MS/MS Scan Modes. Eötvös University, Budapest April 16, MS/MS Scan Modes. Árpád Somogyi. Product Ion Scan Select. Scan. Precursor Ion Scan Scan

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1 MS/MS Modes Árpád Somogyi Eötvös University, Budapest April 16, 2012 MS/MS Modes Product Ion Precursor Ion Neutral Loss Δ ed Reaction Monitoring (SRM) 1

2 modes in a triple quadrupole (QqQ) (one quadrupole shown here) Analyte Mixture Voltage RF DC V m1 V m2 V m3 m/z m1 m/z m2 m/z m3 mass spectrum 2

3 Analyte Mixture Voltage RF DC V m1 V m2 V m Analyte Mixture Voltage RF DC V m1 V m2 V m

4 Analyte Mixture Voltage RF DC V m1 V m2 V m Voltage 200 Analyte Mixture 100 RF DC V m2 Desired Analyte m/z m2 mass spectrum 4

5 Modes of scanning in a Triple Quadrupole (QQQ) scan or select q2 (gas) rf only scan or select Quadrupole is a mass filter QQQ used in this tutorial to describe scan modes and = analyzers q2 (middle quadrupole) used for CID (dissociation) Ways to set quadrupoles:, & rf only Other instruments are used A variety of instruments are used for MS/MS To name a few 5

6 QQQ q2 Benefits: Simple, ion filter Good for quantification Q-TOF q2 TOF Benefits: Higher resolution & mass accuracy All ions recorded in parallel Ref: Chemushevich,

7 Q-Linear Ion Trap (Q-trap) q2 LIT Benefits: Quadrupole-like CID spectra with ion trap sensitivity No ion trap low mass cutoff Ref: Hopfgartner, 2003 LT-Orbitrap (pictured with ETD source) API ion source quadrupole Mass linear filter Ion trap C-trap q2 HCD collision cell reagent Ion source orbitrap Orbi reagent reagent Benefits: LTQ: Ion trap sensitivity Orbi: High dynamic range & high resolution & mass accuracy 7

8 Trapping Instruments q2 Benefits: Sensitivity MS^n (most) MS/MS Modes Product Ion Precursor Ion Neutral Loss Δ ed Reaction Monitoring (SRM) 8

9 Product Ion q2 (gas) Qualitative structural information is used to select one m/z This parent ion is dissociated in Q2 (Rf only) Q2 in Rf only mode is high transmission device Fragments (product ions) are formed by collisions Product ions are scanned through Prerequisite: Produce an MS spectrum for selection Output = MS/MS spectrum Tandem in Space (QQQ) Product Ion Source (gas) Detector one m/z (fixed V ac /V dc ) 9

10 Tandem in Space (QQQ) Product Ion Source (gas) Detector Products (collide with gas) (scan V ac /V dc ) MS select MS/MS MS select MS/MS 10

11 MS/MS of a Peptide (YGGFL, m/z = 556.2) a/b 4 Y G G F L y 3 y 2 b Relative Intensity y 2 y a H 2 O m/z Multiple stages of MS in a trapping instrument MS n of trocade (a drug metabolism study) MS MS 2 MS 3 MS 4 Ref: Hopfgartner,

12 Product Ion s may be Software Controlled Goal: collect MS/MS spectra for complex mixtures Complex mixture can be separated by HPLC HPLC linked directly to analyzer by ESI source Mass analyzer collects continuous MS spectra At pre-determined intensity of a precursor ion, MS/MS spectra acquired Data Dependent acquisition Dynamic Exclusion = exclude repeats Ion Current over 60 min MS/MS MS 12

13 Advantages for product ion scan NOTES QQQ Q-trap Q-TOF TOF-TOF Ion Trap (3D, LT) ICR Q or Trap-ICR LT-Orbitrap Orbitrap Animation 13

14 MS/MS Modes Product Ion Precursor Ion Neutral Loss Δ ed Reaction Monitoring (SRM) Precursor Ion q2 (gas) Screen for precursor ions that produce a given product ion is scanned All precursor ions collide with target gas (in CID) Fragments (product ions) are formed allows transmission of one fragment ion m/z Run as HPLC-MS/MS experiment Prerequisite: Determine expected product ions by MS/MS Output = chromatogram showing time/intensity of precursors of interest and reconstructed spectrum 14

15 Precursor Ion Detection of Source (gas) Detector Precursors (sequential rf/dc) Precursor Ion Detection of Source (gas) Detector rf/dc 1 at rf/dc 1 (collide with gas) 15

16 Precursor Ion Detection of Source (gas) Detector fragment at rf/dc 1 (fixed rf/dc ) Precursor Ion Detection of Source (gas) Detector rf/dc 2 at rf/dc 2 (collide with gas) 16

