Proteomics Flashcards
(38 cards)
Define The Proteome
Profiling of the entire set of proteins within a system/cell at a given time and defined
conditions.
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 how a Quadrupole works?
A quadrupole is a type of ion trap that 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.
The quadrupole can be tuned to allow only ions of a specific mass-to-charge ratio to pass through while others are trapped or deflected.
Commonly used in mass spectrometry 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 that utilizes three quadrupoles in sequence to perform a specific type of analysis known as multiple-reaction monitoring (MRM).
1.The first quadrupole acts as a mass filter, selecting ions of a specific mass-to-charge ratio for further analysis.
- The second quadrupole, also called the collision quadrupole, is used to perform collision-induced dissociation (CID) on the selected ions, breaking them down into smaller fragments.
- The third quadrupole, known as the detection quadrupole, is used to analyze the fragment ions generated in the second quadrupole.
Explain how a Quadrupole Time of flight (TOF) functions?
A quadrupole time-of-flight (QTOF) mass spectrometer is a type of mass spectrometer that combines the capabilities of a quadrupole mass filter and a time-of-flight (TOF) detector.
A QTOF instrument uses a quadrupole mass filter to selectively pass ions of a specific mass-to-charge ratio and then uses a TOF detector to measure the mass of the ions based on the time it takes them to travel a fixed distance.
- Ions are first generated and accelerated into the quadrupole mass filter, where they are focused and separated according to their mass-to-charge ratio.
2.The ions then pass through a collision cell where they may undergo collision-induced dissociation (CID) to produce fragment ions.
3.The fragment ions are then accelerated into the TOF detector where they are detected based on the time it takes them to travel a fixed distance.
Explain how Time-of-flight spectrometry is measured.
Time-of-flight (TOF) is a measurement technique used to determine the mass-to-charge ratio (m/z) of ions in a mass spectrometer. In a TOF mass spectrometer, ions are first generated and accelerated into a flight tube. The ions are then allowed to travel a fixed distance through the flight tube before they are detected by a detector at the end of the tube. The 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.
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. The ions are then trapped and oscillate in an electric field within the Orbitrap analyzer. The oscillating motion of the ions generates a current, which is measured and used to determine the mass-to-charge ratio of the ions.
Explain the role of mass resolution within mass spectrometry?
the ability to resolved closely related adjunct mass peak. The bigger the number the higher the
mass resolution?
High mass resolution can also be useful in applications where the sample contains isomers, which are molecules with the same chemical formula but different structural arrangements.
High mass resolution means that the peaks are well separated and it is easy to distinguish between them, while low mass resolution means that the peaks are closer together and it is difficult to distinguish between them.
What are the units for mass spectometry and what does this represent?
In mass spectrometry, m/z (mass-to-charge ratio) is a measure of the mass of a molecule or ion relative to the number of charges it carries.
When a sample is introduced into a mass spectrometer, it is first ionized, which means that electrons are removed from the molecules or atoms in the sample. The resulting ions have a positive charge, and the m/z value is calculated by dividing the mass of the ion by the number of charges it carries.
Define Scan Speed?
How fast can the mass analyzer scan the entire range of the mass spectrum
The higher the number, the faster it can scan = Good (Speed range 0.5scan/sec-1 to 30 scan/sec-1 )
Explain MS acquisition modes, MMR, PRM, DDA and DIA.
1.MRM (Multiple Reaction Monitoring): MRM is a targeted acquisition mode that is used to detect and quantitate specific 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.
2.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.
3.DDA (Data-Dependent Acquisition): 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.
4.DIA (Data-Independent Acquisition): DIA does not rely on the relative abundance instead splitting chamber and indistrciminatley fragmenting of precursor ions, thus it allows for detection of low-abundance precursors.
Explain the role of s Matrix assist laser desorption ionization (MALDI) and its drawbacks?
MALDI is a soft ionization method used in mass spectrometry to analyze large biomolecules such as proteins.
The sample is mixed with a small molecule called a “matrix” and applied to a metal plate. The 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. The resulting ions are then analyzed by a mass spectrometer.
+ 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, also known as “precursor ion scanning” is used to determine the mass-to-charge ratio (m/z) and the relative abundance of each precursor ion are determined.
