Ion Mobility Mass Spectrometry Fundamentals What is it

Ion Mobility Mass Spectrometry: Fundamentals What is it? Why use it? Hannah Florance Field Applications Specialist LCMS LMN Spring Workshop 26 April 2019 1 September 10, 2020 Title Confidentiality label Regulatory statement (if applicable)

Drift (Time Resolved) Ion Mobility – A Brief History… Clemmer, Jarrold Applications to clusters & biomolecules 2014 Agilent 2006 Mason-Schamp Equation 1950’s Mass Spectrograph Aston & Thomson 1919 1905 IM-QTOF 1997 1965 Mc. Daniel & Mason Transport of Ions in Gases Clemmer, Smith Drift Tube – with Analytical Merit Ion mobility theory 300 200 Paul Langevin 100 0 1872 - 1946 Anal. Chem. , 2015, 87 (3), pp 1422– 1436 Agilent Restricted # Journal Publications/ year

Ion Mobility – Commercial and Custom Instrumentation Drift Tube (DTIM) Travelling Wave (TWIMS) Trapped (TIMS) High-Field Asymmetric Waveform (FAIMS) 4 September 10, 2020 Title Confidentiality label Regulatory statement (if applicable)

Ion Mobility - Gas Phase Separation of Ions Common Uses • Portable (In the Field) – Drugs and Explosives – Explosive screening (Airports/ Military) • Mass Spectrometer (Research) – Proteomics – Protein Conformations/ Structure – Pesticides and Clinical – Metabolomics/ Lipidomcs • Controlled Temperature and Pressure 5 September 10, 2020 Title Confidentiality label Regulatory statement (if applicable) Anal. Chem. 2014, 86, 2107 • Atmospheric Pressure

Ion Mobility – Gas Phase Separation of Ions • Size (mass) • Shape (structure) • Charge • Mobility through a carrier buffer gas Increasing Surface Area Increasing Interaction with Buffer Gas Arrival Time Distribution (ATD) td (ms) 6 September 10, 2020 Title Confidentiality label Regulatory statement (if applicable)

Ion Mobility – Collisional Cross Section td (ms) CCS Ω (CCS) Å2 7 September 10, 2020 Title Confidentiality label Regulatory statement (if applicable)

Ion Mobility – What does it bring to the table? https: //mcleanresearchgroup. shin yapps. io/CCS-Compendium/ o Orthogonal separation o Reduced noise o More features (? ) 8 September 10, 2020 o Structural Information o Multiple conformations (proteins/peptides/glycans) o Separate isomers o Distinguish between isobars o Unique identifying characteristic o RT, m/z, CCS o Aid to metabolite classification Angew. Chem. Int. Ed. 2017, 56, 8342 – 8349 Title Confidentiality label Regulatory statement (if applicable)

https: //labs. chem. ucsb. edu/bowers/micha el/theory_analysis/ionmobility/index. shtml Current Opinion in Chemical Biology 2018, 42: 51– 59 9 September 10, 2020 Title Confidentiality label Regulatory statement (if applicable)

Basic Principles of DTIM What are we measuring? 10 September 10, 2020 Title Confidentiality label Regulatory statement (if applicable) Drag Force/ Fluid Friction

Basic Operational Principle of Ion Mobility For Conventional DC Uniform Field IMS Analyte Ions Ion Mobility Cell VH VL Detector Gating Optics Electric Field Stacked ring ion guide gives linear field 2015

Low Field Drift Cell 60 msec Analyte Ions VH VL Ion Mobility Cell Detector Gating Optics tdrift t 0 Agilent Restricted

Basic Principles of DTIM Measuring mobility, K VH ΔV VL t=td t=0 Length, L 13 September 10, 2020 Title Confidentiality label Regulatory statement (if applicable)

Basic Principles of DTIM Influential Parameters Reduced Mobility to correct for collision frequency Temperature and Pressure where 14 September 10, 2020 Title Confidentiality label Regulatory statement (if applicable) (perfect gas)

Basic Principles of DTIM Influential Parameters Reaching the detector ΔV td t=0 t. A +t 0 Length, L 15 September 10, 2020 Title Confidentiality label Regulatory statement (if applicable)

Basic Principles of DTIM y = c + mx Influential Parameters Reaching the Detector ΔV t. A td t=0 t. A +t 0 Length, L 16 September 10, 2020 Title Confidentiality label Regulatory statement (if applicable) t 0