17 Precursor Ion Detection of Source (gas) Detector fragment at rf/dc 2 (fixed rf/dc ) Precursor Ion Detection of Source (gas) Detector at rf/dc 3 rf/dc 3 (collide with gas) 17

18 Precursor Ion Detection of Source (gas) Detector fragment at rf/dc 3 (fixed rf/dc ) Precursor Ion Spectrum Reconstructed by software Software stores memory of the rf/dc voltages that coincide with fragments striking the detector! rf/dc 3 Relative Intensity rf/dc 2 These rf/dc voltages equal specific m/z values m/z 18

19 Precursor ion result precursor of Coming into NOT DETECTED all ions In mixture (TIC) In, at one rf/dc ratio, m/z = m/z = Precursor ion is Fragmented in q2 fixed to detect m/z = hits the detector Reconstructed chromatogram total ion Current m/z Learning Check: FACT SHEET Precursor Ion Consider identification of a mixture of halogenated compounds by MS/MS Describe a Precursor Ion that might be used to identify all monohalogenated benzenes in a sample What is the m/z that hits the detector? What happens in, q2,? Draw the spectrum F Br I Cl C 12 Cl 35/37 H 1 Br 79/81 F 19 I

20 Learning Check: PROBLEM SOLVER Precursor Ion 1) Calculate the mass of one precursor ion, for example, fluorobenzene 2) Draw a likely fragment ion common to all of these analytes? (assume a simple fragment from M + is formed) 12 = 1 = 19 = Total = 3) Calculate the mass of the common fragment 12 = 1 = Total = Learning Check: PROBLEM SOLVER Precursor Ion 1) Calculate the mass of one 6 12 = 72 precursor ion, for example, 5 1 = 5 fluorobenzene 1 19 = 19 Total = 96 2) Draw a likely fragment ion common to all of these analytes? (assume a simple fragment from M + is formed) H H F.+ H H H 3) Calculate the mass of the common fragment = 1 = Total = 20

21 Learning Check: Precursor Ion 6 12 = = 5 What m/z hits the detector? m/z = 77 H H. + H H H What happens in q2? q2 all ions sequential CID Fix: m/z 77 Cl Br FI / Draw the spectrum Relative Intensity = m/z Precursor Ion : A literature example Combinatorial Chemistry Combinatorial libraries result from the simultaneous synthesis of a great number of compounds. analytical challenge to characterize Purpose: Determine purity and identity of pooled library QQQ mass spectrometer PROBLEM: MS SCAN IS COMPLEX AND PROVIDES LITTLE INFORMATION Triolo,

22 Precursor Ion : A literature example Combinatorial Chemistry The compound components X, Y, Z are not identified mass of X = 299 mass of Y = 40 mass of Z = 100 Library compounds: X-AA 1 -Y-AA 2 -Z Library compounds, example if AA 1 = Arg, AA 2 = Ala: X-Arg-Y-Ala-Z [mass of Arg = 156, Ala = 71] mass: = 666 for mass spectrometry, add 1 proton to form ion: = 667 When AA1 = Arg, a fragment will form, m/z = 455 m/z = 455 X-Arg-Y-AA 2 -Z Triolo, 2001 Learning Check: FACT SHEET Precursor Ion in Combinatorial Chemistry Alanine ALA 71 Arginine ARG 156 Asparagine ASN 114 Aspartic Acid ASP 115 Cystein CYS 103 Glutamic Acid GLU 129 Glutamine GLN 128 Glycine GLY 57 Histidine HIS 137 Isoleucine ILE 113 Leucine LEU 113 Lysine LYS 128 Methionine MET 131 Phenylalanine PHE 147 Proline PRO 97 Serine SER 87 Threonine THR 101 Tryptophan TRP 186 Tyrosine TYR 163 Valine VAL 99 R? X AA1 Y AA2 Z ? 100 Sum ? AA2 + 1 = Precursor Mass Library compounds: X-AA 1 -Y-AA 2 -Z A fragment ion will form for cleavage at this bond when aa 1 = Arginine m/z = 455 X-AA 1 -Y-AA 2 -Z 22

23 Learning Check: precursor scan results Precursor ARG? [M+H]+ X AA1 Y H+ AA2 Z The precursor ion results of this experiment are shown in the left column find the amino acid for each of these compounds Learning Check: FACT SHEET Precursor Ion in Combinatorial Chemistry Consider identification of a mixture of these library compounds by MS/MS Describe a Precursor Ion that might be used to determine that all amino acids are represented in position 2 in the compounds (AA 2 ) if position 1 = Arg What is the m/z that hits the detector? What happens in, q2,? Draw the spectrum Library compounds: X-AA 1 -Y-AA 2 -Z A fragment ion will form for cleavage at this bond when aa 1 = Arginine m/z = 455 X-AA 1 -Y-AA 2 -Z 23

