1. Orbitrap Mass Analyzer
Dr. Michaela Scigelova
Bremen, Germany

2. 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 z φ r
Then ions move along a strange spiral which has 3 components:
Rotational movement with frequency of rotation
Radial movement with this frequency
Axial oscillations along the central electrode
Only one of them is completely independent on energy and position of ions- that is the axial frequency. Therefore, only this frequency could be used for mass analysis. This is where “ideal Kingdon trap” becomes a mass analyzer- the Orbitrap.
A. Makarov, Anal. Chem 2000, 2

3. Ion Injection and Formation of Ion Rings
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
(r,φ) (r,z)

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

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

7. Orbitrap Analyzer - the ‘Heart’ of a Mass Spectrometer
Q Exactive Orbitrap Elite
1.5 x
The Orbitrap Elite is now the first instrument that uses the new high-field Orbitrap analyzer.
The high-field Orbitrap is actually smaller in size compared to the standard Orbitrap:
The outer electrode diameter shrank by a factor of 1.5 whereas the diameter of the central electrode only shrank by a factor of 1.2, making the central electrode relatively thicker. This arrangement creates a higher field making the ions going forth and back at an almost 2 times higher frequency resulting in an almost 2 times higher resolution.
And, because the little hole where the ions enter the Orbitrap also shrank, an additional lens was implemented to focus the ions more effectively into the Orbitrap, resulting in a higher ion transmission.
1.2 x
Standard Orbitrap
High-field Orbitrap

8. Resolution – Key Performance Characteristics
Q Exactive Orbitrap Elite
Res Setting
m/z 400 Hz
12.500 12
25.000 7
50.000 3
1.5
Res Setting
m/z 400 Hz
15,000
7.7
30,000
6.9
60,000
4.0
120,000
2.3
240,000
1.2
480, Developer’s Kit

9. Resolution – Why Is It Important?
Enables accurate mass
Increases confidence of identification
Improves quantitative accuracy
Gives access to qualitatively different information
Figure shows the detection of two species with a mass difference as small as amu at different resolution settings on the Q Exactive instrument. The two species are not resolved at 35,000 FWHM, which results in a mass shift and an inaccurate detection and quantitation for both species. The two species are separated to 50% valley at 70,000 resolution, and baseline-separated at 140,000 resolution. Therefore, higher resolution (in this case 140,000 FWHM) allowed for more reliable identification and accurate quantitation.
At such high resolution, more than two hundred species can be separated in a unit-mass window. This translates into resolving, in theory, >100,000 species in the mass range of amu at any one point in time. This greatly reduces background interference and enables confident identification and quantitation of the target peptides in the presence of very complex backgrounds.
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. Mass Accuracy – What for?
N =
O =
S =
C =
Mass measured
Tolerance [Da]
Suggestions
Calc Mass
32.0
+/- 0.2 O2
CH3OH
N2H4 S
32.02
+/- 0.02 +/
But here is the interesting thing – only the mass of 12C is a nice round number (12). All other elements are either a tiny bit larger (H, N) or a tiny bit smaller (O, S). They show a so called mass defect. If we can measure with enough accuracy, then this mass defect can effectively exclude some of the compound suggestions.
In ideal case, just a single elemental composition ( a particular combination of selected elements considered within certain limits) will remain.
Mass accuracy is thus a powerful filter. A large number of elemental compositions (peptide candidates) can be excluded just by applying a simple filter; maximum allowed mass deviation.
Accurate mass is a powerful filter

11. HR/AM Analysis – Identification
Increases confidence in identification
[M+H]
Mass accuracy
Number of hits*
200 ppm
265
100 ppm
133
30 ppm 39
10 ppm 14
5 ppm 5
3 ppm 4
340
350
360
370
380
390
400
410
m/z 10 20 30 40 50 60 70 80 90
100
110
120
130
140
Relative Abundance
* Compounds containing CNOH

12. HR/AM Analysis - Quantitation
Improves quantitative accuracy by effectively removing background
Selectivity is built into the high resolution analysis
m/z
+/- 2.5 ppm
m/z
GPCho (18:3/0:0)
High resolution ensuring selectivity in phospholipid analysis. Extracted ion chromatograms for m/z and m/z The top two traces are shown for 2.5 ppm windows, and bottom traces for 7.5 ppm. Only using the 2.5 ppm windows (top 2 traces), the extracted ion chromatogram for m/z is specific for GPCho (18:3/0:0). Increasing the uncertainty allowance yielded the incorporation of other masses. After increasing the ppm window to values >2.5 ppm, it was no longer possible to distinguish two important M+22 species, generated by either sodiation or the addition of two carbons and a further unsaturation site. Data courtesy of Gary Woffendin, Thermo Fisher Scientific.
m/z
+/- 7.5 ppm
GPCho (18:3/0:0)
GPCho (16:0/0:0) + Na
m/z
Koulman et al., Rapid Commun. Mass Spectrom.2009; 23: 1411–1418

