Methods in Mass Spectrometry Dr. Noam Tal Laboratory of Mass Spectrometry School of Chemistry, Tel Aviv University
Sample Engineering Chemistry Biology Life Science Medicine Industry IDF / Police
Sample Mixture Pure Organic/Biologic Reaction Extract (Organism) Complex mixture Known MW Unknown MW Screening or Separation Preparation: Centrifugation Extraction Filtration Confirmation Analysis Mode: POS/NEG/Both Ionization Method(s) Conditions Additives
Ionization Method 1000000 100000 MALDI ESI 10000 1000 100 APPI APCI EI/CI Non Polar Polarity Ionic
TAU MS LAB Autospec: EI, CI, (GC), FAB SYNAPT: ESI, APCI, APPI, (LC), MALDI TQD: ESI, APCI, MRM (LC)
What is a Mass Spectrometer? Mass spectrometer measures the ratio between mass and charge of Ions m/z structural Information Identification Quantification Very fast High Sensitivity (Subpicomole) Samples: HPLC grade chemicals - Tissue, polluted waters, Blood Mass range: from proton to protein Resolution: M/DM 10 3-10 6 Elemental composition Very expansive.
What is a Mass Spectrometer? Source Analyzer Detector Vacuum pump Source Ionization M o M +, M -, [M-H] -, [M+H] + Analyzer - Mass Separation - Resolution - Mass Range Detector - Ion counter -SNR, -Sensitivity
Ionization Methods EI CI FAB MALDI ESI APPI APCI
Above 3000 Da Sample 100-3000 Da No MALDI No APCI No ESI Yes APPI ~15% Yes ~80% EI/CI ~1% ~4%
Electron Ionization (EI) M o + e - (70 ev) M + (~5eV) +2e - (~65eV) Fragment Ions Neutral Fragments M o + e - (70 ev) M - (only ~1%)
Electron Ionization (EI)
Electron Ionization (EI) Advantages Sensitivity: Picomole Database: ~100,000 compounds Fragments: Structure + Fingerprints GC, Analytical chemistry Disadvantages Mass range: ~1000 Da Fragments: Decomposition
Chemical Ionization (CI) CH 4 +e - CH 4 + + 2e- CH 4 + CH 3 + + H CH 4 + + CH 4 CH 5 + + CH 3 CH 5 + + M MH +
Chemical Ionization (CI) Advantages Less Fragmentation Suitable for fragile molecules Disadvantages No library Mass range ~700 Da Less Sensitive
Fast Atom Bombardment (FAB) Liquid Secondary Ion Mass Spectrometry CI: CH 5 + + M MH + FAB: Matrix + M MH + Matrix + M [M-H] - O 2 N NBA Glycerole HOH 2 C CH 2 OH OH C CH 2 OH H TEA N(CH 2 CH 2 OH) 3
Fast Atom Bombardment (FAB) Advantages Mass range ~7000 Da Soft Ionization Massive Cluster Impact Disadvantages Less Sensitive Only for Polar compounds
Matrix Assisted Laser Desorption Ionization (MALDI) Matrix + Energy + M [MH] +, [M-H] -
3,4-Dihydroxycinnamic acid Matrix Assisted Laser Desorption Ionization (MALDI) α-cyano-4 -hydroxycinnamic acid Dithranol trans-3-indoleacrylic acid 2,5-Dihydroxybenzoic acid Sample plate 3,4-Dihydroxycinnamic acid 9-Aminoacridine Tetraoctylammonium bromide
Matrix Assisted Laser Desorption Ionization (MALDI) FAB MALDI Matrix: liquid Solid Energy: Atoms/ions LASER Mass Range: 70,000 Da 300,000Da Sensitivity: Low Femptomol Background: yes <1000 Da
Electro Spray Ionization (ESI)
Electro Spray Ionization (ESI) Polar compounds in MeOH / H 2 O / MeCN Sensitive: Femptomole Multiple Charge Mass Range: ~100,000 Da Proteomics Atmospheric Pressure Interface: Direct / HPLC Soft Ionization: Biology, Proteomics, Non covalent compounds
Atm. Pressure Chemical Ionization (APCI) Inlet Nebulizing Gas Vaporizer (Heater) Corona Discharge Needle To MS
Atm. Pressure Chemical Ionization (APCI) Less polar compounds Sensitivity: Pos >> Neg Single charge Volatile compounds Mass range: ~1500 Da Atmospheric Pressure Interface: Direct / HPLC Soft Ionization
