Master Thesis Questions

What is the DC-protein assay based on?

The reaction is similar to the well-documented Lowry assay, but with the following improvements: The reaction reaches 90% of its maximum color development within 15 minutes thereby saving valuable time, and the color changes not more than 5% in 1 hour or 10% in 2 hours after the addition of reagents.

What is FDR and what is the difference between 1% and 5% FDR?

False Discovery Rate (FDR) is a statistical method used to control the expected proportion of false positives (incorrectly identified significant results) among all results deemed significant in multiple hypothesis testing.

What FDR Levels Mean

• 1% FDR: Among all results called significant, you expect that on average 1% are actually false positives (i.e., truly null but reported as significant).

• 5% FDR: Among all results called significant, you expect that on average 5% are actually false positives.

Practical Implications

• Stricter Control: A 1% FDR is more stringent than a 5% FDR. It means you are willing to tolerate fewer false positives among your significant findings.

• Statistical Power: Lowering the FDR threshold (e.g., from 5% to 1%) generally reduces the number of results you call significant, because you require stronger evidence to declare significance. This can decrease the chance of false positives but may also reduce the chance of detecting true positives (lower power).

• Trade-off: A higher FDR threshold (like 5%) allows more discoveries but accepts a higher proportion of them being false. A lower threshold (like 1%) is more conservative, leading to fewer discoveries, but with higher confidence that those called significant are true positives.

Take away

• 1% FDR is more conservative, resulting in fewer but more reliable significant findings.

• 5% FDR is less conservative, resulting in more findings but with a higher proportion of false positives among them.

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.

LFQ

LFQ intensity is similar to iBAQ but applies normalization to exclude outliers, providing more accurate ratios of protein abundance between different samples. This normalization is crucial for ensuring reliable comparison of protein levels across biological samples. Takes into account retention time and aligns peptide peaks between different samples.

Andromeda search engine

A peptide search engine based on probabilistic scoring