Mass spectrum_L3 Flashcards
What are the significance of fragmented ions in mass spectrum analysis?
The fragmented ions are particularly informative in deducing the construction of molecule by using the signals they produce
What is the MS/MS spectrum?
(1) In MS/MS experiments two (or more) analysers are connected in tandem, two dimensional spectrum is allowed to be produced as a result.
(2) Ions produced in the source are selected by the first analyser based on the preference are COLLISIONALLY ACTIVATED by exposure to a collision gas(usually inert gas, such as hydrogen and argon) as they pass through a collision chamber located between the two analysers, kinetic energy is transfered to generate fragmented ions
(3) Fragment ions plus any non-fragmented molecular ions are separated by the second analyser according to the mass to charge ratio and detected to give a MS/MS spectrum
Give examples of MS/MS spectrum?
(1) three quadrupoles connected: the first quadrupole selects the ions of interest, the second quadrupole allows inert gas collision, and the last quadrupole separates the ions by m/z ratio
(2) quadrupole is the lowest performance instrument, its mass data is not so accurate
MS and MS/ modes of Orthogonal Acceleration Time-of-Flight Mass Spectrometer (Q-TOF)
(1) Q-TOF MS mode: The quadrupole is set to allow all ions through and the TOF is the mass analyser; pulses of ions are accelerated into the TOF by the “pusher” and the MS spectrum is recorded
(2) Q-TOF MS/MS mode: The quadrupole is set to allow selected ions through the collision cell which contains the collision gas; the TOF is the mass analyser and pulses of fragment ions are accelerated into the TOF by the “pusher” and the MS/MS spectrum is recorded. The data quality has been improved, new omic technology like proteomics is pushed forward.
describe ES MS/MS sequencing
(1) Multiply charged ions (2+ or 3+) selected where possible, these ions require less collisional energy than singly charged ions, in addition the majority of impurity-derived signals are singly charged. Therefore peptide-derived fragment ions will predominate in the MS/MS data even if singly charged contaminants are present at the same m/z value as the molecular ion of the peptide
(2) Multiply charged ions are readily recognised by the interval between the isotope peaks: e.g. C-12 and C-13 peaks are separated by 1 for singly charged ion, separated by 0.5 for doubly charged ion, 0.333 for triply charged ion……
(3) Trypsin is the enzyme of choice for digesting proteins because tryptic peptides have a minimum of two charges (N-terminus plus C-terminal K or R), the sample is started with protein followed by proteolysis to fragmentise protein to fragments, trypsin acts on the bond formed between any amino acid at N-terminus and lysine or arginine(positively charged AAs), therefore, the digested peptides carry one unit positive charge.
If a series of signal data is observed as 460.4, 461.4, 461.9 and 462.4, what conclusion can you draw from the observatioin?
The doubly charged ion peaks at 461.9, due to its multicharge property, it is ideal for the 2D MS analysis
describe the b-ion formation pathway
The peptide bond at the N-terminus is attacked by the ionising proton which generates a neutral and undetectable molecule and a positively charged ion with a triple bonded oxygen, the mass to charge ratio should be the residual mass plus one which accounts for the proton
describe the a-ion formation pathway
A-ion formation is a secondarry pathway for b-ion fragmentation, usually due to excessive energy, the reaction does not take place every time, once generated, a loss of carbon monoxide is derived from the ion, in other words, the total weight will be substracted by 28. The a-ions are structurally informative, confirming the correct identification of b-ions.
describe the y-ion formation pathway
y-ion is formed by the attack of hydrogen from N-terminal residue which sits next to the peptide bond to be broken, as well as an ionisation proton. The separated N-terminus ion is neutral and undetectable by analyser, the y-ion is singly charged at the N atom, with the addition of two hydrogens and the uncounted CO group at the very C-terminal end, the mass to charge ratio should be residual mass + 19
Will every cleavage give rise to peaks of a-/b-/y-ions?
No, for example, cleavage at N-G does not favour the peak formation owing to mirror chemistry
What points should be taken into account when interpreting the mass spectrum?
(1) the every first AA at N-terminus should either be lysine or arginine
(2) b-ion and y-ion complementarity
(3) total molecular ion mass should be total residual mass plus one
(4) a-ion mass is 28 fewer than b-ion mass
What are the factors in proteomices?
What proteins are expressed and when?
What amounts?
How are they modified -phosphorylation, glycosylation, prenylation etc?
How do they interact with each other?
How do levels/types change during differentiation/disease etc?
What step is usually taken as a crude separation and visualization of proteins of interest from cell extract?
Two-dimensional gel electrophoresis (2DGE) separates the crude sample based on two properties
What are the pros of using gels?
(1) Quick
(2) Relatively Inexpensive
(3) Good resolving technique for complex mixtures
(4) Works with crude samples
(5) Moderately tolerant to salts and detergents
(6) Good visual representation of entire sample
(7) Easy comparison
(8) Relatively reproducible
What are the common detection stains for 2DGE?
Coomassie blue, silver stain, radiolabelling, fluorescent stains for some proteins, silver stain is more sensitive.
