Chemical Aspects of Mass Spectrometry Abraham Badu Chemistry and Biochemistry, OSU July 12, 217 Spread of MS by Discipline https://masspec.scripps.edu/ mass spectrometry 2 1
Current Challenges in Mass Spectrometry? Quantitation Ionization Mass Range Ion Activation Resolution Cost Sample Preparation 3 Current Challenges in Mass Spectrometry Quantitation MS really good for answering what is there, but not how much is there Ionization Contributes to quantitative challenges Itself a challenge if (i) analyte cannot be charged effectively or (ii) integrity of analytes changed during ionization Mass Range Molecular weight info cannot be missing in MS Ion Activation Structural info that reflects that native state of the analyte Resolution Cost No single instrument/approach can solve all challenges Cost grows rapidly as we try combine different MS instruments 4 2
Electrospray-based Ionization In ESI, analyte solution undergoes three major processes before gas-phase ion production: (a) Production of charged droplets (b) Solvent evaporation and droplet disintegration resulting in smaller charged droplets (c) Mechanism by which the gas-phase ion is formed 5 Electrospray-based Ionization 6 3
Electrospray-based Ionization Why use such as a high voltage (5 kv) in Electrospray Ionization? How much time do analytes spend in charged droplet? 7 Different molecules compete for 1. Charge proton affinity 2. Space hydrophobicity Xin, Bian and Cooks Angew. Chem. Int. Ed. 216, 55,1296 8 4
Ion Suppression [DDAH] 186 CORTISONE 361 m/z Equimolar mixture: dodecyl amine (DDA) and cortisone mixture separation ionization Removing Ion Suppression 9 time Contained-Electrospray Ionization N 2 kv cavity Reaction Mixture headspace vapor Droplets velocities:.2 2 mm/s Discontinuous formation of liquid films:.1 1 s mixing times in cavity 1 5
Re-configurable Outlet (A) Contained-ES Apparatus (B) Different Operation Modes N 2 (i) TYPE I Reaction Mixture kv cavity N 2 (ii) TYPE II Cavity (1 cm) headspace vapor N 2 11 Conventional ESI Removing Ion Suppression Reactive ESI 9 -THC OPGP 12 6
Relative Abundance Relative Abundance 7/11/217 Removing Ion Suppression Conventional ESI methamphetamine 1 methamphetamine 15 5 Benzoylecgonine 29 315 Δ9-THC Benzoylecgonine 15 25 35 45 Reactive-ESI 1 29 315 15 trans-δ 9 Tetrahydrocannabinol (Δ9-THC) equimolar mixture 5 15 25 35 45 m/z 13 Removing Ion Suppression Ion Intensity in contained-esi MS reflects analytes composition in solution GT: Cortisone (mole ratio) Girard T (GT) Reagent 1:2 2:1 Cortisone (M) 3:1 14 7
Relative Abundance 7/11/217 Quantitative Electrospray Ionization? Ion Intensity in contained-esi MS reflects analytes composition in solution 15 Protein Analysis - Myoglobin 1 Myoglobin in pure water 1% Holo 9 8 Heme-containing protein Binds to oxygen in the muscle tissue of vertebrates 1 21 19 17 23 15 Myoglobin in 1% acetic acid 1% Apo Very sensitive to ph changes 25 13 Heme group is lost with 1-3 s upon contact with acid 6 1 14 18 22 26 m/z 16 8
Relative Abundance Relative Abundance 7/11/217 Analytes and Reagents Analytes Mass (kda) pi N 2 Myoglobin 17. (17.6) 7.4 Ubiquitin 8.57 5.2 Carbonic Anhydrase II 29.1 5.9 Cytochrome C 12.4 1.4 Protein Solution kv cavity Reagent pka Vapor pressure (mm Hg) Acid Vapor HCl <1 114.8 Acetic Acid 4.76 11.7 Formic Acid 3.75 44.8 17 17 Detecting Native State Myoglobin in High Abundance 1 9 8 Acetic Acid High pka 9 8 Low vapor pressure Modest effect 18 9
Normalized Intensity 7/11/217 Detecting Highly Charged Intact Myoglobin No Acid Relative Abundance Relative Abundance 1 HCl vapor, Type I 22 1 24 26 m/z 2 13 18 11 6 1 14 18 m/z 9 8 9 Relative Abundance 1 Myoglobin in 1% acetic acid 1 21 19 17 23 15 25 13 6 1 14 18 22 26 m/z 22 6 65 7 75 8 85 9 95 1 m/z Holo (69%) Apo (31%) HCl Lower pka High vapor pressure Fast droplet modification Protein unfolds faster than the heme cofactor can escape (~1 ms in solution) 19 Ion Mobility Unfolded Distribution Folded Distribution 1% 8% 6% ESI in Pure H2O 4% ESI in 1% Acetic Acid 2% % 1 2 3 4 5 6 7 8 9 1 Drift Time (ms) 2 1