13. Mass Accuracy across the Elution Profile
21 scans per elution peak
External calibration
RT:
1.75
1.80
1.85
1.90
1.95
Time (min) 10 20 30 40 50 60 70 80 90
100
Relative Abundance

14. HR/AM - Another ‘Dimension’ in your data
Reveals fine isotopic structures
Separation of isobaric species in standard LC MS/MS analysis. The 34S isotope containing peak is clearly resolved from the
13C2 isotope.
Michalski et al., MCP 2012, /mcp.O

15. Intact Protein Analysis
Complete charge state envelope of IgG ‘Humira’
Major glycosylation forms are baseline separated
Relative intensity reproducibility within a few percent
Five micrograms of mAb were desalted and eluted from a ProSwift™ RP-10R
monolithic column using a 15min gradient and analyzed using ESI-MS on the
Q-Exactive. The was mAb eluted over one minute as shown in (A). The
average spectrum over the elution time shows a nicely distributed complete
charge envelope of the mAb (B). A zoom-in view of each charge state reveals
five major glycosylation forms that are baseline separated (C).
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

16. Intact Protein Analysis
Mass measurement accuracy
Average error for 34 measurements 6.9 ppm
Standard deviation 6.4 ppm
mass deviations from expected target masses
for the 5 most abundant glycoforms
The average ppm error for all 34 measurements was 6.9 ppm with a standard
deviation of 6.4 ppm. This indicates that the Q Exactive is a very powerful platform
for confirmation of protein primary structure.
Confirmation of protein primary structure
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

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)
not identified, possibly and internal fragment ion
Even 240,000 might not be enough for resolving this complex MS/MS spectrum.
Sw needs to subtract intensities, the same way as Xtract is doing it.
Spectrum is an average of 10 spectra, each with 10 microscans. 7.5 s for each scan, add overhead/fill time. About 2 min averaging. Separated by HPLC, BioBasic C4, 100 ul/min, 10 cm, 1 mm ID.
Load: 5 ug on column
Gradient: 20-80% B 10 min. 0.1% FA. Column overloaded, elution time about 5 min.
Dissolve in water, 1 ug/ul, injected 5 ul.
Markus Kellmann, Orbitrap Elite data

18. Spatially Resolved HDX
Structural dynamic studies by measuring backbone amide HDX
Problems:
back-exchange under quench conditions
gas-phase hydrogen scrambling
Figure 1. Schematic drawing of the cooling device adapted to a chipbased
nanoflow electrospray ionization (ESI) setup. A flow of nitrogen
gas cooled to subzero temperature is generated by passing it through a
coil of copper tubing immersed in dry ice. The inset shows how the
cold gas flow is directed to the front of the pipet tip (containing the
sample solution) that is in direct contact with the ESI chip. The
labeled parts are (a) entrance cone of Orbitrap mass spectrometer, (b)
holder for ESI chip, (c) ESI chip, (d) holder for conductive pipet tip,
(e) conductive pipet tip, (f) outlet tubing for cold nitrogen gas, (g)
copper tubing, (h) dry ice, and (i) thermoinsulated box. Only the front
end parts of the NanoMate are shown.
Figure 4. Dissecting the solution structural dynamics of the Nterminal
half of ubiquitin. Deuterium levels of the c fragment ions of
ubiquitin after native state D-to-H exchange for 60 min measured by
electron-transfer dissociation mass spectrometry (filled circles) and
NMR (red line). Error bars represent average ± SD for measurements
made in triplicate. NMR deuterium levels were obtained from
exchange rate constants determined by Johnson et al. (ref 39). The
theoretical deuterium content in the case of 100% gas-phase H/D
scrambling is indicated (dotted gray line). The inset displays the
crystal structure of ubiquitin (PDB 1UBQ). The turn between β-
strands 1 and 2 and the loop between the central α-helix α1 and β-
strand are highlighted in red (see text for further information).
Regions of secondary structure of the N-terminal half are illustrated
above the figure for reference.
A nearly perfect correlation
is observed between the deuterium content of the c ion series
and the graph showing the cumulative deuterium content
obtained from known NMR exchange rate constants39
(compare filled circles with red graph in Figure 4). The
cumulative deuterium graph exhibits a distinctive profile, where
the horizontal region of the graph from c6 to c11 corresponds to
a flexible region in ubiquitin that is devoid of deuterium (a turn
between β-strands 1 and 2), whereas the neighboring protected
regions (β-strands 1 and 2) retain deuterium and exhibit a
nearly linear increase.
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. HK97 bacteriophage capsomers 253 kDa
Analysis of Protein Complexes
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