Atm. Pressure Photo Ionization (APPI) Inlet Nebulizing Gas Vaporizer (Heater) UV Lamp ~ 10 ev To MS Direct APPI Dopant/Solvent assisted APPI D = Photosensitizer: toluene, Acetone
Atm. Pressure photo Ionization (APPI) Conjugated compounds Organometallic Complexes Conditions: Solvent. Flow rate, Temp, Dopant
APCI APPI Sensitivity: Pos >> Neg Pos ~= Neg Mass range ~1200 Da ~2500 Da Aliphatic: Yes Limited Conjugated: Yes Excellent Organometallic: Limited Excellent Conditions: Sensitive less sensitive Solvent: Toluene, DCM, Hexane, MeCN, MeOH
Analyzer Source Analyzer Detector Analyzer: separate Ions Mass range Resolution: The Ability of a Mass Spectrometer to separate two masses (M1, M2) R=M/DM FWHM = full width at half maximum
Analyzer Magnetic sector Quadrupole TOF Q-TOF
Magnetic Sector Analyzer Optical Prism Magnetic sector
Magnetic Sector Analyzer m z B 2 V * K
Quadropole Combination of DC and RF on the rods Ions moving into the Quad oscillate depending on m/z Only one m/z can pass A mass spectrum can be obtained by scanning the RF
Time Of Flight (TOF) E p zev v t L L t E 1 2 k mv m 2zeV 2 Sample Sample Pusher Detector Detector m z 2 t 2eVL 2 Reflectron
Time Of Flight (TOF)
Q-TOF
MS Analysis MW Elemental composition (HRMS) Charge state Isotope pattern Adducts POS vs NEG Fragmentation
MS MH + Erythromycin C 37 H 67 NO 13 = 733 gr/mol
Resolution Mass Accuracy HRMS
Elemental composition (HRMS)
MS Analysis Carbon Isotope 12 C 12.00000 98.9% 13 C 13.00335 1.1%
ESI
ESI z=3 z=2 z=1 0.33Da 0.50Da 1.0 Da
ESI
ESI [MNa] + [2MNa] +
ESI C 138 H 177 BN 6 O 79
Protein Analysis ESI
ESI Human Insulin C 257 H 383 N 65 O 77 S 6 5807.57
ESI Horse myoglobin 16,951.49
ESI C 32 H 36 Fe 2 N 10 O 2 Na
ESI C 18 H 21 Br 4 N 2 O 2
ESI RuC 20 H 16 N 4 Cl
ESI C 35 H 30 BO 9
ESI C 30 H 34 NFPd
ESI
ESI [MH] + [MNa] +
LCMS Liquid Chromatography - Mass Spectrometry HPLC UPLC MS UV Detector MS Detector Sample Column Autosampler Pump Solvents
LCMS UV NEG POS
LCMS NEG POS
LCMS
LCMS APCI APPI Water extract In MeCN ESI
APPI 9-cis-b-carotene C 40 H 56
APPI
APPI
APPI [M-H+Na] + C 32 H 48 PtP 2 F 6 [M-F] +
Pt Complex APPI
APPI [M-Cl] + [M] +
APPI
APPI
Ref APPI
APCI 300 o C
APCI
APCI
FAB Insulin C 254 H 377 N 65 O 75 S 6 5733.49
FAB
FAB Cyanocobalamin - B 12 C 63 H 88 CoN 14 O 14 P =1355 [M-H] -
MALDI
MALDI C 168 H 162 O 16 Na
MALDI C 36 H 36 N 24 O 12
MALDI
MALDI PEG
MALDI Cyanobacteria
MALDI
Tandem Mass Spectrometry Source Analyzer Detector Energy Source Analyzer 1 Analyzer 2 Detector Gentle ionization do not produce a significant amount of fragment ion Fragments are Important for identification and structure study. Tandem MS Induce fragmentation by collision with gas molecules (Argon)
MS/MS Collision Energy
Source MS/MS Analyzer 1 Collision cell Analyzer 2 Filter Fragmentation Scanner M1 or Filter M2 M2 F1, F2, F3 Fn F3 M3 Detector Compound Fragments
Source MRM Multiple Reaction Monitoring Analyzer 1 Collision cell Analyzer 2 Parent 500 (A) 500 (B) Daughter 406 (20) 322 (100) 158 (56) 478 (100) 188 (37) Detector Parent (500) (A) or (B) Fragments
MRM MRM of Toxin 250 pgr 1 ngr 2.5 ngr 100 ngr
MRM 2 ngr ESI-LCMS/POS/ MRM Toxin 50 ngr ESI-LCMS/POS/ MRM Toxin ESI-LCMS/POS/SCAN 100-2000
Summary Ionization Method MS/ HRMS Isotope pattern Adducts Fragmentation MRM Conditions Solvent, additives, temp, flow, potential etc.