Normalized Intensity Normalized Intensity 7/11/217 Ion Mobility Unfolded Distribution Folded Distribution 1% 8% 6% 4% ESI in Pure H2O ESI in 1% Acetic Acid Type I Mode, HCl Vapor 2% % 1 2 3 4 5 6 7 8 9 1 Drift Time (ms) 21 Ion Mobility Unfolded Distribution Folded Distribution 1% 8% 6% 4% 2% ESI in Pure H2O ESI in 1% Acetic Acid Type I Mode, HCl Vapor Type II Mode, HCl Vapor % 1 2 3 4 5 6 7 8 9 1 Drift Time (ms) 22 11
Normalized Intensity Normalized Intensity 7/11/217 Specific Charge States 1% 22 Charge State 8% 6% 4% 2% %.5 1 1.5 2 2.5 3 3.5 4 Drift Time (ms) 1% 2 Charge State 8% 6% 4% 2% %.5 1 1.5 2 2.5 3 3.5 4 Drift Time (ms) ESI in 1% acetic acid Type I Mode with HCl Vapor Type II Mode with HCl Vapor 23 Ubiquitin C-terminus N-terminus Lysine 48 Lysine 63 Small tightly folded protein (76 residues) Very resistant to denaturation (up to ph 2.3 water) Condition for denaturation: MeOH/H 2 O (1:1) at ph 2.3 13 basic sites Katta and Chait J. Am. Chem. Soc. 1993, 115, 6317-6321 24 24 12
Relative Abundance Relative Abundance 7/11/217 Ubiquitin in Water ESI, no acid vapor 8 7 6 9 1 7 acid vapor 8 7 1 8 1 9 13 7 Combination of Type II Operation Mode with Heat Allow Protonation of all 13 Basic sites 25 25 Online Processing of Protein at Atmospheric Pressure Neutral ph Native Protein Ionization Lowly Charged Protein Ion Mobility Collisional Cross Section Intact Protein Mid Rang ph Low ph Slightly basic ph With Cross-linking Reagent Intermediate Protein Denatured Protein X-linked Protein Ionization Ionization Ionization Moderately Charged Protein Highly Charged Protein Charged X-linked Protein Fragment Fragment 26 13
TIC 7/11/217 Type II Mode of Operation N 2 5 psi N 2 5 kv ES-emitter 1.E8 5 psi; 5 kv 5 psi; kv psi; 5 kv 1.E6 5 psi N 2 kv 1.E4 1.E2 psi N 2 5 kv 1.E 1 2 3 4 Time (sec) 27 Expanding the Mass Range of Portable Mass Spectrometers with Chemistry On-demand Protein Biomarker Detection Minimal sample preparation Stable ION with small mass PAPER as inexpensive substrate CELL PHONE for results communication 28 14
Advances in Ionization vacuum vacuum Ion Source Mass Analyzer Detector Ion Source Mass Analyzer Detector Ambient Ionization Methods EXTRACTION Analyte Sampling from their Native Environment without Sample Pre-treatment Mass Analyzer vacuum Detector Some Sample Types Human Tissue Whole Plant Drug Tablet Cooks et. al. Science 24, 36, 471-473; Science 26, 311, 1566 Badu-Tawiah et. al. Annu. Rev. Phys. Chem. 213, 64, 481 55 29 Desorption Electrospray Ionization (DESI) 1 µm 15 m/s 3 15
Advances in Mass Spectrometry Ambient Ionization Methods Analyte Sampling from their Native Environment without Sample Pre-treatment Mass Analyzer vacuum Detector Desorption Electrospray Ionization Low Temperature Plasma Ionization Laser-assisted Ionization Paper Electrospray Mass Spectrometry 32 16
Expanding the Mass Range of Portable Mass Spectrometers with Chemistry On-demand Protein Biomarker Detection Minimal sample preparation Stable ION with small mass PAPER as inexpensive substrate CELL PHONE for results communication 33 Our Approach: Ionic Probes N n O O NCS Charge-tag Cleavable site Conjugate unit 34 17
The Approach: Ionic Probes N Charge-tag O O n Cleavable site Conjugate unit NCS On-demand Analysis: Interruptible Storable Restorable Two Separate End Points Central lab 35 Plastic Cover The Device Plasma Separation Membrane Splitter Water Sensitive Paper Reagent Storage Disease Biomarker Dwell Layer Capture Layer Hydrophobic Barrier Collimating Layer Detection Layer Blotting Paper 36 18
A/IS 7/11/217 Detection of Malaria HRP-2 Antigen Probes LoD in PBS (/zone) LoD in Serum (/zone) n = 3 5 pm (1 fmol) 75 pm n = 1 5 pm (1 fmol) 1 nm WHO Recommended LoD: 2 pm Mean: 2 parasites ul -1 = 28 nm On-chip: 1 pm (2 fmol) Enzyme amplified (ELISA): 5 1 pm (1 fmol) 37 Detection after Storage Paper chips were stored for 3 days before analysis Intensity Ratio of m/z 87 / 59 6 4 2 a) Control Antigen 1 d 3 d 7 d 14 d 3 d Time (day) 38 19
A/IS Optical Density 7/11/217 Detection after Storage Enzyme not active after 2 h of storage under dry conditions Intensity Ratio of m/z 87 / 59 6 4 2 1. a) Control Antigen 1 d b) 3 d 7 d 14 d Time (day) 3 d Tris buffer solution Dry O.D..5. 1 168 Time (h) 39 2 2
The Ohio State University Start-up funding National Science Foundation US Department of Energy BES (basic energy sciences) funding American Society for Mass Spectrometry 21