N-terminal Sequencing

N-Terminal Sequencing uses a chemical process based on the technique developed by Pehr Edman in the 1950’s where by;

The N-terminal amino acid reacts with phenylisothiocyanate (PITC).
The derivatizing process results in a phenylthiohydantoin (PTH) – amino acid.
This amino acid is then sequentially removed while the rest of the peptide chain remains intact.
Each derivatization process is a cycle.
Each cycle removes a new amino acid.
The amino acids are sequentially analyzed to give the sequence of the protein or peptide.

Instrument on site is an Applied Biosystems Procise Protein Sequencer - this instrument is well suited to high sensitivity low picomole analyses.
Routinely used for the analysis of membrane bound gel electrophoresis separated proteins, HPLC separated tryptic digest fragments.
Multiple sample cartridges facilitate 24/7 operation, allowing sequencing of several proteins with low turn around time.
Optimized sequencing chemistry allows complete sequencing of small proteins such as toxins and chemokines
Compatible with proteins and peptides prepared from a variety of sources, such as HPLC separation, PVDF blotting following electrophoresis, and peptide synthesis
Minimum of 12 picoMoles of a purified protein per run is required. In practice, due to normal losses that occur during subsequent processing, this may require 100pM or more to be loaded per lane on a 1D acrylamide gel
Acetylated or biotinylated samples will not be accepted since they cannot be sequenced (the N-terminus will usually be blocked
Nitrocellulose is not compatible with the Edman chemistry.
No sequence will be seen if the protein is N-terminally blocked either naturally (e.g. by formyl, acetyl or pyroglutamyl groups) or accidentally during isolation and storage (see above Note regarding in solution samples).
Cys will give a blank result unless reduced and alkylated 4-vinyl pyridine is the recommended alkylating agent. Specify how Cys was modified.

PVDF Blots: Electroblotted proteins on PVDF membranes are stained by coomassie R250 or Poncaue red and then loaded directly on the sequencer.
Dry Sample: The sample will be reconstituted in 0.1% TFA/20% acetonitrile

Sample amount / concentration
Sample format: Solid (dried, blot (stain used), or gel), liquid (buffer composition)
Number of residues requested.

Liquid Lyophilized samples should be provided as concentrated as possible and in low salt buffers.
Protein can be submitted dry, in solution, as an SDS or native polyacrylamide gel piece, or blotted to PVDF membrane. Nitrocellulose is not compatible with the Edman chemistry.

Dry Sample: The sample will be reconstituted in 0.1% TFA/20% acetonitrile so it should be soluble in this buffer.

PVDF blotted samples:

Blotting is done as for a Western, but without blockers or antibodies and must be thoroughly washed.
Stain with Coomassie R-250 only or Poncaue Red, not G-250.
Destain blots enough to leave target bands visible, wash with distilled and deionised water and dry. They can then be stored @4 oC for several weeks.
As the protein is to be extracted from SDS gels, urea used as an electrophoresis reagent must be very pure and should have no cyanate ions present as these will carbamylate the proteins and block them. Cyanate ions form particularly at an alkaline pH.
Urea solutions used in gel preparation or solubilisation should be made from ultra high purity urea.
To avoid further carbamylation, samples containing urea should not be stored for long periods and should not be heated.
Ampholines used to create pH gradients can also contain amines and reduce the efficiency of sequencing. IEF strips pre-prepared by Bio-Rad or Pharmacia do not present this problem.
Protein can be passively eluted from polyacrylamide in an overnight procedure but is generally less efficient than electroblotting and does not work with high mw proteins, ideally proteins should be less than 60kDa.

Samples in solution:

Must be supplied with a minimum protein concentration of 1ug/ml with a sufficient volume to obtain at least 10pM/run.
The buffer composition must be provided with the sample since certain solutes may need to be removed before sequencing can be done.
Sample should be free from interfering buffers and salts. Buffers containing primary amines, such as TRIS and HEPES, should in particular be avoided, although low concentrations of SDS (up to 0.3%) and PBS can be tolerated.
If interfering buffers/salts are unavoidable it should still be possible to sequence the material after clean up.