20. Ligand Binding Stoichiometry
2000
4000
6000
8000
10000
12000
14000
m/z 10 20 30 40 50 60 70 80 90
100
Relative Abundance
R=2048
R=2161
R=2002
R=1873
R=2186
R=1600
R=1353
R=1342
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. 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
High resolution MS scan at three different transient lengths
followed by 20 CID MS/MS scans in the linear ion trap. Note that cycle time is unaffected by the resolution of the full scan. Preview refers to
the portion of the survey scan that is used to select precursor ions for fragmentation. B, LC MS heat map of peptides eluting over a 3 min elution
time interval in a 40 Th range. More detail is visible in the ultra high resolution setting (left panel) compared with the normal resolution setting
(right panel).
3 min
40 Th
148,000 peptide clusters x 93,000 peptide clusters
Michalski et al., MCP 2012, /mcp.O

22. Data Dependent Acquisition - Issues
Run-to-run reproducibility
Inability to effectively target peptides of interest
The DDA sampling strategy offers an elegant simplicity and
has proven highly useful for discovery-driven proteomics. Of recent
years, however, emphasis has shifted from identification to
quantification-often with certain targets in mind. In this context,
faults in the DDA approach have become increasingly evident.
There are two primary limitations of the DDA approach: First,
is poor run-to-run reproducibility and, second, is the inability to
effectively target peptides of interest (8). Hundreds of peptides
often coelute so that low-level signals often are selected in one
run and not the next, and selecting m∕z peaks to sequence by
abundance certainly does not offer the opportunity to inform
the system of preselected targets.
Overlap between various combinations of duplicate analyses of
yeast peptides. (a–d) Venn diagrams displaying the overlap of different trials
of the same dissociation method: CAD and CAD (a), ETD and ETD (b)
CID x CID ETD x ETD
Swaney DL, McAlister GC, Coon JJ: Nature Methods 2008, 5,

23. Proteome Analysis in a Single LC/MS Run
Rapid and robust proteome analysis
50 cm column length
4 h gradient
35 oC (nano-UHPLC)
Near complete yeast proteome
More than 4,000 proteins/run (1% FDR)
Median sequence coverage 23%
Figure: C. The median sequence coverage of individual runs after matching is around 17%. The median sequence coverage from the combined run for 4,206 proteins was 23.4 % as shown. D. The conjoint circles represent the frequency of identification of proteins in the 6 runs. Proteins identified in all six runs are designated as core proteome in the innermost circle.Single runs of FASP-prepared and LysC digested lyzates
Proxeon Easy NanoLC use of long columns with linear velocity of 250 nl/min
in the temperature range of 35 oC
50 cm column with 75 μm inner diameter, packed in-house with
1.8 μm C18 particles (Dr Maisch GmbH, Germany)
To facilitate deep sampling of the proteome, we employed relatively long columns and small particle
sizes (50 cm, 1.8 μm). This was readily accommodated by the UHPLC pump, which produced a stable
flow of 250 nL/min at 500 bar. Another advantage of the UHPLC system is its ability to load sample at a
higher flow rate and to equilibrate columns more quickly, leading to a shortening of overhead times. We
found the combination of a 50 cm column and 4 h gradients to be a good combination for standard use.
Minimum undersampling
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

24. Data Dependent Decision Tree
Decision tree–driven tandem mass spectrometry for shotgun proteomics
Now, let’s have a look at the behaviour of phosphopeptides at harsher collisional conditions, such as those experienced in a quadrupole or multipole (Qtof, HCD on Orbitrap). The above examples are fragmentation by HCD, when the high resolution tandem mass spectrum of the phosphopeptide was obtained in
the orbitrap analyser of an LTQ Orbitrap Velos (Thermo Fisher Scientific). The presence of a complete series of fragments
at the peptide bonds, each measured with mass deviations of a few parts per million, and the presence of fragments that
include the phosphogroup, unambiguously identified the peptide sequence shown, as well as the phosphorylation site at
Ser225. an and bn ions are fragments at the nth peptide bonds that contain the amino-terminal part of the peptide, whereas
yn ions contain the carboxy-terminal part. NL, normalized intensity level (counts per second).
What you do not find in the spectrum is the peka of the dephopshorylated intact peptide (neutral loss peak) which was so prominent for ion trap-based spectra. The next slide shows another example confirming this behavour of pS/T peptides under HCD conditions.
Swaney DL, McAlister GC, Coon JJ: Nature Methods 2008, 5,

