BIOC12 Lec 4

Question 1

how is Edman Degradation able to identify residues (overview)

Answer 1

allows identification of one residue at a time from
the N-terminus, thus protein sequencing

Question 2

how is a ptc peptide formed

Answer 2

PITC reacts with the free N-terminal residue of the
amino acid chain

Question 3

how is Anilinothiazolinone
derivative of the residue produced

and how does it happen

why is this residue significant

Answer 3

treat PTC-peptide with anhydrous acid (TFA,
trifluoroacetic acid) the peptide bond

When the PTC-peptide is treated with an anhydrous acid, such as trifluoroacetic acid, the peptide bond of the N-terminal residue is selectively cleaved, releasing an anilinothiazolinone derivative of the residue

This derivative can be extracted with an organic solvent, such as butyl chloride, leaving the remaining peptide in the aqueous phase

Question 4

describe the Edman degradation procedure (slide 14)

Answer 4

1. The N-terminal residue of a polypeptide chain reacts with phenylisothiocyanate to give a phenylthiocarbamoyl–peptide.
2. Treating this derivative with trifluoroacetic acid releases an anilinothiazolinone derivative of the N-terminal amino acid residue.
3. The anilinothiazolinone is extracted and treated with aqueous acid, which rearranges the derivative to a stable phenylthiohydantoin derivative that can then be identified chromatographically.
4. The remainder of the polypeptide chain, whose new N-terminal residue was formerly in the second position, is subjected to the next cycle of Edman degradation.

Question 5

why is removing disulfide bonds important during edman degradation and what compound is used to do this

Answer 5

When a protein contains one or more cystine residues, the disulfide bonds must be cleaved to permit release of the cysteine residues as PTH–amino acids during the appropriate cycles of Edman degradation. Thiol compounds, such as 2-mercaptoethanol, are often used to cleave disulfide bonds. Thiols reduce cystine residues to pairs of cysteine residues

Question 6

How does tandem mass spectrometry work (overview)**

Answer 6

*Use of cleavage agents and proteases
to generate short amino acid chains for
use in Edman or Mass Spec for
sequencing
(describe image too)

Question 7

why is cyanogen bromide
needed to reduce the residues to allow Edman
degradation (sl 20)***

Answer 7

Most proteins contain too many residues to be completely sequenced by Edman degradation proceeding only from the N-terminus. Therefore, proteases (enzymes that catalyze the hydrolysis of peptide bonds in proteins) or certain chemical reagents are used to selectively cleave some of the peptide bonds of a protein. The smaller peptides formed are then isolated and subjected to sequencing by the Edman degradation procedure

Question 8

describe the reaction in slide 20

Answer 8

The chemical reagent cyanogen bromide (BrCN) reacts specifically with methionine residues to produce peptides with C-terminal homoserine lactone residues and new N-terminal residues (Figure 3.20). Since most proteins contain relatively few methionine residues, treatment with BrCN usually produces only a few peptide fragments. For example, reaction of BrCN with a polypeptide chain containing three internal methionine residues should generate four peptide fragments. Each fragment can then be sequenced from its N-terminus.

Question 9

what is the purpose of trypsin and what can the next amino acid not be?

Answer 9

trypsin catalyzes hydrolysis of peptide bond on carbonyl
side of lysine and arginine (positive charge amino acids)
as long as the next amino acid ( C-terminal to
lysine/arginine) is not proline

Question 10

what is the purpose of Staphylococcus aureus V8

Answer 10

cleaves on the
carbonyl side of negatively charged residues (glutamate
and aspartate)

Question 11

what is the correct buffer and what bonds does it cleave for Staphylococcus aureus V8

Answer 11

can be designed using correct buffer ( 50 mM
ammonium bicarbonate) to cleave only glutamyl
bonds

Question 12

what type of bonds is chymotrypsin cleave

Answer 12

hydrolysis of
peptide bonds on carbonyl side of uncharged residues
with aromatic or bulky hydrophobic side chains (e.g.,
phenylalanine, tyrosine, tryptophan)

Question 13

what is the goal for fragmenting

Answer 13

goal is to generate many fragments that can be
separated and sequenced by Edman degradation or
Mass spec

Question 14

important take aways for slide 23

Answer 14

(a) Trypsin catalyzes cleavage of peptides on the carbonyl side of the basic residues arginine and lysine. (b) Chymotrypsin catalyzes cleavage of peptides on the carbonyl side of uncharged residues with aromatic or bulky hydrophobic side chains, including phenylalanine, tyrosine, and tryptophan. (c) By using the Edman degradation procedure to determine the sequence of each fragment (highlighted in boxes) and then lining up the matching sequences of overlapping fragments, one can determine the order of the fragments and thus deduce the sequence of the entire oligopeptide.

blocked N-terminal ends cannot be sequenced
(post translational modification, bacterial proteins
6/7/2024 Brunt 2024 (formylated methionine) effect use of Edman

Question 15

what are the differances between edman and mass spec (slide 25)

Answer 15

refer to slide 25

Question 16

explain an overivew of how protein identifacation works with mass spec

Answer 16

refer to slide 26

Question 17

what are the steps of mass spec in slide 26

Answer 17

Identification of simple protein mixtures from IEF- 2D gel electrophoresis is carried out by enzymatic

the peptide mixture is then analyzed by mass spec

the MS analysis is then carried out for the database

Question 18

slide 27 flagged

Question 19

define the protein structures on slide 31

Answer 19

(a) The linear sequence of amino acid residues defines the primary structure. (b) Secondary structure consists of regions of regularly repeating conformations of the peptide chain, such as helices and sheets. (c) Tertiary structure describes the shape of the fully folded polypeptide chain. The example shown has two domains. (d) Quaternary structure refers to the arrangement of two or more polypeptide chains into a multisubunit molecule.

Question 20

why is primary strcure equvilant to dna

Answer 20

Primary structure is the unique sequence of amino acids encoded
by DNA, read N terminal to C terminal (equivalent to 5’ and 3’ in
DNA/RNA)

Question 21

what are the forces in the terterary strcure

Answer 21

include H bonds, ionic bonding between charged side groups of the
amino acids, Van der Waal forces between hydrophobic side groups
of amino acids where water is excluded (the electrons of one atom
are attracted to the positive charge of another atom but only up to a
certain distance)

Question 22

describe the bonds in the quatinary strcutre

Answer 22

The bonds are the same as in tertiary
structure but may include stronger covalent
bonds such as the disulfide (S-S) bonds
formed between two cysteines within or
between the polypeptide chains. e.g. These
are found in antibody molecules holding the
four chains together.

Question 23

what are the two types of point mutations and what are they

Answer 23

Point mutations refer to those changes in DNA which
occur within a single gene and includes mutations in the
control regions and introns as well as exons.

• Point mutations can be divided into
  deletions/insertions which cause frameshift mutations
  and substitution mutations where one nucleotide is
  substituted for another