24 Learning Check: Precursor Ion in Combinatorial Chemistry What m/z hits the detector? m/z = 455 X-Arg- + (299) + (156) What happens in, q2,? q2 all ions sequential CID Fix: m/z 455 Draw the spectrum for a few compounds Gly Pro Phe X-Arg-Y-Gly-Z X-Arg-Y-Pro-Z X-Arg-Y-Phe-Z ( = 653) m/z Precursor Ion of m/z 455 of a pooled library MS Precursor Ref: Triolo,

25 Advantages for precursor scan NOTES QQQ Q-trap Q-TOF TOF-TOF Ion Trap (3D, LT) ICR Q or Trap-ICR LT-Orbitrap MS/MS Modes Product Ion Precursor Ion Neutral Loss Δ ed Reaction Monitoring (SRM) 25

26 Neutral Loss q2 (gas) (offset from ) Screen for ions that undergo a common loss and are both scanned is offset by the neutral loss selected The precursor ion collides in q2 forming fragments Compounds providing the selected loss are detected Run as HPLC-MS/MS experiment Prerequisite: Determine expected loss by MS/MS Output = chromatogram showing time/intensity of precursors of interest and reconstructed spectrum Δ Neutral Loss Loss of m/z = Source (gas) Detector Precursors (sequential rf/dc) 26

27 Neutral Loss Loss of m/z = Source (gas) Detector rf/dc 1 (collide with gas) Neutral Loss Loss of m/z = Source (gas) Detector for offset m/z (Offset rf/dc) 27

28 Neutral Loss Loss of m/z = Source (gas) Detector rf/dc 2 (collide with gas) Neutral Loss Loss of m/z = Source (gas) Detector for offset m/z (Offset rf/dc) 28

29 Neutral Loss Loss of m/z = Source (gas) Detector rf/dc 3 (collide with gas) Neutral Loss Loss of m/z = Source (gas) Detector for offset m/z (Offset rf/dc) 29

30 Neutral Loss Spectrum Reconstructed by software Software stores memory of the rf/dc voltages that coincide with fragments striking the detector! Relative Intensity offset rf/dc 2 The rf/dc voltages equals a specific m/z value m/z Learning Check: Neutral Loss Consider identification of a mixture of halogenated compounds by MS/MS Describe a Neutral Loss that might be used to identify all Chlorine containing compounds What is the m/z that hits the detector? What happens in, q2,? Draw the spectrum F Br I Cl C 12 Cl 35/37 H 1 Br 79/81 F 19 I

31 Learning Check: Neutral Loss What m/z hits the detector? What happens in q2? q2 Draw the spectrum Relative Intensity m/z Learning Check: Neutral Loss. + Neutral loss of 35 or 37 H m/z of less 35 H What m/z hits the detector? for example: H chloro-benzene: = 77 What happens in q2? q2 all ions sequential CID scan offset 35 amu Draw the spectrum Relative Intensity Cl m/z H H 31

32 Neutral Loss : A literature example Drug Metabolite Early stages in design of a drug metabolism study Want to Fish out relevant metabolites Metabolites are in human urine after administration of tolcapone tolcapone is a catechol-o-methyl transferase inhibitor Possible metabolite is a glucoronide of tolcapone metabolites are structurally related to parent drug but, product ion spectra may be energy dependent tolcapone Hopfgartner, 2003 Neutral Loss : A literature example Drug Metabolite researchers expect a metabolite that is a glucuronide of tolcapone tolcapone Mass = 273 Expected conjugate: mass 273 = tolcapone 176 = glucuronide add n 449 HO OH O OH HO O tolcapone glucoronide Mass = 449 OH glucuronide conjugates commonly provide mass loss of

33 Neutral Loss : A literature example Q-trap ( = Linear ion trap) Hopfgartner, 2003 Metabolite of tolcapone:lc-ms/ms Analysis of human urine TIC of neutral loss of 176 Da m/z (Neg ion): tolcapone conjugate = 449 loss of H = -1 neutral loss spectrum at t = 5.8 min MS/MS spectrum 30 ev same fragments as MS/MS of tolcapone MS/MS spectrum 50 ev Hopfgartner,

34 Advantages for neutral loss scan NOTES QQQ Q-trap Q-TOF TOF-TOF Ion Trap (3D, LT) ICR Q or Trap-ICR LT-Orbitrap MS/MS Modes Product Ion Precursor Ion Neutral Loss Δ ed Reaction Monitoring (SRM) 34

35 ed Reaction Monitoring (SRM or MRM) q2 (gas) Single (SRM) or Multiple (MRM) reaction monitoring Quantitative target analyte scan is fixed to allow transmission of one precursor m/z This precursor ion collides in q2 forming fragments is fixed to allow transmission of one fragment m/z Run as HPLC-MS/MS experiment Prerequisite: Determine expected product ions by MS/MS Output = chromatogram showing time/intensity of precursors of interest and reconstructed spectrum ed Reaction Monitoring Source (gas) Detector one m/z (fixed V ac /V dc ) 35

