Sequences of Proteins

Question 1

The Sequence of Amino Acids
in a Protein is…

Answer 1

• is a unique characteristic of every protein
• is encoded by the nucleotide sequence of DNA
• is thus a form of genetic information
• is read from the amino terminus to the carboxyl
  terminus

Question 2

MALDI Mass Spectrometer:

Answer 2

• Ions are generated by a LASER firing at the
  target plate
• The time of firing of the LASER and the
  arrival time of the ions at the detector are
  known, the relative masses can then be
  calculated
• Only singly charged ions are generated, other
  types of spectrometer may generate multiply
  charged ions

Question 3

Peptide Mass Fingerprinting:

Answer 3

A mass spectrum of the peptide mixture resulting
from the digestion of a protein by a proteolytic
enzyme.

• Choice of Enzyme
• Search Masses
• Constraining the Protein Molecular Weight
• Which masses to include in a search
• Modifications

Question 4

Sequence Databases:

Answer 4

• International databases of protein sequences are
  maintained
• Many of these databases are accessible via the
  internet
• Examples:
• GenBank
• Protein Identification Resource (PIR)
• European Molecular Biology Data Library
  (EMBL)

Question 5

Synthetic Peptides:

Answer 5

: Essential Tools for Bioscience

Synthetic peptides have become essential tools in all areas of:

-antigen-antibody research

-epitope mapping

• pharmaceutical development
• structure-function studies.

Question 6

Solid-phase peptide synthesis:

Answer 6

A cyclical, three-step process
Solid-phase peptide synthesis, pioneered by Dr. R.B. Merrifield in 1963.

It involves the successive addition of amino acids to create a linear
peptide chain.

The C-terminus of the growing peptide is covalently bound to a
solid support, or resin, during synthesis.

Question 7

Three chemical reactions are repeated for…

Answer 7

each amino acid that
is added to the peptide chain:

1. deprotection
2. activation
3. coupling

Question 8

During coupling, the active ester forms an…

Answer 8

amide bond with the
deprotected amino group on the end of the peptide chain.
After coupling, a new cycle of synthesis begins with the next deprotection.
When automated synthesis is complete, chemical “cleavage”
removes side-chain protecting groups from the peptide and the
synthetic peptide from the resin support.

Question 9

Synthetic peptides have a…

Answer 9

wide variety of uses as biological
probes and in the pharmaceutical industry.

The most popular synthetic approach is the Merrifield solid
phase synthesis technique.

Question 10

Merrifield solid
phase synthesis technique:

Answer 10

This method utilizes an inert, insoluble polymer as the
support for repetitive, stepwise condensation of the amino
acids to the growing chain.

In outline, the Merrifield procedure entails:

1. Anchoring C-terminal amino acid to the support resin
2. Deblocking and Condensation (repeat as necessary)
3. Release of peptide from the resin