25. Product Dependent Trigger: HCD PD ETD
ZIC HILIC separation of a glycoprotein digest
targeted analysis of N-linked glycopeptides in complex
mixtures that does not require prior knowledge of the glycan
structures or pre-enrichment of the glycopeptides. Despite the
complexity of N-glycans, the core of the glycan remains constant,
comprising two N-acetylglucosamine and three mannose units.
Collision-induced dissociation (CID) mass spectrometry of Nglycopeptides
results in the formation of the N-acetylglucosamine
(GlcNAc) oxonium ion and a [mannose+GlcNAc] fragment (in
addition to other fragments resulting from cleavage within the
glycan). In ion-trap CID, those ions are not detected due to the low
m/z cutoff; however, they are detected following the beam-type CID known as higher energy collision dissociation (HCD) on
the orbitrap mass spectrometer. The presence of these product ions following HCD can be used as triggers for subsequent
electron transfer dissociation (ETD) mass spectrometry analysis of the precursor ion. The ETD mass spectrum provides peptide
sequence information, which is unobtainable from HCD.
full FT-MS scan (m/z
380−1600) and subsequent HCD MS/MS scans of the 40
most abundant ions above an absolute signal intensity threshold
of 500 counts. If peaks at m/z (HexNAc oxonium ions)
or (HexHexNAc oxonium ions) (±m/z 0.05) were
within the top 20 most abundant peaks, a supplemental
activation (sa) ETD MS/MS scan of the precursor ion in the
linear ion trap was triggered.
Figure 1. Online ZIC-HILIC liquid chromatography HCD product
ion-triggered ETD MS/MS of Lys-C digest of ribonuclease B. (a)
Survey scan mass spectrum recorded in the orbitrap at retention time
(RT) min. (b) HCD MS/MS spectrum of precursor ions with
m/z (c) Supplemental activation ETD MS/MS of precursor
ions with m/z
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. Product Dependent Trigger: HCD PD ETD
HCD fragmentation spectrum of m/z
Oxonium ions observed among top 20 peaks
Figure 1. Online ZIC-HILIC liquid chromatography HCD product
ion-triggered ETD MS/MS of Lys-C digest of ribonuclease B. (a)
Survey scan mass spectrum recorded in the orbitrap at retention time
(RT) min. (b) HCD MS/MS spectrum of precursor ions with
m/z (c) Supplemental activation ETD MS/MS of precursor
ions with m/z
Singh et al., JPR 2012, doi: /pr300257c

27. Product Dependent Trigger: HCD PD ETD
ETD fragmentation triggered
Peptide sequence information
Glycosylation site localization
Figure 1. Online ZIC-HILIC liquid chromatography HCD product
ion-triggered ETD MS/MS of Lys-C digest of ribonuclease B. (a)
Survey scan mass spectrum recorded in the orbitrap at retention time
(RT) min. (b) HCD MS/MS spectrum of precursor ions with
m/z (c) Supplemental activation ETD MS/MS of precursor
ions with m/z
Singh et al., JPR 2012, doi: /pr300257c

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
The DDA sampling strategy offers an elegant simplicity and
has proven highly useful for discovery-driven proteomics. Of recent
years, however, emphasis has shifted from identification to
quantification-often with certain targets in mind. In this context,
faults in the DDA approach have become increasingly evident.
There are two primary limitations of the DDA approach: First,
is poor run-to-run reproducibility and, second, is the inability to
effectively target peptides of interest (8). Hundreds of peptides
often coelute so that low-level signals often are selected in one
run and not the next, and selecting m∕z peaks to sequence by
abundance certainly does not offer the opportunity to inform
the system of preselected targets.
Bailey et al, Instant spectral assignment for advanced decision tree-driven mass spectrometry.
PNAS 109, 8411–8416, (2012) .

29. The Newest in Data Dependent Acquisition
Posttranslation modification site localization
Ambiguous position from HCD
Triggered ETD resolving the ambiguity
The DDA sampling strategy offers an elegant simplicity and
has proven highly useful for discovery-driven proteomics. Of recent
years, however, emphasis has shifted from identification to
quantification-often with certain targets in mind. In this context,
faults in the DDA approach have become increasingly evident.
There are two primary limitations of the DDA approach: First,
is poor run-to-run reproducibility and, second, is the inability to
effectively target peptides of interest (8). Hundreds of peptides
often coelute so that low-level signals often are selected in one
run and not the next, and selecting m∕z peaks to sequence by
abundance certainly does not offer the opportunity to inform
the system of preselected targets.
Bailey et al, Instant spectral assignment for advanced decision tree-driven mass spectrometry.
PNAS 109, 8411–8416, (2012) .

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
MSn capability
Quan&Qual experiments done on a single platform
Orbitrap Elite - the most comprehensive system for proteomics
Speed and sensitivity
Choice of multiple fragmentation techniques
Suitable for ID, PTM, label-free, TMT/iTRAQ, SILAC, AIMS, top-down
Q Exactive - benchtop system designed with ease-of-use in mind
Suitable for all the above plus:
Unique spectrum multiplexing feature for targeted quantitation