Basic Principles of DTIM Drift Time to Ω (Momentum Transfer Collision Integral) Charge on ion Elementary charge Buffer gas density no. 17 September 10, 2020 Title Confidentiality label Regulatory statement (if applicable) Observed Temp, K Boltzmann Constant

Basic Principles of DTIM Calculating the Collisional Cross Section Electric field Elementary charge Ion charge state Corrected drift time Boltzmann Constant Temp, K No. density of drift gas Mass of ion Mass of drift gas Drift gas pressure Drift tube length 18 September 10, 2020 Title Confidentiality label Regulatory statement (if applicable)

DTIM - Challenges Practical challenges: Theoretical challenges: o Increasing the resolution o longer tube o higher p o higher ΔV o alternative gas o Widely used algorithms such as Mob. Cal use He o o o 19 Gas purity with no net gas flow Measuring p inside the tube Measuring T inside the tube Determining the tube length L Measuring the actual voltages o ΔV applied September 10, 2020 o Hard Spheres o Met. CCS/ Lipid. CCS o Calc using chemical info o no structural information o Account for polarizability of gas o Trajectory method for other gases (N 2) o Density Function Theory Title Confidentiality label Regulatory statement (if applicable)

DTIM - Gases Nitrogen Helium Anal. Chem. 2014, 86, 2107− 2116 20 September 10, 2020 Title Confidentiality label Regulatory statement (if applicable)

DTIM - Gases Ubiquitin o Native (N) state (charge states 6+ to 8+) o Unfolded A-state (charge states 8+ to 11+) o PSA theoretical cross section Anal. Chem. 2015, 87, 7196− 7203 21 September 10, 2020 Title Confidentiality label Regulatory statement (if applicable)

DTIM - Gases o Agilent tune mix o Alprenolol o Ondansetron o Clozapine N-oxide o Colchicine o Verapamil o Reserpine Analyst, 2015, 140, 6834– 6844 22 September 10, 2020 Title Confidentiality label Regulatory statement (if applicable)

Drift Tube Ion Mobility Mass Spectrometer Front Trapping Funnel 23 September 10, 2020 Drift Tube Rear Funnel Title Confidentiality label Regulatory statement (if applicable)

Ion Mobility – Why use it? Applications 24 September 10, 2020 Title Confidentiality label Regulatory statement (if applicable)

Ion Mobility – Collisional Cross Section https: //mcleanresearchgroup. shin yapps. io/CCS-Compendium/ o Orthogonal separation o Reduced noise 25 September 10, 2020 o Structural Information o Multiple conformations (proteins/peptides/glycans) o Separate isomers o Distinguish between isobars o Unique identifying characteristic o RT, m/z, CCS o Aid to metabolite classification Angew. Chem. Int. Ed. 2017, 56, 8342 – 8349 Title Confidentiality label Regulatory statement (if applicable)

Orthogonal Separation Signal Response y ilit am r g to Chromatographic Minutes Seconds Ion Mobility ~5 -100 milliseconds 1 -2 milliseconds Time of Flight Mass Spectra ~150 microseconds nanoseconds Peak Width Mass Spectrum a om r Ch Full Time Scale ob M n o I Separation Agilent Restricted

Compound Classification using CCS Anal. Chem. 2014, 86, 2107− 2116 27 September 10, 2020 Title Confidentiality label Regulatory statement (if applicable)

Applications - Isobars © Agilent Technologies, Inc. , 2015 5991 -5723 EN 28 September 10, 2020 Title Confidentiality label Regulatory statement (if applicable)

Applications - Isomers Citrate (C 6 H 8 O 7) 29 September 10, 2020 Isocitrate (C 6 H 8 O 7) Title Confidentiality label Regulatory statement (if applicable) © Agilent Technologies, Inc. , 2015 5991 -5723 EN

Applications – Lipid Classification o 456 Singly charged lipids o 7 classes o 2 categories Phosphatidylethanolamines 30 September 10, 2020 Title Confidentiality label Regulatory statement (if applicable) Nature Communications, Vol. 10, 985 (2019)

Applications – Lipid Classification Nature Communications, Vol. 10, 985 (2019) 31 September 10, 2020 Title Confidentiality label Regulatory statement (if applicable)

Round-Up Ion Mobility – Why use it? • Orthogonal Separation for Hyphenated MS techniques – Improved S/N – Increased numbers of metabolites – Separation isomers/ isobars • Provides an extra dimension of information for identifying known and unknown metabolites – Method for classifying compounds 32 September 10, 2020 Title Confidentiality label Regulatory statement (if applicable)