L10-12

Question 1

T or F:

Mass spectrometry can be used for protein identification as well as protein quantification

Answer 1

True.

Question 2

Mass spectrometers are typically composed of

Answer 2

1. An ion source
2. Mass analysers (e.g. quadrupole, orbitrap)
3. Fragmentation chambers
4. Detector.

Question 3

What do mass spectrometers measure?

Answer 3

Mass spectrometers measure the mass-to-charge ratio (m/z) of ionized particles, which can be peptides, peptide fragments, or whole proteins.

Question 4

High-Performance Liquid Chromatography (HPLC)

Answer 4

Pressurised sample and solvent is passed through a solid phase column
Different molecules interact differently with the column, and therefore have different retention times
HPLC is used as aninitialseparation stage before MS
No information is provided on protein/peptide identity, only the rough amount of material present at each time point

Question 5

Quadrupole Mass Selection

Answer 5

Quadrupole basics:
• A combination of an RF and DC electric field is applied across quadrupole
• Only ions with a certain mass/charge ratio are stable through the quadrupole –all other ions are deflected
• Ions with a particular m/z ratio can be selected by varying the DC and RF fields
• The number of ions collected within each m/z window is recorded (output signal is proportional to the number of ions reaching the detector)

Question 6

Orbitrap mass detection

Answer 6

• Ion packets are injected into the orbitrap mass analyser
• Ions orbit around a central electrode
• Orbiting ions induce a current in the outer electrode, which is recorded and analysed to yield m/z ratios for all ions
• Orbitrap measures the full m/z range at the same time (unlike the quadrupole)