Ionization Methods. Ionization Methods

Transcription

1 Ionization Methods Árpád Somogyi Associate Director CCIC, Mass Spectrometry and Proteomics Laboratory OSU August 17, 2015 Ionization Methods Neutral species Charged species Removal/addition of electron(s) M + e - (M +. )* + 2e - electron ionization Removal/addition of proton(s) M + (Matrix)-H MH + + (Matrix) - chemical ionization (CI) atmospheric pressure CI (APCI) fast atom bombardment (AB) Electrospray/nanospray ionization (nesi) matrix assisted laser desorption/ionization (MALDI) desorption electrospray ionization (DESI) direct analysis in real time (DART) native spray 1

2 Electron Impact (EI) Ionization Still widely used in orensic Environmental Drug Metabolism, etc. 2

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4 Internal Energy Distribution P(E) and k(e) Curves Direct bond cleavage Rearrangement RRK (simplified) k(e) = (1 E 0 /E) s-1 7 4

5 Chemical Ionization Ion-molecule reaction(s) between a reagent gas and the sample at a relatively high pressure Most common reagent gases methane, isobutane, ammonia Mechanisms CH 4 + e - CH 4 +., CH 3+, CH 2 +., CH 4+ + CH 4 CH 5+ + CH 3 CH 3+ + CH 4 C 2 H 5+ + H 2 CH 5+ + M [M+H] + + CH 4 C 2 H M [M-H] + + C 2 H 6 C 2 H M [M+ C 2 H 5 ] + 5

6 Protonation is one type of ionization M + AH+ MH+ + A CH3CH2NH2 + (NH3)nNH4+ CH3CH2NH3+ + (n-1) NH3 The extent of fragmentation depends on the exothermicity of the reaction Proton affinity (PA): M + H + MH + -ΔH r = PA Proton affinity (PA): M + H+ MH+ - ΔHr = PA PAs of common CI reagents (kcal/mol) methane (131) < water (173) < methanol (185) < CH2=C(CH3)2 (197) < ammonia (205) If the analyte has a much higher PA than that of the unprotonated reagent, the protonation of the analyte will be (very) energetic (fragment rich CI spectra, semi-ci ) 6

7 Sources still being developed DESI- desorption ESI (sample not in solution) 7

8 DART DART Direct Analysis in Real Time Penning Ionization M* + S S+. + M + electron (but also allows MH+, M-H-, etc) 8

9 Can we teach elephants to fly? John enn Yes, of course!!! Nobel Price in Chemistry, 2002 John B. enn Koichi Tanaka 9

10 Electrospray Ionization ESI Soft Laser Desorption SLD Matrix Assited Laser Desorption/Ionization MALDI Matrix-Assisted Laser Desorption\ Ionization Pulsed Laser Extraction Grid to TO Desorbed plume of matrix and analyte ions Analyte Sample Plate Matrix Cation (H +, Na + etc) MATRIX - A small acidic organic molecule (matrix) is mixed with a low concentration of analyte in a common solvent and allowed to co-crystallize on a sample plate to form a solid solution by which the analyte molecules are isolated from each other. ASSISTED matrix assists the desorption and ionization of the analyte(s) LASER Typically a Nitrogen laser (351 nm) or Yag/Nd laser (334 nm) DESORPTION Energy from the laser desorbs the matrix into the gas-phase and carries the analyte with it. IONIZATON - Detect [M+H]+ by transferring a proton from the matrix to the analyte Choose a matrix based on the molecular weight, solubility and chemical structure of the analyte. Excellent for intact molecular weight determination for both polar and non-polar molecules with mass > 500 amu. 10

11 MALDI Matrices Matrix Abbrev Sample Type 2,5-dihydroxybenzoic acid DHB Peptides < 5,000 polymers, dedrimers Good universal matrix cold matrix 3,5-dimethoxy-4-hydroxycinnamic acid (Sinapinic acid) SA Peptides and Proteins > 10,000 hot matrix -cyano-4-hydroxycinnamic acid HCCA Excellent for peptides, digestion products and proteins 50% ACN in 0.1% TA, TH, 2:1 chloroform:meoh 50-70% ACN in 0.1% TA 50% ACN in 0.1% TA Dithranol Non-polar polymers TH, Methylene chloride Indoleacrylic acid IAA Non-polar polymers TH, methylene chloride 3-hydroxypicolinic acid HPA DNA and negative ion samples Trihydroxyacetophenone THAP DNA and negative ion samples See me for more specific procedure See me for more specific procedure Nor-harmane Universal 50% ACN, TH, chloroform 11

12 The MALDI Plate with Different Samples in Different Matrices 2 GHz time digital converter 1000 Hz laser (acquisition of 1000 spectra in 1 second 12

13 MALDI-TO spectrum of a pure protein (linear mode) [M+H] Intensity [M+2H] [2M+H] m/z a.i. Exercise 2 This is a MALDI-TO spectrum of two proteins. Identify the proteins by molecular weights and assign the labeled ions m/z /export/home/tof/data/mslab/091705/pro_6/1lin/pdata/1 unknown Thu Oct 6 14:27:

