Amino Acid Sequence Flashcards
How can we identify the first amino acid (first method)
- tag with fluorodinitrobenzene at N term (bright yellow)
- when amino group is deprotonated, it can react with the fluorodinitrobenzene and react with the florine
- HF is a good “leaving group”
What is at the N term of amino acid
free amino group
How can you make the N term reactive
- at high pH, amino group becomes NH- (a nucleophone)
- has free electrons and is reactive
-
What is the big snag
- hydrolysis: realseases the N-terminal amino acid with yellow chain attached
- hydrolysis destroys the rest of the polypeptide side chain
- note this only identifies the N term amino acid
How did Per Edman refine identifying amino acid sequence
- method does not have hydrolysis, do not lose the rest of the protein
- method allowed N-term aa to be to be reacted and identified
- part 1: coupling: required base (makes amino group a nucleophilic), reaction must be complete before next step (labels N-term AA with PITC)
Part 2: requires an acid (cleaves first peptide bond)
-must be carried in anhydrous mixture, or else other bonds will be broken
*know reagent: phenylisothiocynate (PITC)
**just understand whats going on **
** method can only be carreid for about 50 cycles, to much error*
What is the molecular bio approach for determining amino acid sequence?
-sequence DNA then get amino acid sequence (automated)
How do you identify long protein chains
- take long protein and cut into shorter oligopeptides by selective hydrolysis
- bigestive enzyme trypsin binds and recognizez Arg and Lys side chains, will only hydrolyse bonds on C term end of these aa (made so only arg or lys can make it into the typsin “pocket”
- carboxylate group of Arg or Lys is positioned next to catalytic unit of trypsin, targets hydrolysis of the peptide bond
- not if attached to a proline, no hydrolysis occures if lys/arg are attached to a pro
- NOTE: at actual cterm end will not have am arg or lys
What is selective hydrolysis
cuts polypeptide at specific locations to yeild a limited number of oligopeptides of definite size
What is chymotrypsin
- identifies phe and trp, and can hydrolyse those bonds
What is cyanogen bromide?
- reagent which cuts polypeptide chains at methionine residues
- attacks S atom of Met
- polypeptide chain is broken on carboxylate side, and met is conversted to homoserine, Hse (serine with extra CH2)
How many peptides wil be generated by treating the peptide below with CNBr?
SETKLMRTILWMPDDTASDMHTPIIN
- hydrolyses at C terminal end even if there is a proline
- cuts after each M
- make 3 cuts and have 4 fragments
How are along aa idenfitied using mass spec?
- use mass spec if known (tandem mass spec)
- uses tiny amounts
1) hydrolyzed by protease (hydrolyse the multiple samples of protein differently)
2) First Ms-1: separate peprides of diferent masses (separates the fragments
3) Collision cell: each fragment broekn into 2, one peptide bond is broken in every fragment that goes in (where it is broken is random)
4) second MS-2: measure fragment masses - somehow need to break every bond
What is the overlap mathod
- two samples treated with different hydrolysis methods
- sequences from one set of oligopeptides are lined to loverlap with oligopeptides from another set to deduce how initially joined
*just match up the two sets of same amino acids*
Why is how peptides are generated important?
- when generated with trypsin, each peptide has K or R at C term
- peptides generated at low pH
- acid residues have no charge on side chain (COOH)
- basic residues have +1 charge (NH3+)
- peptides with CHARGES produce highest signal ie: ones with K or R at C term *want to be reading in order of N-C, remember to flip
- mass spec will better regognize fragment with charge
What is Ninhydrin?
reacts with amino groups to give purple colour
detect ad measure quantitiy of aa in chromotography
