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Orbitrap Mass Analyzer Dr. Michaela Scigelova Bremen, Germany.

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Presentation on theme: "Orbitrap Mass Analyzer Dr. Michaela Scigelova Bremen, Germany."— Presentation transcript:

1 Orbitrap Mass Analyzer Dr. Michaela Scigelova Bremen, Germany

2 2 z φ r Orbitrap Analyzer – An Electrostatic Trap Ions trapped in an electrostatic field Central electrode kept on high voltage Outer electrode is split and able to pick up an image current induced by ion packets moving inside the trap A. Makarov, Anal. Chem 2000,

3 3 Ion Injection and Formation of Ion Rings (r,φ) (r,z) An ion packet of a selected m/z enters the field Increasing voltage squeezes ions Voltage stabilises and ion trajectories are also stabilized Angular spreading forms a ROTATING RING

4 4 Detection of Ions Ion packets enter the analyzer slightly off axis The field inside the trap effects an oscillation of the ion packets/rings The moving ion rings induce an image current on outer electrodes The frequency of harmonic oscillations is proportional to ions’ m/z

5 5 Fourier Transform Baron Joseph Fourier Mathematical operation transforms frequency signal into a time domain spectrum Orbitrap is a Fourier transform-based mass analyzer Scigelova et al. Mol. Cellular Proteomics 2011, 10: M

6 6 From an Analyzer to a Mass Spectrometer... Detection of ions Fragmentation Ultra-high resolution Accurate mass + + ? ?

7 7 Orbitrap Analyzer - the ‘Heart’ of a Mass Spectrometer Q ExactiveOrbitrap Elite 1.2 x 1.5 x Standard OrbitrapHigh-field Orbitrap

8 8 Resolution – Key Performance Characteristics Res Setting m/z 400 Hz 15, , , , , ,000 Developer’s Kit Q ExactiveOrbitrap Elite Res Setting m/z 400 Hz

9 9 Resolution – Why Is It Important? Enables accurate mass Increases confidence of identification Improves quantitative accuracy Gives access to qualitatively different information More on the topic: N. Cortes-Francisco et al., Accurate mass measurements and ultrahigh-resolution: evaluation of different mass spectrometers for daily routine analysis of small molecules in negative electrospray ionization mode. Anal. Bioanal.Chem. 2011, 400:

10 10 Mass measured Tolerance [Da]SuggestionsCalc Mass 32.0+/- 0.2O 2 CH 3 OH N 2 H 4 S /- 0.02CH 3 OH N 2 H / CH 3 OH C = H = N = O = S = Mass Accuracy – What for? Accurate mass is a powerful filter

11 11 Increases confidence in identification HR/AM Analysis – Identification [M+H] Mass accuracyNumber of hits* 200 ppm ppm ppm39 10 ppm14 5 ppm5 3 ppm4 * Compounds containing CNOH

12 12 HR/AM Analysis - Quantitation Improves quantitative accuracy by effectively removing background Selectivity is built into the high resolution analysis m/z m/z /- 2.5 ppm +/- 7.5 ppm m/z m/z GPCho (18:3/0:0) GPCho (16:0/0:0) + Na Koulman et al., Rapid Commun. Mass Spectrom.2009; 23: 1411–1418 GPCho (18:3/0:0)

13 13 Mass Accuracy across the Elution Profile 21 scans per elution peak External calibration

14 14 HR/AM - Another ‘Dimension’ in your data Reveals fine isotopic structures Michalski et al., MCP 2012, /mcp.O

15 15 Intact Protein Analysis Complete charge state envelope of IgG ‘Humira’ Major glycosylation forms are baseline separated Hao Z., A Complete Workflow Solution for Intact Monoclonal Antibody Characterization Using a New High-Performance Benchtop Quadrupole-Orbitrap LC-MS/MS. Thermo Application Note 2012 Relative intensity reproducibility within a few percent

16 16 Intact Protein Analysis Mass measurement accuracy Average error for 34 measurements 6.9 ppm Standard deviation 6.4 ppm Hao Z., A Complete Workflow Solution for Intact Monoclonal Antibody Characterization Using a New High-Performance Benchtop Quadrupole-Orbitrap LC-MS/MS. Thermo Applicaiton Note 2012 Confirmation of protein primary structure