36 ed Reaction Monitoring Source (gas) Detector (collide with gas) ed Reaction Monitoring Source (gas) Detector one m/z (Fixed rf/dc) 36

37 Learning Check: ed Ion Monitoring Consider identification of a mixture of halogenated compounds by MS/MS Describe a SRM that might be used to identify fluorobenzene What is the m/z that hits the detector? What happens in, q2,? Draw the spectrum F Br I Cl C 12 Cl 35/37 H 1 Br 79/81 F 19 I 127 Learning Check: ed Ion Monitoring H. + H What m/z hits the detector? m/z = 77 H H H What happens in q2? q2 Fix: m/z 96 CID Fix: m/z 77 F 96 Draw the spectrum Relative Intensity m/z 37

38 MRM example: Detection of an antiviral drug and it s metabolite in human plasma herpes virus replication inhibited by action of acyclovir but low bioavailability valacyclovir metabolizes to acylovir with high bioavailability Goal: accurate detection in plasma QQQ mass spectrometer, MDS SCIEX API-4000 Studied fragmentation of compounds by CID acyclovir (ACV) valacyclovir (VCV) fluconazole (internal std - IS) Ref: Yadav, 2009 Product ion mass spectra VCV 325.2/152.2 ACV 226.2/152.2 IS 307.1/

39 MRM chromatograms VCV & IS in plasma VCV ACV IS blank IS only VCV & IS plasma VCV & IS plasma (subject) Mean pharmacokinetic profile after oral administration of 1000 mg VCV tablet to 41 healthy subjects Ref: Yadav,

40 MRM example: Improve Sensitivity for Corticosteroid Detection Used illegally as growth promoters in cattle Purpose: detect low residue levels in biological matrices QQQ mass spectrometer (QuattroLC, Micromass) Studied fragmentation of corticosteroids by CID Determined negative mode to produce more specific ions Evaluated 3 acquisition methods in negative mode Product ion Neutral loss Multiple reaction monitoring Ref: Antignac, 2000 Improving Sensitivity for Corticosteroid Detection 40

41 Comparison: Product Ion, Neutral Loss, MRM 1 ng Total ion current Chromatograms 100 pg 10 pg Neutral Loss 10X more sensitive than MS/MS MRM 10X more sensitive than N.Loss blank Ref: Antignac, 2000 Improving Sensitivity for Corticosteroid Detection MRM = best method requires setting many transitions for mixture analysis set for multiple [M+acetate]- set for 2 products of each (-60 and -30 from M+acet]- 41

42 MRM chromatograms of mixture of 11 steroids Advantages for selected ion monitoring NOTES QQQ Q-trap Q-TOF TOF-TOF Ion Trap (3D, LT) ICR Q or Trap-ICR LT-Orbitrap 42

43 MS/MS Modes Summary Product Ion Qualitative Structural Information Precursor Ion Screen for compound types that lose a detectable fragment Neutral Loss Screen for compound types that lose a neutral ed Reaction Monitoring (SRM) Identify specific compounds MS/MS Modes Strategy: Phosphorylation of serine, threonine or tyrosine = 79 serine phosphorylation added to serine: = 80 could be lost from serine (as an ion): 79 (PO 3- ) C 12 H 1 O 16 P 31 could be lost from serine (as a neutral): (H 2 O)= 98 threonine tyrosine 43

44 Suggested Reading List & References Precursor Ion and Neutral Loss s Hopfgartner G., Husser C., Zell M.; Rapid Screening and characterization of drug metabolites using a new quadrupole-linear ion trap mass spectrometer, JMS, 2003; 38: Triolo A, Altamura, M., Cardinali, F., Sisto, A., Maggi C., Mass spectrometry and combinatorial chemistry: a short outline, JMS, 2002; 36: Chemushevich, I.V., Loboda A.V., Thomson B.A., An introduction to quadrupole time-of-flight mass spectrometry, JMS, 2001, 36: Multiple Reaction Monitoring Antignac, J.P., Bizec, B.L., Monteau, F., Poulain, F., Andre, F., Collision-induced dissociation of corticosteroids in electrospray tandem mass spectrometry and development of a screening method by high performance liquid chromatography/tandem mass spectrometry, RCMS, 2000, 14, Yadav, M., Upadhyay, V., Singhal, P., Goswami, S., Shhrivastav, P.S., Stability evaluation and sensitive determination of antiviral drug, valacyclovir and its metabolite acyclovir in human plasma by a rapid liquid chromatography tandem mass spectrometry method, J.Chrom.B, 2009, 877(8-9),