Lecture 6 Flashcards
Name a few Mass Analysers
- Time-of-Flight Analyzer
- Quadrupole Analyzer
- Orbitrap Analyzer
What does TOF analyzer measurer?
- Time of flight mass spectrometer measures the mass-dependant time it takes ions of various masses to move from the ion source to the detector.
How does TOF analyzer work?
- The starting time must be well defined.
- Ions are formed in pulses, small ions reach the detector before large ones and the detector measures the time it takes
Quadrupole Mass Analyser
- A quadrupole mass filter consists of four parallel metal rods with different charges
- Two opposite rods have an applied positive potential and the other two rods have a negative potential
- Applied voltages affect ions travelling down flight path.
Orbitrap Mass Analyser
- Ions are trapped between a central & an outer electrode
- Ions oscillate to produce a current
- Current is transformed into a m/z (Fourier Transform)
- High sensitivity, good range
- Very High Resolution & Accuracy
What is the highest peak called?
Base peak - relative arbitrary value of 100
Abundance Measure - how is it measured?
- Ions are trapped between a central & an outer electrode
- Ions oscillate to produce a current
- Current is transformed into a m/z (Fourier Transform)
- High sensitivity, good range
- Very High Resolution & Accuracy
Peptide Mass Fingerprinting
the peptides are analysed by mass spectrometry and the masses generated are then used to search against a sequence database.
Tandem Mass Spec (MS/MS) Peptide Identification
A peptide is fragmented in a mass spectrometer and the fragment ion masses are then used to search a sequence database. Proteins can be correlated with the fragmentation of a single peptide using this technique.
Is reverse phase liquid chromatography necessary with peptide mass fingerprinting?
8 Because the peptides are generated from protein resolved by 1D/2D gel electrophoresis –no need to use reverse phase liquid chromatography to separate peptides
* Instead can be directed spotted onto MALDI target plates and placed in the MALDI-TOF instrument
Reasons why the number of experimental peptides does not match the number of theoretical peptides (in-silicodigest)
- Poor ionization
- Poor digestion (large peptides)
- Peptides too low in abundance to be detected
Factors Affecting Performance of PMF
- Sample Purity
- Specificity of the digestion
- Employing the highest mass accuracy availble in the measurement of peptide masses.