17 17 Sequence Confirmation of mAB ETD fragmentation of an intact IgG ‘Humira’ Resolution settings 240,000 for fragment detection Increased sequence coverage Localization of modifications (deamidation) Markus Kellmann, Orbitrap Elite data

18 18 Spatially Resolved HDX Structural dynamic studies by measuring backbone amide HDX Problems: 1)back-exchange under quench conditions 2)gas-phase hydrogen scrambling Subzero temperature in the ion source Not compatible with pepsin digestion Perform top-down analysis in HR MS NO gas-phase scrambling with ETD! D assigned to individual residues HR ETD – black circles; NMR – red line Amon et al., Anal. Chem. 2012, 84, 4467

19 19 Extending the mass range Protein assemblies up to 1 million Da IgG antibody 150 kDa HK97 bacteriophage capsomers 253 kDa Yeast proteasome 730 kDa E. coli GroEl 801 kDa Analysis of Protein Complexes

20 20 Ligand Binding Stoichiometry m/z Relative Abundance R= R= R= R= R= R= R= R= R=2252 E. coli GroEl 801 kDa Orbitrap mass spectrometry of native protein complexes up to 1 MDa Rebecca Rose, Eugen Damoc; Eduard Denisov; Alexander Makarov; Albert J.R. Heck: ASMS 2012

21 21 Orbitrap Elite and the Proteome Heat map of peptides eluting over 3 min interval in a 40 Th range Parallelization of data acquisition: a survey scan of 240,000 resolution with 20 CID scans all within a 2.7 s cycle time Michalski et al., MCP 2012, /mcp.O min 40 Th 148,000 peptide clusters x 93,000 peptide clusters

22 22 Data Dependent Acquisition - Issues Run-to-run reproducibility Inability to effectively target peptides of interest CID x CIDETD x ETD Swaney DL, McAlister GC, Coon JJ: Nature Methods 2008, 5,

23 23 Rapid and robust proteome analysis 50 cm column length 4 h gradient 35 o C (nano-UHPLC) Near complete yeast proteome More than 4,000 proteins/run (1% FDR) Median sequence coverage 23% Nagaraj et al.,Systems-wide perturbation analysis with near complete coverage of the yeast proteome by single-shot UHPLC runs on a bench-top Orbitrap. MCP 2011, M Proteome Analysis in a Single LC/MS Run Minimum undersampling

24 24 Data Dependent Decision Tree Swaney DL, McAlister GC, Coon JJ: Nature Methods 2008, 5, Decision tree–driven tandem mass spectrometry for shotgun proteomics

25 25 Product Dependent Trigger: HCD PD ETD ZIC HILIC separation of a glycoprotein digest Singh et al., Higher Energy Collision Dissociation (HCD) Product Ion-Triggered Electron Transfer Dissociation (ETD) Mass Spectrometry for the Analysis of N ‑ Linked Glycoproteins JPR 2012, doi: /pr300257c

26 26 Product Dependent Trigger: HCD PD ETD HCD fragmentation spectrum of m/z Oxonium ions observed among top 20 peaks Singh et al., JPR 2012, doi: /pr300257c

27 27 Product Dependent Trigger: HCD PD ETD ETD fragmentation triggered Peptide sequence information Glycosylation site localization Singh et al., JPR 2012, doi: /pr300257c

28 28 The Newest in Data Dependent Acquisition Instant spectral assignment Algorithm processes tandem mass spectra in real-time Takes ∼ 16 ms to execute Enables autonomous, real-time decision making by the MS system Real-time prediction of peptide elution windows en masse Significant improvement of quantitative precision and accuracy Boosted rates of posttranslational modification site localization Bailey et al, Instant spectral assignment for advanced decision tree-driven mass spectrometry. PNAS 109, 8411–8416, (2012).

29 29 The Newest in Data Dependent Acquisition Posttranslation modification site localization Ambiguous position from HCD Triggered ETD resolving the ambiguity Bailey et al, Instant spectral assignment for advanced decision tree-driven mass spectrometry. PNAS 109, 8411–8416, (2012).

30 30 Summary High resolution is a key characteristics of MS data enabling Mass accuracy Confident identification Reliable quantitation Data dependent acquisition offers an elegant simplicity and has proven highly useful for discovery-driven proteomics Mass spectrometry technology enables comprehensive analysis of proteomics samples Multiple fragmentation techniques MS n capability Quan&Qual experiments done on a single platform


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