Methods in Mass Spectrometry. Dr. Noam Tal Laboratory of Mass Spectrometry School of Chemistry, Tel Aviv University

Transcription

1 Methods in Mass Spectrometry Dr. Noam Tal Laboratory of Mass Spectrometry School of Chemistry, Tel Aviv University

2 Sample Engineering Chemistry Biology Life Science Medicine Industry IDF / Police

3 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

4 Ionization Method MALDI ESI APPI APCI EI/CI Non Polar Polarity Ionic

5 TAU MS LAB Autospec: EI, CI, (GC), FAB SYNAPT: ESI, APCI, APPI, (LC), MALDI TQD: ESI, APCI, MRM (LC)

6 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 Elemental composition Very expansive.

7 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

8 Ionization Methods EI CI FAB MALDI ESI APPI APCI

9 Above 3000 Da Sample Da No MALDI No APCI No ESI Yes APPI ~15% Yes ~80% EI/CI ~1% ~4%

10 Electron Ionization (EI) M o + e - (70 ev) M + (~5eV) +2e - (~65eV) Fragment Ions Neutral Fragments M o + e - (70 ev) M - (only ~1%)

11 Electron Ionization (EI)

12 Electron Ionization (EI) Advantages Sensitivity: Picomole Database: ~100,000 compounds Fragments: Structure + Fingerprints GC, Analytical chemistry Disadvantages Mass range: ~1000 Da Fragments: Decomposition

13 Chemical Ionization (CI) CH 4 +e - CH e- CH 4 + CH H CH CH 4 CH CH 3 CH M MH +

14 Chemical Ionization (CI) Advantages Less Fragmentation Suitable for fragile molecules Disadvantages No library Mass range ~700 Da Less Sensitive

15 Fast Atom Bombardment (FAB) Liquid Secondary Ion Mass Spectrometry CI: CH 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

16 Fast Atom Bombardment (FAB) Advantages Mass range ~7000 Da Soft Ionization Massive Cluster Impact Disadvantages Less Sensitive Only for Polar compounds

17 Matrix Assisted Laser Desorption Ionization (MALDI) Matrix + Energy + M [MH] +, [M-H] -

18 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

19 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

20 Electro Spray Ionization (ESI)

21 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

22 Atm. Pressure Chemical Ionization (APCI) Inlet Nebulizing Gas Vaporizer (Heater) Corona Discharge Needle To MS

23 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

24 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

25 Atm. Pressure photo Ionization (APPI) Conjugated compounds Organometallic Complexes Conditions: Solvent. Flow rate, Temp, Dopant

26 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

27 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

28 Analyzer Magnetic sector Quadrupole TOF Q-TOF

29 Magnetic Sector Analyzer Optical Prism Magnetic sector

30 Magnetic Sector Analyzer m z B 2 V * K

31 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

32 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

33 Time Of Flight (TOF)

34 Q-TOF

35 MS Analysis MW Elemental composition (HRMS) Charge state Isotope pattern Adducts POS vs NEG Fragmentation

36 MS MH + Erythromycin C 37 H 67 NO 13 = 733 gr/mol

37 Resolution Mass Accuracy HRMS

38 Elemental composition (HRMS)

39 MS Analysis Carbon Isotope 12 C % 13 C %

40 ESI

41 ESI z=3 z=2 z=1 0.33Da 0.50Da 1.0 Da

42 ESI

43 ESI [MNa] + [2MNa] +

44 ESI C 138 H 177 BN 6 O 79

45 Protein Analysis ESI

46 ESI Human Insulin C 257 H 383 N 65 O 77 S

47 ESI Horse myoglobin 16,951.49

48 ESI C 32 H 36 Fe 2 N 10 O 2 Na

49 ESI C 18 H 21 Br 4 N 2 O 2

50 ESI RuC 20 H 16 N 4 Cl

51 ESI C 35 H 30 BO 9

52 ESI C 30 H 34 NFPd

53 ESI

54 ESI [MH] + [MNa] +

55 LCMS Liquid Chromatography - Mass Spectrometry HPLC UPLC MS UV Detector MS Detector Sample Column Autosampler Pump Solvents

56 LCMS UV NEG POS

57 LCMS NEG POS

58 LCMS

59 LCMS APCI APPI Water extract In MeCN ESI

60 APPI 9-cis-b-carotene C 40 H 56

61 APPI

62 APPI

63 APPI [M-H+Na] + C 32 H 48 PtP 2 F 6 [M-F] +

64 Pt Complex APPI

65 APPI [M-Cl] + [M] +

66 APPI

67 APPI

68 Ref APPI

69 APCI 300 o C

70 APCI

71 APCI

72 FAB Insulin C 254 H 377 N 65 O 75 S

73 FAB

74 FAB Cyanocobalamin - B 12 C 63 H 88 CoN 14 O 14 P =1355 [M-H] -

75 MALDI

76 MALDI C 168 H 162 O 16 Na

77 MALDI C 36 H 36 N 24 O 12

78 MALDI

79 MALDI PEG

80 MALDI Cyanobacteria

81 MALDI

82 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)

83 MS/MS Collision Energy

84 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

85 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

86 MRM MRM of Toxin 250 pgr 1 ngr 2.5 ngr 100 ngr

87 MRM 2 ngr ESI-LCMS/POS/ MRM Toxin 50 ngr ESI-LCMS/POS/ MRM Toxin ESI-LCMS/POS/SCAN

88 Summary Ionization Method MS/ HRMS Isotope pattern Adducts Fragmentation MRM Conditions Solvent, additives, temp, flow, potential etc.