Protein Sequencing Flashcards
protease
enzyme that hydrolyzes peptide bond
protease examples
Trypsin
Chymotrypsin
Elastase
what AA does trypsin work on
lysine
arginine
what AA does chymotrypsin work on
phenylalanine
tryptophan
tyrosine
what AA does Elastase work on
Alanine
Glycine
Serine
Valine
zymogens
inactive form of an enzyme
enteropeptidase
cleaves digestive enzymes at specific peptide bonds
what AA residue helps hold fragments together? how?
Cysteine. it can form disulfide bonds
what enzyme activates trypsin
enteropeptidase
what enzymes does trypsin activate
Elastase
Carboxypeptidase
Chymotrypsin
Lipase
what are the 2 methods of protein sequencing
Edman Degradation
Mass Spec
edman degradation 8 step strategy
-separate chains
-cleave disulfide bonds
-determine amino acid composition
-end group determination
-cleave chain into smaller fragments
-repeat previous step
-sequence peptides
-reconstruct sequences
methods to separate polypeptide chains
extreme pH
8M urea
6M guanidine HCl
high salt concentrate
what reagents cleave disulfide bridges
2-mercaptoethanol: cleave bond
Iodoacetate: cap new ends to keep bond from reforming
what is used to analyze AA composition
HPLC
what is AA composition
how many of each AA are present
what is used to determine the N terminal
Dansyl chloride OR FDNB
what is used to cleave chain into smaller fragments
Trypsin
Chymotrypsin
CNBr-cleaves Met
what is used to determine the C terminal
carboxypeptidases
-A: aromatic and aliphatic(not P,K,R)
-B: + charge(K and R)
T/F when repeating step 5 you should use a different cleaving agent than the one previously used
TRUE
what is used to sequence peptides produced from steps 5 and 6
Edmans reagent(PTH)
what is the disadvantage of using edmans reagent to sequence proteins
extremely time intensive(we’re talking years)
how can you determine what is the last/leftover fragment
it will not end with the AA targeted by the cleaving agent
how many AA residues can mass spectrometry measure
up to 25
