BIOL 4350 Lecture Notes - Lecture 10: Tandem Mass Spectrometry, Gibbs Free Energy, Mass Spectrometry

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9 Feb 2017
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FEBRUARY 8 PPT 5 (15-) LECTURE 10
- Presentations
o source of bonus questions for exams
- Mass spectrometry
o Taking ions and measuring their time of flight in a tube
Time of flight= between the time the ion enters the tube and hits a detector at the end
Gives the mass to charge ratio for the ion that gives an incredibly precise mass
for the ion
o Revolutionized what we can do in terms of analyzing proteins
o Example of a non-fragmented protein
Peaks for insulin and lactoglobulin
2 peptides were subjected to this type of analysis
2 peaks that correspond to beta lactoglobulin
o mass to charge ratio is equal to the mass
o there is a +1 peak
o 18,364
o +2 peak for the same peptide is around 9000 because the mass is divided
by 2 because it is a +2 ion
You can do the same analysis for the insulin (+1 and +2 peaks)
Fragmented protein with a known nuclease
o Nuclease/proteases
Like restriction enzymes
Cleave at specific spots of the polypeptide sequence
Ex:
Enterokinase will only cleave at AspAspAspAspLys
If you take a polypeptide like insulin and cleave it with a known protease, it will cleave
only on the spots and you can do mass spec on the peptide fragments
Those fragets are soeties alled a proteolyti figerprit
You can take the fragments that were generated from mass spec of a known
protein and match it to a protein in the database for human genomes
o Every polypeptide is going to have a characteristic weight
Can be measured very precisely
- Tandem mass spectrometry
o First flag whole peptide through the first tube
o In the second tube, you do some high speed collision with a gas such as a noble gas (unreactive)
o Breakages our at the peptide id ad it’s a o-hydrolytic cleavage even
o On average, a single polypeptide chain will be broken one time
Get an amino terminal fragment and a carboxyl terminal fragment and you can know
the exact mass of these fragments
o Very specific mass to charge ratio
o Get a bunch of peptide fragments and use a computer to figure out which fragments are made
up with which amino acids
- Cleavages
o A biologist may have a 2D gel and have a particular spot of interest
Figure what is varying in the spot of the two biological samples
o Cut the gel and use an electrical method to elute the peptide from the gel and into solution
o Then, incubate it with a protease, typically trypsin
Cleaves the c terminal between arginine and lysine (except when the next is a proline)
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FEBRUARY 8 PPT 5 (15-) LECTURE 10
o Do mass spec to get a bunch of peaks
Peaks correspond to the molecular weights of the trypsin fragments
- Nuclear Pore complex
o Made up of 45-50 different proteins
o Mass spec
Purified proteins and got a bunch of peaks
Many corresponded to known nuclear pore complex but some were unknown proteins
Technology is so evolved and advanced that it can identify the proteins of complex
nuclear complexes
o Finding phosphorylations
You can chop it up into bits and find out which of the predicted molecular fragments is
raised in weight by one phosphate group
- Enzymes
o Speeds up reactions
o Have enzymes because life would progress so slowly that life would not be possible
o The spontaneous reactions may be 1 molecule per hundred years
o First macromolecule that evolved in lift
o How to make reactions go faster
Increase concentration of reactants
Increase the temperature
Reactions depend 100% on collisions
o Transition state theory
in between reactants and products, you must first go through a transition state
old covalent bonds are partially unformed and new covalent bonds are partially formed
unstable which is why it is higher in energy than the reactants and the products
so fleeting that it has never been observed
reaction coordinate diagram
free energy diagram
Gibbs free energy changedifference in free energy between substrate and
product
Activation energydifference between reactants and transition state
o Equilibrium constant
[product] / [substrate]
higher number = reaction more driven to go to the products
mathematically related to the gibbs free energy reaction
o Exergonic
Downhill
Spontaneous
-G
o Endergonic
Uphill
Unfavorable
+G
o enzymes are protein catalysts (makes protein go faster)
lower reactions by a specific mechanism
lowers transition state energy
make the activation energy required much lower
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