MS2, also known as “product ion scanning” selects a specific precursor ion, and it fragment the precursor ion using collision-induced dissociation (CID) or other fragmentation methods. The resulting product ions are then analyzed to provide additional information about the structure of the precursor ion.
MS2 is useful for isobaric compounds
where they have exact same chemical
formula but different chemical
structure
What are the current limitations of MS based proteomics?
-Detection limit as it can be difficult to detect low-abundance proteins or post-translational modifications.
-Sample complexity: MS-based proteomics can be limited by the complexity of the sample, as it can be difficult to identify and quantitate all of the proteins present in a complex mixture.
-Data analysis: MS-based proteomics can be limited by the complexity of data analysis, as it can be difficult to accurately identify and quantitate proteins using the large amount of data generated by MS-based experiments.
What preparation does MS1 & 2 samples require?
Conventional proteomics required the samples to be trypsinized and turn
into peptide mixture and analysed using both MS1 and MS2 level for chemical ID.
Explain how Protein estimation works?
Multiple methods available for the estimation of protein.
- Bradford assay
- Lowry method.
- Bicinchoninic acid assay.
- Fluorescent based assay.
Essentially A calibration is created using a set of known concentration standards (such as BSA) and a minimum of 6 measurements are taken.
This will in turn will generate a 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)
Explain why protein estimation is crusial for the trypsinization step?
trypsinization is a stichometrical reaction meaning we need a very specific ratio to fragment proteins correctly.
How would you solve for X within a calibration curve for protein estimation using (Y = Ax+B). Additinally the sample was in 10ul solution and underwent a 50 fold dilution?
- Subtract B (Y = Ax+B) = (Y-B = Ax)
- Divide by A (Y-B = Ax) = (Y-B)/A=x
- Divide by solution volume %10 = xuL = units
- Multiply Dilution x50
Explain Gel base method for protein fractionation
SDS-Page?
SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) is a gel-based method for protein fractionation. It is based on the principle that proteins will migrate through a gel matrix under an applied electric field.
In SDS-PAGE, proteins are first denatured by the addition of a detergent, sodium dodecyl sulfate (SDS), which coats the proteins and gives them a uniform negative charge. This allows for proteins to be separated based on their size, rather than charge. The proteins are then applied to a polyacrylamide gel, which acts as the matrix through which the proteins will migrate under an applied electric field
Explain the usefuleness of gel electrophoresis (DiGE) ?
In-gel electrophoresis (DiGE) allows for detection of
changes in protein modification
(band profile and expression).
while changes in any type of modification can potentially be detected, there is still the need to identify the nature of that modification, usually by MS-based approaches.
Describe the steps of Shot gun (Bottom up) proteomic analysis and the aim?
(1) Extraction of protein content from the biological samples
(2)Subsequent determination of approximate protein (Bradford
assays)
(3) Fractionation (SDS gels) and trypsin digestion
(4) Clean up step and lyophilization
5) Chemical analysis (DIA or DDA)
(6) Database matching and peak table generations
(7) Data analysis and interpretation
Explain Trypsin digestion
The process of trypsin digestion begins with the addition of trypsin to the protein sample. Trypsin is a serine protease, which means it cleaves peptide bonds by hydrolyzing the peptide bond adjacent to the carboxyl group of the lysine and arginine residues, generating peptides of varying lengths.
pH of 8.3, which is the optimal pH for trypsin activity. The reaction is carried out at 37°C for several hours
These cuts are predictable allowing standardisation. Lastly Tripsin is inactivated using Urea and washed using
List all the stages of protein analysis
1.Protein collection/ estimation techniques
2. Gel electrophoresis
3. tripsinisation + urea + wash to end reaction
4. Ionisation source - Electrospray ionization
Convert liquid samples to
gas phase ions
5 mass analyzer MS1
6. fragmentation
7. MS2 fragment analysing to tell structural component
8. Database matching (Mascot, Global proteome machine (GPM)
* Peakatable (Common proteins detected, ID, relative abundance)
* Quality of data, reproducibility, QCs
Describe the function and mechanisms of Liquid chromatography system?
Main purpose is separate the complex protein mixture into simpler ordered system
Through chromatographic separation using :
Analytical LC proteomic column and solvent gradient profile to separate complex mixture in both time and space.
(C18 reverse chromatography is exclusively used)
Nanoflow system low internal diameter and low flow rate increases peak resolution and improves measurement
