Chapter 9 Mass spectrometry Flashcards
Objectives
Describe the principle of Mass Spectrometry (MS)
From the MS diagram, determine the original mass of the whole protein
With examples, explain how MALDI-TOF MS and ESI MS work.
MS usage
MS is used extensively in clinical labs involved in the determination of protein expression levels and disease biomarker discovery
Workflow in proteomic analysis between chromatography and peptide
Sample separation and visualization
Comparative analysis
Digestion
Mass spectrometry principles
A mass spectrometer creates +ve charged particles (ions) from molecules. It then analyzes those ions to provide information about the molecular weight of the compound and its molecular structure.
A mass spectrometer does not actually measure the molecular mass (gram, Dalton) directly, but rather the mass-to-charge ratio (m/z) of the ions formed from the molecules.
All substances have to be converted into gas state (usually by heat) before ionization
The mass spectrometry analysis introduced in this lecture is to confirm identity of a suspected proteins
MS principle step 1 ionisation
Analyte (protein) needs to be converted into gas phase (vaporized)
Depending on the type of MS, the analyte will be turned into charged molecules
by adding or taking away electrons
or by adding or taking away protons
the 2 types of fragmentation in MS
Soft ionisation: less fragmentation
Hard ionisation: fragmented into basic elements
How to choose between which fragmentation
Depending on the energy levels, analyte may be fragmented
Step 1 depending on energy levels
Depending on energy levels, the ionisation process may fragment (break up covalent bonds of) the analyte
Step 2 acceleration principles
The positive ions are repelled away from the very positive ionization chamber and pass through three slits, the final one of which is at zero volt. All ions are accelerated into a finely focused beam.
ionization chamber at <10000 volts produce a ionization beam between intermediate plate and final plate at 0 volts
Step 3 deflection principle
Different ions are deflected by the magnetic field by different amounts, which depends on the mass of the ion.
Lighter ions are deflected more than heavier ones.
the charge on the ion.
Ions with 2 or more +ve charges are deflected
than ones with only 1 +ve charge.
Concept checkpoint
How do you tell which ion is the heaviest if they all have the same charge ?
How do you tell which ion has the highest charge if they all have the same mass ?
for qn above, the ion stream that curves up to the most top
for qn below, the ion stream that curves down to the most bottom
Step 4 Detection
The gas phase ions are sorted in the mass analyzer according to their mass-to-charge (m/z) ratios and then collected by a detector.
In the detector the ion flux is converted to a proportional electrical current.
The data system records the magnitude of these electrical signals as a function of m/z and converts this information into a mass spectrum.
Mass spectrometry workflow
Ionization (ESI/MALDI)
Acceleration/sorting
Detection
Mass to charge ratio (m/z)
Ionization principles
Analyte has to be converted into gas-phase ions:
Analyte is the material to be analyzed e.g. peptide from in-gel digestion or whole protein.
Movement of gas-phase ions can be precisely controlled by electromagnetic fields.
Large macromolecules like peptides or whole proteins are polar and non-volatile i.e. difficult to convert to gas-phase ions.
Soft ionization methods used in electrospray ionization (ESI) and matrix-assisted laser-desorption ionization (MALDI) enable conversion of whole proteins/peptides to gas-phase ions.
Ionization process
Soft ionization process ionizes peptides into gas-phase ions with less fragmentation (less covalent bonds broken).
