chapter 5: protein sequencing Flashcards
primary structure of protein
linear sequence of amino acids that contain disulfide (S-S) bridges
Frederick Sanger
-won the nobel prize for sequencing bovine insulin
-won nobel prize for DNA sequencing
Steps to study a proteins primary structure
- Separate into individual polypeptide chains by cleaving disulfide bonds
- End terminus analysis (C and N)
- Sequencing
- Cleaving disulfide bonds: Method 1
Oxidize with performic acid
- Cleaving disulfide bonds: Method 2
- Reduce with XS Thiol/mercaptoethanol
- Cap thiols with iodoacetic acid
*can reverse
- End terminus analysis (C)
*Carboxypeptidases and exopeptidases are exoproteases that cleave at the C-Terminus
-Exopeptidases: cleave protein peptide bonds from end inwards
- Carboxypeptidase B: cleave at Lysine and Arginine only
-Carboxypeptidase A: cleave everything else
- end terminus analysis (N pt.1)
-Fluorodinitrobenzene (DNFB)
-Dabsyl Chloride
Dansyl Chloride
1.Undergo NAS to get one of the three above to join the polypeptide on the N-term. due to reacting with a weak base (OH-)
2. reacts with a strong acid to get acid catalyzed hydrolysis to break all peptide bonds
3. have your N-terminus
*disadvantage: destroy protein sequence, except for N terminus
*Lysine give a false positive bc of its NH2 side chain
- End terminus analysis (N pt.2: Edman Reagent/Degregation)
Edman reagent= PITC (phenyl isothiocyanate) * look at structure
- Uses a weak base (OH-) to add PITC to the polypeptide chain on the N terminus-PTC polypeptide
- use anhydrous F3CCOOH (trifluooroacetic acid, TFA) to break polypeptide bond- Thiazoline derivative
- uses a mild acid (h+) to rearrange
*TFA rsn is self cleaving
*leaves remaining polypeptide in aq soln. for another round of rsn with PITC
*Rxn is 98% efficient but after 50-100 cycle is error prone
*100 AA is the upper limit
*if chain has more than 100 AA then need to fragment into more segements
Fragmentation Methods: Enzymatic Fragmentation with Proteases
- Trypsin: only cuts lysine and arginine
- Chymotrypsin: cuts after bulky aromatic side chains (Phe, Trp, Tyr)
- Elastase: cuts after neutral aa
*if R group next to it is proline the proteases won’t be able to cut
Fragmentation Methods: Cyanogen Bromide (CNBr)
-CNBr cleaves on the side of Methionine
-will cleave regardless of what R group is next to it
- Determination of protein seq by Mass Spectrometry: MALDI-TOF
Matrix Assisted Laser Desorption Ionization- Time of Flight
-use trypsin digestion to determine protein sequence by Peptide Mass Fingerprinting (PMF)
-use vaporization technique to get protein fragments into a gas phase (ESI also can do this)
-Time of Flight (TOF) MS then uses a long tube where each ion has the same electrostatic force applied
* smallest fragments accelerates faster and hit detector 1st and larger molecules hit the detector giving a fingerprint fragment masses
- Determination of protein seq by Mass Spectrometry: Electrospray Ionization (ESI)
Can also use electrospray ionization (ESI) instead of MALDI to get protein into ions gas phase
- Determination of protein seq by Mass Spectrometry: Tandem Mass Spectrometry
-sep and seq mixtures of proteins
-purifies and fragments proteins
-useful for studying proteomics
invaraint
aa do not change
-must have it for function
conserved regions
aa have same polarity
-polarity is imp for function
hypervariable region
aa of varied polarity
-polarity not imp
