Master Thesis

Describe the working principle, key characteristics, and technical requirements of Electrospray Ionization (ESI) in mass spectrometry. Include three advantages and two limitations of this technique.

• Application of a strong electric filed (1-4 keV) -→ Redox chemistry at interface between solution and electrode → charging of solution and formation of Taylor Cone → Repulsion of the ions → nebulisation (small droplets are formed an there is a fast flos outwards the capillary) → Countercurrent of (heated) gas, perpendicular to the spray → evaporation of the solvent within the droplets → Smaller droplets (Coulomb explosions)

• Atmospheric Pressure Ionisation (API)

Key Characteristics

1. Soft Ionization: Minimizes fragmentation, preserving intact biomolecules like proteins

2. Multiply Charged Ions: Reduces the effective mass-to-charge ratio (m/z), enabling analysis of large molecules (>1 MDa).

3. Solution Compatibility: Directly interfaces for online linkong with liquid chromatography (LC) or capillary electrophoresis (CE).

Advantages

1. High Sensitivity: Detects analytes at femtomole levels.

2. Broad Mass Range: Analyzes molecules from small organics (m/z 100) to macromolecules.

3. Compatibility with LC/CE: Ideal for high-throughput workflows.

Limitations

1. Ion Suppression: Co-eluting matrix components can reduce ionization efficiency.

2. Complex Spectra: Multiply charged ions complicate data interpretation.

3. Limited Structural Data: Requires tandem MS (e.g., CID, HCD) for fragmentation.

Technical requirements

• Sample concentration is usually 1-10 pmol/µl

• Typical solventia: 50:50 ACN:water + TFA 0.02%. The acid stimulates ionisation, the organic solvent stimulates droplet formation and evaporates easily.

• Low pH: positive ionisation of basic side chains and terminal NH2 group

• Higher pH: negative ionisation of COOH groups, but also of phosphate and sulphate groups.

• Flow rate can vary, depending on LC-system: from ± 100 nl/min till 1 ml/min. Lower flowrates (100-500 nl) are possible with nanopumps and do not require a curtain gas.

Explain how a quadrupole mass filter selectively transmits ions based on their mass-to-charge ratio (m/z). Include the roles ofRF/DC voltages and ion trajectory stability in your answer.

A quadrupole mass analyzer uses electric fields to separate ions by their mass-to-charge ratio (m/z).

1. Physical Structure

• Four parallel metal rods (hyperbolic or cylindrical) arranged symmetrically around the ion path.

• Opposing rod pairs are electrically connected:

  • One pair receives a positive DC voltage (+U)

  • The other pair receives a negative DC voltage (-U)

• Both pairs have a superimposed radio frequency (RF) alternating voltage (AC) (±Vcos(ωt))148.

2. Electric Field Dynamics

• The combined RF/DC voltages create a quadrupolar electric field between the rods.

• The field oscillates rapidly (~1–3 MHz), generating alternating focusing/defocusing forces on ions49.

• Ion trajectories depend on:

  • m/z ratio

  • RF frequency (ω)

  • DC/RF voltage amplitudes (U and V)26.

3. Ion Trajectory Stability

• When passing through the rods, ions undergo oscillating movement, dependent on charge and mass, so the Ions experience transverse oscillations in the oscillating field.

• For a given U and V, only ions with specific m/z values develop stable trajectories (confined near the central axis). Each pair makes up a filter. For instance: horizontal pair: only ions allowed above 1030 m/z (high pass) and vertical pair: only ions allowed under 1031 m/z (low pass).

• Unstable ions collide with rods and are neutralized or thrown out of the quadrupole.

4. Mathieu Equation & Stability Diagram

Ion motion is governed by the Mathieu equation.

Parameter	Effect on Ion Motion
Increased RF (V)	Transmits heavier ions (higher m/z)
Increased DC (U)	Filters lighter ions (lower m/z)

5. Mass Scanning

• To detect different m/z values:

  1. RF amplitude (V) and DC offset (U) are linearly ramped while keeping U/V constant.

  2. This moves the operating point along the scan line in the stability diagram.

  3. Ions sequentially attain stable trajectories based on their m/z9.

6. Key Advantages

• Fast scanning: Full mass spectrum acquisition in milliseconds.

• Compact design: No magnetic fields required (unlike sector instruments).

• Tunable resolution: Adjusting U/V ratio changes peak width and transmission efficiency.

• Compatible with ESI sources since these provide a continuous flow of ions that is necessary for the scanning process of the quadrupole.

• Disadvantage/ Scanning limits the times to analyze ions within a chromatographic peak (peak saturation).

Explain how an Orbitrap mass analyzer measures ions' mass-to-charge ratios (m/z). Include the roles of electrostatic trapping, harmonic axial motion, and Fourier transform signal processing in your answer.

The Orbitrap mass analyzer determines m/z ratios by trapping ions in a stable electrostatic field and measuring their oscillatory motion.

1. Physical Structure

• Electrode configuration:

  • Outer electrode: Barrel-shaped, grounded.

  • Inner electrode: Spindle-shaped, charged with a high DC voltage (~3–5 kV).

• The gap between electrodes creates a quadro-logarithmic electrostatic potential: ϕ(r,z)=k2(z2−r22)+Cr2ϕ(r,z)=2k(z2−2r2)+r2C

  where kk is the field curvature constant and CC adjusts radial confinement.

2. Ion Trapping and Motion

• Trapping mechanism:

  • Ions with a high velocity (kinetic energy, 1600 eV) injected tangentially into the field experience two forces:

    1. Radial attraction toward the inner electrode.

    2. Centrifugal force from their angular momentum.

  • Balanced forces create stable helical trajectories (orbital motion around the spindle + axial oscillations). Inner electrode is adjusted to ± -3200V -> ions under influence of a
    centrifugal and an electrostatic force (in the case of + ions) -> ions circulate around the inner electrode along elliptical trajectories such as a satellite moves around the earth (orbit). The ions also move back and forth along the axis of the central electrode so that their trajectories in space resemble
    helices.

• Axial harmonic motion:

  • Ions oscillate along the spindle’s axis with a frequency v:

    Angular velocity = ω=2πν=√(q/m)k (q=charge in Coulomb, k= constant).

    where kk is a constant determined by the electric field geometry.

  • Frequency depends only on m/z, independent of ion energy or injection angle.

3. Ion Injection

• C-trap preparation:
  Ions from external sources (e.g., electrospray) are accumulated and cooled in a curved linear trap (C-trap).

• Pulsed injection:

  • A DC voltage pulse ejects ions into the Orbitrap.

  • The inner electrode’s voltage is ramped to "squeeze" ions into stable orbits.

4. Signal Detection

• Image current detection:
  Oscillating ions induce a time-dependent image current on the split outer electrodes.

• Fourier transform (FT) processing:
  The current signal is converted to a frequency spectrum via FT, revealing ion m/z values.

Parameter	Impact on Performance
Voltage stability	Directly affects mass accuracy (up to 1 ppm)7
Detection time	Longer acquisition → higher resolution (>240,000)6

5. Key Advantages

• Ultra-high resolution: Resolves isotopes and subtle mass differences (e.g., 0.001 Da).

• No magnetic field: Compact design compared to FT-ICR systems.

• Robustness: Minimal maintenance due to static electric fields.

IBAQ

Normalization: The protein intensities are divided by the amount of theoretical tryptic peptides of those proteins with 6 to 30 aminoacids.