Gregor Overney Bill Barry Nathan Sanders Lane Howard Mikhail Ugarov Gene Wong Yergeny Kaplun George Stafford Bill Frazier Bruce Wang Huy Bui Crystal Cody John Fjelsted Ken Imatani Robert Kincaid Robin Scheiderer Collaborators: PNNL NIH Texas A&M Vanderbilt University Boston University 37

38 38

39 Drift time (ms) Standard Procedure for Calculating CCS (Drift Time versus P/V Curves) Colchicine Reserpine Ondansetron t D = experimental drift time t 0 = time ions spend out side the drift cell t d = corrected drift time y = 8,019x R² = y = 6258x Ondansetron R² = y = 5,410x R² = Reserpine Colchicine P/V (Torr/V) Compound Drift time (ms) Slope Intercept t 0 (ms) Ondansetron Colchicine Reserpine

40 Standard Procedure for Calculating CCS - Auto Stepping the Low Field Gradient Time segments with different drift fields 40

41 Collision Cross Section (Å 2 ) Conformational Space Occupancy of Biomolecules Hypothetical Ordering of Biomolecular Classes lipids peptides carbohydrates oligonucleotides Mass (Da) 41

42 Ion Mobility System Design Uniform DC electric field (no RF voltage applied) Desolvation chamber at 0 V ~1700 V on chamber Ion source maintained at ground potential (no voltage offset) Uniform low static electric field across drift cell 80 cm long, approximately 20 V/cm with 4 Torr Nitrogen buffer gas Mobility resolution approaches theoretical limit Minimizes ion excitation or heating (helps to maintain ion structures and conformations) Uniform low field ion mobility allows direct determination of accurate CCS (Ω) 42

43 Ion Mobility Hardware Design Drift Cell Uniform field drift cell 80 cm Front funnel Trapping funnel Drift Cell (80 cm) Rear funnel Ion funnel technology improves sensitivity 43