Tandem MS = MS / MS. ESI-MS give information on the mass of a molecule but none on the structure

Transcription

1 Tandem MS = MS / MS ESI-MS give information on the mass of a molecule but none on the structure In tandem MS (MSMS) (pseudo-)molecular ions are selected in MS1 and fragmented by collision with gas. collision induced decay CID electron transfer decay ETD (= ECD) The fragment ions are analyzed in a second MS.

2 Quadrupole ESI ion source Entrance optics Mass analyzer Q 0 Q 1 detector MS Separation of primary ions

3 ESI ion source Entrance optics Triple Quadrupole Collision cell Q 2 Q 0 Q 1 Q 3 detector MS ion transfer just a longer flight path, nothing gained Separation of primary ions MS / MS tandem MS Precursor ion selection Difference: Collision energy Separation of fragment ions

4 ESI ion source HPLC Entrance optics Target analysis by MS-MS Collision cell Q 2 Q 0 Q 1 Q 3 detector MS / MS Precursor ion selection In LC-MS: mass selected for compounds eluting in a certain time window Analysis of fragment ions

5 ESI ion source HPLC Entrance optics Target analysis by MS-MS Collision cell Q 2 Q 0 Q 1 Q 3 detector MS / MS Precursor ion selection - fixed in time window Example: dexamethazon m/z 393 Transitions Reaction monitoring SRM / MRM Detection of specific fragment ions Only m/z 147 Or: 121,147,237

6 Tandem MS = MS / MS Target analysis: mass of analyte and mass of fragments are known beforehand. MS1 and MS2 are preset on target masses maximum dwell time, maximum sensitivity In proteomics applicationsnothing is known. Precursor mass is determined by survey scan Precursor mass is selected by operator (off-line) or PC (on-line; data-dependent acquisiton ) according to abundance, charge state and additional information

7 Tandem MS = MS / MS Precursor Ion scan: Fragment masses indicate structural details e.g. 365 reveals glycopeptides Neutral loss scan: Loss of a certain mass by removal of chemical group, e.g. 18 by H 2 O Loss of 98 indicates phosphorylation Requirement for proteomics applications: Resolution of multiply charged isotope clusters, high accuracy of MSMS Q-TOF, ion trap, IT-ICR

8 Hybrid-instruments: Quadrupole-TOF (Q-TOF) entrance lenses MS1 quadrupole Collision cell octapole MS2 TOF rotary vacuum pumps rough pumps turbomolecular pumps for high vacuum inside instrument PC for control and data aquisition Server for databank searches N 2 -generator(and oil-free compressor) Argon (collision gas) Waters Synapt II: R in V-mode: R in W-mode: Bruker Maxis 4G: up to TOF as MS2 allows higher resolution, accuracy and upper mass limit.

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10 Hybrid-instruments with Orbitrap analyzers Combination of ion trap and Orbitrap analyzer Newest option: Combination of quadrupole with Orbitrap analyzer

11 Applications of MS-MS Hybrid instruments or Trap: Exact mass analysis of unknown compounds over a wide mass. Typical application: peptide identification by MS-MS structural analysis of biochemicals > fast "scan" rate of TOF or Trap

12 Q-TOF in MS Mode entrance lenses MS1 octapole MS2 Primary ions are collected and sent to MS1

13 Q-TOF in MS Mode MS1 MS2 MS1 does not filter, all ions pass through

14 Q-TOF in MS Mode MS1 MS2 collision cell is inactiv (ions are slow) ions pass unaltered

15 Q-TOF in MS Mode MS1 MS2 TOF analyses primary ions

16 Q-TOF in MS/MS Mode entrance lenses MS1 octapole MS2 Primary ions are collected and sent to MS1

17 Q-TOF in MS/MS Mode entrance lenses MS1 Collision cell MS2 MS1 selects parent ionof a certain mass (m/z); Others cannot pass

18 Q-TOF in MS/MS Mode entrance lenses MS1 Collision cell MS2 Collision with gas atoms (e.g.ar) causes fragmentation of ions (collission induced dissocation = CID) Collision energy is controlled by kinetic energy of the analyte ions.

