|
|
|
|
|
Protein Sequence
Analysis and Sample Preparation
|
ABI Procise
|
N-Terminal Sequence Analysis
- 20 pmol or more of sample is required for routine analysis
- samples can be submitted in lyophilized/liquid form in Eppendorf tube or on a PVDF
membrane
- turn around times: 1-3 working days
Minimum of 5 residues required. Prices include desalting and adsorptive binding
of the sample to PVDF membrane, if necessary Guidelines
for Sample Preparation can be downloaded.
|
|
Procedure: |
Figure 1: Overview
Sequence Analysis is performed on new, state of the art
instrumentation (Procise 494 HT, Perkin-Elmer) using Edman chemistry. Edman degradation
can be used to sequence proteins rapidly, and with little starting material. The process
is easily automated, and sequencers can operate sometimes with as little as a few
picomoles of sample. The machine performs the coupling, cleavage, conversion and
identification steps for each amino acid residue, and researchers often choose to identify
10-15 residues from the protein. The protein may then be identified by searching a
database to match the sequence and properties of the protein.
See description of the
process followed.
|
Figure 2: Chemical Description
In Edman sequencing, a protein is attached to a
solid support such as a chemically modified glass disk or a porous polyvinylidene fluoride
membrane in the reaction cartridge. It is then coupled to phenylisothiocyanate
(PITC) at
pH 8 and 45ºC. The free N-terminal amino group reacts with the carbon of the
isothiocyanate group to give the phenylthiocarbamyl (PTC) derivative of the peptide. The
next step is cleavage of the PTC derivative using anhydrous trifluoroacetic acid to give
the anilinothiozolinone (ATZ) derivative of the N-terminal amino acid, and the peptide
with one fewer amino acid, which is free to undergo further couplings and cleavages.
The
ATZ residue is then filtered into the conversion flask, where it is converted to the
phenylthiohydantoin (PTH) amino acid. This is a two step process. First, the ATZ
derivative is hydrolyzed under aqueous, acidic conditions to give the PTC amino acid. The
acid then cyclizes to give the stable PTH derivative. These derivatives are then injected
into an HPLC column where its retention time is compared with that of known PTH amino acid
standards. |
|
|
|
