Proteomics I

Pregunta	Respuesta
What is the consequence of a block at the start of the citric acid cycle knowing that involved proteins have strong epistatic interactions?	The proteins in such a cycle work in coordinated fashion (optimized clusters). A block at the start has severe consequences for the function of the cycle.
Explain the relationship between 'linear dependence' and 'the ribosome'	There is need for co-regulation as there should be balance between the production of subunits (there is a strong epistatic interaction)
Argue why it can be complicated to find a certain protein.	Proteins have different life times, localization and concentration in the cell. The protein of interest may have a short life time and is degraded rapidly.
What makes detection of all proteins at the same time difficult?	The proteins are present in an extremely wide range of concentrations. They cannot be captured with the same machine settings.
What are techniques to determine protein structure?	X-ray crystallography, NMR, single-particle EM, homology modelling
What is the principle of X-ray crystallography?	X-ray beams hit a molecule after which a diffraction pattern is generated. This gives information about the protein structure by means of an electron density map.
What is the principle of NMR? What is a benefit?	A chemical shift of the H-atoms in a molecule is induced and measured. A benefit is that movements of the molecule can be captured.
What is the principle of single particle EM?	Proteins are hit with a beam resulting in a diffraction pattern. Many diffraction patterns are bundled to generate the protein structure.
Which two MS techniques are most used?	ESI-MS and MALDI-TOF
What is the principle of ESI-MS?	Charged particles are selected on charge. Prediction occurs with TOF - time of flight is directly related to the size of a molecule.
What is the principle of MALDI-TOF?	A laser beam is used to release molecules from a solid state. This generates fragments. These are selected on charge and mass.
What is the difference is mass of various isotopes?	1 Dalton
Can a protein be identified based on its mass only? What is the solution?	No, not all proteins have different masses. Proteins are fragmented into peptides and as sequences differ, breaking points do as well.
Name the steps for protein identification with mass spectrometry.	Proteins --> separation (HPLC) --> digestion (trypsin) --> ioniser --> selection of double-charged ions --> selected fragmented (collision-induced decay) --> singly charged daughter ions analyzed
Why are double charged peptides preferred?	When the peptide breaks, the two daughter peptides will have a single charge (while if charge = 1 only one daughter ion will be charged).
What is the name of the ions generated when a peptide is broken at its peptide bond?	b-ions and y-ions.
Name the use of the quadrupole mass analyzer.	Four electrodes park molecules. Only molecules with a certain m/z are flown through while other ones are attached to the electrodes (+ or -).
We end up with b-ions and y-ions. What do the 'b' and 'y' part refer to?	After breakage: b stands for the part BEFORE the peptide bond. Y stands for the part AFTER the peptide bond.
What do the numbers indicate in the following situation? b2, y2	The numbers indicate the amount of amino acids.
Peptide identification is difficult. Name an approach other than de novo identification.	Make in silico database (need a well-annotated genome for this) --> predict all b and y fragments (knowing the cut sites) Now let the computer compare fragments to the database --> identification.
You have a sample with 6000 proteins which vary in copy number. Which technique is appropriate?	Use LC-MS to separate and simplify the spectrum.

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	Creado por Dominique M hace casi 6 años