Define the proteome
A proteome is the complete set of proteins expressed by an organism // mechanistic expression of the gene expression. It differs from cell to cell and changes over time.
How many amino acids have the potential to make a protein?
20 potential amino acids that can be
combine together to create a protein
Explain the different levels of protein organisations (4)
Primary structure – sequence
Secondary structure – alpha helix or pleated sheets
Tertiary structure – how sheets or helixes fold together
Quaternary structure - multiple tertiary structures
when interpreting mass spectra, which isotope should be used in calculations?
Use the lightest and most abundant isotope of the element when doing calculation – don’t use the average molecular mass!
Explain the role of mass resolution within mass spectrometry?
- the ability to resolve closely related adjacent mass peak
- the bigger the number the higher the resolution
- higher resolution is better but more expensive
Define Scan Speed?
How fast can the mass analyser scan the entire range of the mass spectrum (again the higher the number, the faster it can scan)
what are the different types of MS acquisition modes?
MRM, PRM, DDA, DIA
what is MRM?
Multiple reaction monitoring.
- targeted acquisition mode that is used to detect and quantitate specific, known compounds in a sample
- uses two mass spectrometer scans
- one to select a specific precursor ion, and a second to detect the corresponding product ions after fragmentation
what is PRM?
Parallel Reaction Monitoring.
- similar to MRM but it allows to monitor multiple transitions
- useful in cases where multiple target compounds are present in a sample and need to be quantified simultaneously
what is DDA?
Data-Dependent Acquisition.
Looks for things in DISCOVERY.
- selects and analyzes a series of precursor ions from a sample based on their relative abundance
- the most intense ions are selected and fragmented, and the resulting product ions are analyzed
what is DIA?
Data-Independent Acquisition.
- DIA does not rely on the relative abundance
- instead splitting chamber and indistcriminatley fragmenting of precursor ions, thus it allows for detection of low-abundance precursors
Explain how a Quadrupole works
- a quadrupole is a type of ion trap
- consists of four parallel metal rods arranged in the shape of a square
- by applying different voltages to the rods, an electric field is created that can trap and manipulate the ions
- quadrupole can be tuned to allow only ions of a specific mass-to-charge ratio to pass through; others are trapped or deflected
- used in mass spec. to separate and analyze different ions based on their mass-to-charge ratio
Explain how a A triple quadrupole (QT) functions
- a triple quadrupole (QT) is a type of mass spectrometer
- utilizes three quadrupoles
- performs MRM
- first quadrupole acts as a mass filter, selecting ions of a specific mass-to-charge ratio
- second quadrupole, also called the collision quadrupole, is used to perform collision-induced dissociation (CID) on the selected ions - FRAGMENTS them
- third quadrupole, known as the detection quadrupole, is used to analyze the fragment ions generated in the second quadrupole
Describe an Orbritrap
The Orbitrap is a type of mass spectrometer that utilizes ion trapping.
- ions are first generated and then introduced into the instrument
- ions are then trapped and oscillate in an electric field within the Orbitrap analyzer
- oscillating motion of the ions generates a current, which is measured and used to determine the mass-to-charge ratio of the ions
explain how a Quadrupole Time of flight (TOF) functions
A type of mass spectrometer that combines the capabilities of a quadrupole mass filter and a time-of-flight (TOF) detector.
- uses a quadrupole mass filter to selectively pass ions of a specific mass-to-charge ratio
- then uses a TOF detector to measure the mass of the ions based on the time it takes them to travel a fixed distance
Explain how Time-of-flight spectrometry is measured
- a measurement technique used to determine the mass-to-charge ratio (m/z) of ions in a mass spectrometer
- ions are first generated and accelerated into a flight tube
- ions are then allowed to travel a fixed distance through the flight tube
- they are detected by a detector at the end of the tube
- time it takes for the ions to travel the fixed distance is measured, and this time is used to calculate the mass-to-charge ratio of the ions
Explain the role of MALDI and how it works
MALDI is a soft ionization method used in mass spectrometry to analyze large biomolecules such as proteins.
- sample is mixed with a small molecule called a “matrix” and applied to a metal plate
- matrix is typically a weak acid or a compound that absorbs at the wavelength of the laser
- a laser beam is then directed at the sample, and the energy from the laser causes the matrix to desorb and ionize the sample molecules
- resulting ions are then analyzed by a mass spectrometer
ADV/DISADV of MALDI
+ Useful for intact proteins
- Multiple charges states due to large proteins so hard to derive the original mass.
- not suitable for small ionised molecules
Explain the diffrence between MS1 and MS2
MS1 is used to determine the mass-to-charge ratio (m/z) and the relative abundance of each precursor ion are determined.
MS2 selects a specific precursor ion, and it fragments the precursor ion using collision-induced dissociation (CID) or other fragmentation methods. Analysis shows info about the structure of the precursor ion.
MS2 is useful for compounds
that have exact same chemical
formula but different chemical
structure
What are the current limitations of MS based proteomics?
- detection limited as it can be difficult to detect low-abundance proteins
- can be limited by the complexity of the sample
What preparation do MS1 & 2 samples require?
to be trypsinized and turned
into peptide mixture
what are the methods for protein estimation?
- Bradford assay
- Lowry method
- Bicinchoninic acid assay
- Fluorescent based assa
Explain how protein estimation works
- A calibration is created using a set of known concentration standards (such as BSA) and a minimum of 6 measurements are taken
- This will generate equation of the line (Y = Ax+B)
- Concentration and the response recorded, using the equation is it then
back calculate to estimate the protein content (solve for x)
why protein estimation is crucial for the trypsinization step?
trypsinization is a stichometrical reaction meaning we need a very specific ratio to fragment proteins correctly.