14 Intens. [a.u.] MALDI-TO spectrum (protein profile) of a bacteria m/z Molecular weight distribution defines polydispersity of polymers Zhu, H.; Yalcin, T.; Li, L. JASMS, 1998, 9, Polystyrene with Ag + PD 1.01 Polydispersity (PD) = M w /M n Intens. [a.u.] x * NB_89_M4\0_M16\1\1SRef, "Baseline subt." Trancated Vectra Polymer (Somogyi et. al., unpublished) PD > m/z 14

15 MS is an absolute method for MW determination (but remember possible degradation!) Zhu, H.; Yalcin, T.; Li, L. JASMS, 1998, 9, Problems with polymer analysis Sample preparation Several polymers are not well soluble in conventional solvents Solventless technique Cationizing with Na +, K +, and Ag + by spiking Synthesis of tailor (home) made matrices Degradation during ionization (especially with MALDI) Photodissociation in MALDI (MALDI/ESI comparison desirable) End group analysis reliable but better to have high resolution/accurate mass (T-ICR) 15

16 Some Examples for Tailor-Made Matrices HO N N M-1 COOH HO M-2 COOCH 3 N M-3 N HO N N M-4 OH HO M-5 OH M-6 M-7 COOCH 3 HO N N M-8 COOCH 3 H 3 CO N N M-9 OCH 3 Somogyi, et al., Macromolecules, 2007, 40, Repeating unit masses of polymers Polystyrene (PS) C 8 H 8 ( ) Poly(ethylene terephtalate) (PET) C 10 H 8 O 4 ( ) Poly(methyl methacrylate) (PET) C 5 H 8 O 2 ( ) Poly(vinyl alcohol) (PVA) C 2 H 4 O ( ) - CH2-CH2-O- Poly(ethylene glycol) (PEG) 16

17 Synthetic Polymer Analysis by MS (MALDI-TO) Intens. [a.u.] Intens. [a.u.] m/z m/z CH 3 OH exact mass: u 17

18 Electrospray Ionization (ESI) Metal Capillary 3-5 kv + + used silica Columbic Evaporation Explosion Critical Radius Charge Repulsion > Surface Tension An electric field on the capillary produces a spray of fine charged droplets The presence of a drying gas (Nitrogen) and heat evaporates off the solvent leaving a distribution of multiply charged de-solvated ions. Soft ionization In-line compatible with HPLC 18

19 or any peak j charges away Z 1 = j(p 2 M a ) p 1 p 2 Z 1 = 1( ) ( ) Z 1 = round up to 11 Therefore, p 1 has 11 charges (protons) Pick as p 2 Pick as p 1 Calculate intact molecular weight M r = p 1 z 1 M a z 1 M r = ( * 11) (1 * 11) M r = (Actual for Cytochrome C is ESI-MS of Myoglobin 100 A17 A16 A15 A14 A13 A12 A18 A11 % A19 A20 A21 A m/z 100 A % m/z deconvolutes to mass 19

20 Isotope distribution/charge state by ultrahigh resolution MS E. Nikolaev et al., Initial Characterization of a New Ultrahigh Resolution TICR Cell with Dynamic Harmonization, JASMS, 2011, 22, E. Nikolaev and coworkers, Twelve Million Resolving Power on 4.7T T-ICR Instrument with Dynamically Harmonized Cell Observation of ine Structure In Peptide Mass Spectra, JASMS, 2014, 25, Native (nano)spray - mimic biological environments - prevent unfolding - keep protein complexes intact - narrower charge distributions than with ESI 20

21 (a) Denatured urease was electrosprayed from an aqueous 50% (vol/vol) acetonitrile containing 0.1 % (vol/vol) formic acid solution revealing individual charge distributions from the multiply charged (26.6 kda, orange) and (61.7 kda, magenta) monomers of urease. (b) A mass spectrum of native urease electrosprayed from an aqueous ammonium acetate solution (bottom) displaying multiple ion signals that originate from multiple charged species of the α 12 β 12 intact urease machinery with a measured mass of 1, kda. Insets are close-ups of the indicated regions. The cartoons are adapted from the X-ray structure of the intact α 12 β 12 urease. Pinkse, M.W., Maier, C.S., Kim, J.I., Oh, B.H. & Heck, A.J. Macromolecular assembly of Helicobacter pylori urease investigated by mass spectrometry. J. Mass Spectrom. 38, (2003). Article PubMed ChemPort ESI vs MALDI Choose MALDI when: Peptides or protein with a MW < 5000 Da (MALDI can detect MW > 100KDa, but not very accurately) Complex mixtures (more than 5 compounds) Very little material with higher salt/buffer concentration Choose ESI when: MW > 5000 Da (proteins) Want better mass assignment Want good MS/MS data 21

22 Dual ionization mode on the apex-qe Apollo II with MALDI Option 22