19 Q-TOF in MS/MS Mode entrance lenses MS1 Collision cell MS2 Daughter ions leave the collision cell

20 Q-TOF in MS/MS Mode entrance lenses MS1 Collision cell MS2 MS2 (TOF) analyses fragment ions

21 Proteomics work with ESI-MS/MS De novo sequencing of a peptide of mass from a wasp venom allergen vulgaris PLA MSMS vulgaris_pla_msms MaxEnt 3 68 [Ev4631,It50,En1] (0.040,200.00,0.060, ,2,Cmp) MSMS ES+ AVI Y M A E C IK KI C E A M Y I V A bmax ymax % b y3 y y5 2+ [MH 2] y M/z y y theoret. [MH] AVIYMAECLK + VIYMAECLK IYMAECLK + + YMAECLK + MAECLK + AECLK + ECLK + CLK + LK Doubly charged precursor singly charged fragments

22 100 % Proteomics work with ESI-MS/MS mass 100 % FC+H Y G A C W T T D I S MSMS ES+ 200 M exp : TOF MSMS ES mass I FCISIDTTWCAGYCYTR Doubly charged precursor singly charged fragments

23 Peptide fragmentation Major fragments derived from a peptide (protonated) y 3 y 2 y H + 2 H + 2 H R 1 R 2 R 3 R 4 H N C C N C C N C C N C COOH 2 H O H H O H H O H H a 1 b 1 a 2 b 2 a 3 b 3 Doubly charged precursor singly charged fragments

24 Fragmentation of a singly charged peptide y 6 y 5 y 4 y 2 y 1 NH 3 R 1 R 2 R 3 R 4 R 5 R 6 R H 2 N C C N C C N C C N C C N C C N C C N C COOH O H O H O O H O O H H H H H H H H H H y 3 NH 2 R 1 R 2 R 3 R 4 R 5 R 6 R H 2 N C C N C C N C C N C C N C C N C C N C COOH O H O H O O H O O H H H H H H H H H H H R 1 R 2 H 2 N C C N C C N C C H O H H O H neutral b-fragment R 3 O H H NH 2 R 4 R 5 R 6 R N C C N C C N C C N C COOH H O H H O H O H H H H y 4 -ion

25 Fragmentation of a doubly charged peptide NH 3 R 1 R 2 R 3 R 4 R 5 R 6 R H 3 N C C N C C N C C N C C N C C N C C N C COOH O H O H O O H O O H H H H H H H H H H NH 2 R 1 R 2 R 3 R 4 R 5 R 6 R H 3 N C C N C C N C C N C C N C C N C C N C COOH O H O H O O H O O H H H H H H H H H H H R 1 R 2 H 3 N C C N C C N C C H O H H O H Doubly charged precursor singly charged fragments b 3 -ion R 3 O H H NH 2 R 4 R 5 R 6 R N C C N C C N C C N C COOH H O H H O H O H H H H y 3 -ion peptide mass + H

26 Peptide fragmentation R 1 R 2 H 3 N C C N C C N C C H O H H O H R 3 R 1 R 2 R 3 O H 2 N C C N C C N C C O b 3 -ions peptide mass -17 O O H H H H H acylium ion R 1 R 2 R 3 H 2 N C C N C C N C O O H H H H H a 3 -ion peptide mass -45 R 3 H 2 N C H immonium ion reveal amino acids in peptide

27 Collision energy Collision induced or collison activated dissociation of parent ions (CID or CAD) Triple quads, ion traps, Q-Tofs and similar mass specs can onlyprovide low energy(ev range) fragmentations. can be modulated within certain range (adjusted to mass of peptide) Yields relatively simple fragment spectra. Disadvantage: Leu and Ile cannot be discriminated High energy CID in BE or TOF-TOF instruments.

