Mass Spectrometry Flashcards
What is the basic principles of MS?
- Molecules are bombarded with a beam of energetic electrons
- Molecules are ionised and broken up into many fragments
- Each ion has a particular mass to charge ratio (m/z)
- For most ions, the charge is 1 so m/z is just the molecular mass
- Ions pass through magnetic and electric fields to reach detector
- Spectra generated
What are the FOUR basic steps of MS?
- Ionisation
- Ion Separation by Mass Analyser
- Detection
- Data Processing and Analysis
Explain how a basic MS works - using a magnet sector
- Sample is injected into mass spectrometer
- Molecules are ionised and accelerated
- Ions are separated by mass and charge by mass analyser (electromagnetic deflection)
- Ions that are properly aligned are detected and amplified
- Entire system is in a vacuum
- Generated data reports on relative abundance of each ion based on mass-to-charge ratio (m/z)
Explain the term m/z ratio
A measurement that represents the mass of an ion divided by its charge, used to identify and characterize molecules based on their mass and ionic charge.
What is MS often coupled to and why?
- Coupled with either gas or liquid chromatography
- Results in expanded analytical capabilities with widespread clinical applications
What is the difference between Gas Chromatography-MS and Liquid Chromatography-MS?
GC-MS
* best suited for analyzing volatile and thermally stable compounds, utilizing vaporization of the sample in a gas phase
LC-MS
* used for a broader range of substances, including non-volatile and thermally unstable compounds, with the sample maintained in a liquid solvent.
* better for analysing large biomolecules like proteins and peptides
What formula is used to calculate the mass of an ion in MS?
Rearrange r = mv/qB
where;
* r = radius
* m = mass
* v = velocity
* q = charge
* B = magentic field
So,
m = qBr/v
In MS, what is the Molecular Ion?
- the molecular ion (M+) is the ion formed by the removal of an electron without any fragmentation of the molecular structure.
- Positively charged
- Provides molecular weight of compound
In MS, what is the Base Peak?
Most intense (tallest) peak in a mass spectrum
What are the THREE main components of a mass spectrometer?
- Ionisation Source
- Mass Analyser
- Detector
How are ions produced in MS?
- Removing or adding electrons
- Removing or adding protons (H+)
- Addition of entities such as NH4+ or CH5+
Describe what happens in Electron Ionisation (EI)
- Occurs in gas phase reaction due to samples thermal stability and low molecular weight
- Filament set to 70eV creates stream of high-energy electrons
- Ionisation occurs when collision removes an electron from the sample molecule
- Cleaves covalent bonds, producing repeatable fragments
List FIVE different types of MS
- GC-MS
- LC-MS
- Tandem MS (MS/MS)
- Time of Flight (TOF) MS
- Matrix Assisted Laser Desorption/Ionisation (MALDI) MS
What is MS used for?
For identifying, characterizing, and quantifying molecules in diverse samples by measuring their mass-to-charge ratios
What is the level sensitivity and specificity that can be achieved with MS?
- High sensitivity and specificity
- Can detect compounds at very low concentrations, often in the range of picograms (10^-12 grams) to femtograms (10^-15 grams) per milliliter.
What is ionisation and how is it achieved in MS?
- the process of converting molecules from a sample into charged particles (ions)
- achieved through various ionization techniques, each suitable for different types of samples and analysis goals
Outline the key principles of Gas Chromatography
- Components in a mixture are distributed between two phases; stationary phase and mobile phase (or carrier gas)
- Compounds in the mobile phase interact with stationary phase as they pass through
- Differences in properties of each component causes different retention times, moving out of the column in different orders
Draw a diagram of a GC-MS
What is the difference between using 15eV vs 70eV for ionisation in MS?
- Molecular ions are formed when energy of electron beam reaches 15eV
- Fragmentation of the ion when bombardment energy reaches 70eV
What is MALDI?
