protein sequencing (old school and mass spec)

Question 1

Sanger and protein sequecing

Answer 1

• figured out how to determine the first amino acid in a polypeptide
• identifies N-terminus and molar ratio of aa, but not the sequence
  1. label amino end with FDNB
• binds to N-terminal
  2. hydrolyze the peptide with HCL
  3. recover and analyze the altered N-terminal AA
  4. determine molar ratios of free AA

Question 2

edman degredation

Answer 2

• labels and removes only the amino terminal residue and leaves the rest of the protein intact
  1. add PITC to convert aa to PTC
  2. add trifluoroacetic acid to specifically leave the PTC adduct
  3. process is repeated with shortened polypeptide
• limited to 50 or less polypeptides
• to sequence longer polypeptides, fragment into smaller pieces, isolate the fragments and sequence each one

Question 3

what is mass spec used for

Answer 3

• ## alternative method to determine amino acid sequences

Question 4

m/z

Answer 4

• ratio of the mass of an ion to the charge number
• used in tandem mass spec to figure out mass

Question 5

briefly, how does tandem mass spec work?

Answer 5

1. peptides are separated, ionized, and converted to gas phase
2. the first MS separates ionized peptides so that only one type emerges (many, many copies)
3. the sample of the elected peptides is then collided with an inert gas where it is fragmented further
4. the fragments then travel through the second MS, m/z ratios of the fragments are determined
   
   • only the charged fragments are detected
   • land in certain spot in detector

Question 6

why are proteins digested with protease prior to tandem mass spec

Answer 6

• the protease makes smaller fractions

Question 7

abc vs xyz fragments

Answer 7

• proteins treated with protease can have a charge on either side
• abc has charge on amino fragment
• xyz has charge on carboxyl fragment
• mostly b and y ions because breakage at peptide bond is most common