28 Applications of nano-lc / MS-MS MS BSA 100fmol, Time= min (#339#341), 100%=159692arb MS/MS y5 y6 y9 PBC m/z , all MS b3 y2 y3 y4 b7 b8 y7 b9 y10 b11 b m/z all MS/MS Time [min] 100 fmol BSA injected on column

29 Protein identification by LC / MS-MS Archeal histon Result of the database search of silver stained protein gel spot. Archeal histon was unambiguously found in a Halobacterium salinarum (genome) database using MASCOT TM. Important software packages for protein identification: MASCOT, GPM, SEQUEST. and company derivatives e.g. MassLynx, Proteome Discoverer ProteinScape..

30 Data-dependant aquisition (DDA) At first, the machine works in the MS mode (survey mode) until mass is detected that is: - of sufficient intensity - not in exclude list (background, trypsin, keratin) - doubly or triply charged (-is in include list ) Then, machine switches into MS/MS mode to acquire CID spectrum of this compoundfor e.g. ca. 1 sec Then, this mass is locked for some time to prevent redundancy. Often, the survey mode detects more than one signal MSMS 1, MSMS 2, etc. before switched back to survey.

31 MS/MS specials I.) Dependancy of ion type and collision energy - the larger the more energy required - charges help fragmentation - careful choice of collision energy profile II.) DDA tends to overlook many peptides Solutions: - increase speed of instrument - optimize selection criteria - rerun sample with inclusion list and/or exclusion list Quality criterion: % of identifiable peptide spectra Notorious problem: hybrid spectra (precursor selection not rigorous)

32 MS/MS specials Precursor selection problem turned upside down: MS/MS ALL (in segments: SWATH)

33 MS/MS specials Searching for peptide fragment (and mass) in data banks e.g. Swissprot By MASCOT or SEQUEST and related tools yields list of hits with probability The score depends on: 1.) number of peptides fitting to a particular peptide one hit wonders usually disregarded 2.) number of fragments fitting to theoretical digest of peptide (also: those NOT fitting) 3.) size of peptide (the larger, the better) 4.) size of protein (the smaller the better) 5.) allowance of missed cleavage sites 6.) allowance of modifications the more the worse (search space!) 7.) size of databank (too small is bad!)

34 Sample inlet systems for ESI Syringe pump 5 to 50 µl / min System test Calibration Simple samples Liquid chromatography 50 to 1000 µl / min Most typical LC-MS applications (pharmaceutical, environmental, forensic etc.)

35 Sample inlet systems for ESI Nanospray tips 20 nl /min 40 mm For limited sample amounts in bioscience 1-2 µl of sample give 30 min of analysis time Nanoflow LC 100 to 1000 nl / min For demanding life science applications split

36 Sample inlet systems for ESI Column i.d. Flow rate Technique 4.0 mm 1.0 ml/min Conventional HPLC 2.0 mm 0.25 ml/min Small bore LC 1.0 mm 0,0625 ml/min Micro LC 300 µm 5.6 µl/min Capillary LC 180 µm 2.0 µl/min Capillary LC 75 µm 350 nl/min Nano LC

37 Sample inlet systems for ESI S/N= mm i.d. S/N = ml/min Signal to Noise ratio 75 µm i.d.

38 Sample inlet systems for ESI 4.6 mm i.d 1.0 mm i.d. 300 µm i.d. 75 µm i.d. UV 206 nm ESI has a similar, even stronger concentration dependance 2 pmol digested myoglobin (each column)

39 Chromatographiesäulen im Vergleich 4 mm 0.18 mm 75 µm

40 Nano-Elektrospray

41 Nano-Elektrospray Diameter 1 mm Diameter 0.32 mm Diameter mm Diameter 4 mm, Flow rate 1.6 ml / min Flow rates?