A soft ionisaton technique that involves a laser striking a matrix of small molecules to make the analyte molecules into the gas phase without fragmenting or decomposing them
Describe the principles of MALDI
- Analyte is embedded in a matrix deposited on a solid surface called a target
- UV or IR laser pulse irradiates matrix which becomes vibrationally excited
- Matrix molecules ablate from surface, absorb the laser energy, and carry the analyte molecules into the gas phase
- During ablation, the analyte molecules are ionised by being protonated or deprotonated with nearby matrix molecules
- Most common MALDI ionisation format is for analyte molecules to carry a single positive charge
Draw a diagram of how LC-MS works
Explain the Electrospray Ionisation method
- Soft ionisation technique used to generate ions using electrospray
- High voltage applied to liquid to produce an aerosol
- Opposing flow of heated gas causes charged droplets to evaporate (desolvation)
Explain how Magnetic/Electric Sector mass analysers work
- Magnetic field is used to deflect ions around a curved path
- Radius of curvature of an ion depends on m/z ratio and strength of magnetic field
- Ions with correct m/z pass through the detector
- Ions too heavy or too light do not make it through
- Magnetic field can be varied so that all ions can be detected
- Resolution can be increased further by subjecting ions to an additional electric field
Explain how Quadropole mass analysers work
- Ions are passed through 4 parallel rods which apply varying voltage and radiofrequency
- As the field changes, ions respond by undergoing complex trajectories
- Only ions of a certain m/z ratio will have stable trajectories
- All other ions are lost by collision with rods
- Quadropole analysers have limited resolution and low mass range
Explain how TOF mass analysers work
- Amount of time required for an ion to travel a known distance is measured
- A pulse of ions is accelerated through analyser so that they have identical kinetic energies
- Velocity is directly dependent on mass
- Fastest mass analysers and have unlimited mass ranges
- Best coupled with pulsed ionisation sources such as MALDI
Explain the principle of MALDI-TOF in depth
- Analyte is dissolved and mixed with matrix
- Mixture is spotted onto a metal target plate
- Plate is loaded into MALDI-TOF instrument
- MALDI leads to sublimation and ionisation of sample
- Generated ions are separated depending on m/z through a TOF analyser
- MS profile is then generated
Why is a reflectron used in TOF?
To improve resolution by compensating for kinetic energy variations
Outline the principle of 2D gel electrophoresis
- Used in analysis of complex protein mixtures
- Step 1: Protein is separated into its charges with isoelectric focusing (IEF)
- Step 2: Protein is separated according to mass
Draw a flow chart of protein analysis by MS/MS starting with a cell or tissue
Draw out all possible b-type and y-type ions generated by cleavage and ionisation of an Ala-Gly-Ser peptide
Outline the most common approach for identifying protein by MS (bottom-up approach)
- Proteins are digested with trypsin
- Can be digested in solution or run on SDS-PAGE and gel slices subjected to trypsin
- Tryptic peptides are acidified to give them a positive charge
- Peptide ions are injected into HPLC column which separates the peptides
- Peptides sent to first mass spectrometer and detected
- Peptides from MS1 are then fragmented by CID
- Peptides then sent to MS2
- Able to detect thousands of tryptic peptides from a single sample
Outline the types of post translational modifications that can occur in proteins
- Hydroxylation
- Glycosylation
- Phosphorylation
- Acetylation
- Methylation
- Disulfide bond
Explain the SILAC method in MS
- Cells representing two different biological conditions are grown in either light or heavy medium containing amino acid and stable heavy isotopes
- Cells are harvested and mixed together in equal amounts
- Tryptic digestion
- Peptide separation by LC
- Mass spectrometer
- Tryptic peptide observed as a peak pair representing two sample conditions
- Quantitive comparision of two conditions
Explain the principle of iTRAQ
A) Labelling of four samples with 4-plex iTRAQ reagents
B) Combining four labelled samples into one tube
C) Following LC separation and MS analysis, the iTRAQ reporter is cleaved upon MS/MS fragmentation
D) MS/MS spectra recorded at low m/z ratio and intensity of reporter represents the relative abundance of the peptide in the sample
