N-terminal Protein/Peptide Sequencing
The Protein Facility utilizes a
Perkin Elmer Applied
Biosystems Model 494 Procise protein/peptide sequencer with an
on-line Perkin Elmer Applied Biosystems Model 140C PTH Amino Acid
Analyzer. The chemical process employed by the protein sequencer to
determine the amino acid sequence is derived from the degradation
method developed by Edman.
In this reaction phenylisothiocyanate (PITC) reacts with the amino
acid residue at the amino terminus under basic conditions (provided
by n-methylpiperidine/methanol/water) to form a phenylthiocarbamyl
derivative (PTC-protein). Trifluoroacetic acid then cleaves off the
first amino acid as its anilinothialinone derivative (ATZ-amino acid)
and leaves the new amino terminus for the next degradation cycle. The
ATZ amino acid is then removed by extraction with N-butyl chloride
and converted to a phenylthiohydantoin derivative (PTH-amino acid)
with 25% TFA/water (see figure 1).
Several by-products are also formed during the Edman degradation
chemistry and are shown in figure 2. The
PTH-amino acid is transfered to a reverse-phase C-18 column for
detection at 270nm. A standard mixture of 19 PTH-amino acids is also
injected onto the column for separation (usually as the first cycle
of the sequencing run). This chrmoatogram provides standard retention
times of the amino acids for comparison with each Edman degradation
cycle chromatogram. The HPLC chromatograms are collected using a
computer data analysis system. To determine the amino acid present at
a particular residue number, the chromatogram from the residues of
interest is compared with the chromatogram from the previous residue
gby overlaying one on top of the other. From this, the amino acid for
the particular residue can be determined (see figure
3).This process is repeated sequentially to provide the
N-terminal sequence of the protein/peptide.
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Sample Amount |
10-100 pmols is preferred, although a lower amount is acceptable. |
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Sample Form |
I. In 30-150 microliters of volatile solvents such as water, acetonitrile, propanol, acetic acid, or formic acid. Pure lyophilized peptide/protein is also acceptable. |
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II. On a PVDF membrane. The sample should be as concentrated as possible on the PVDF membrane (e.g. 1 µg/lane). Several bands can be used. The bands should be stained with Coomassie Blue, Ponceau S, or Amido Black. After staining/destaining, a blotted membrane must be rinsed thoroughly with deionized water. The whole membrane may be submitted with the bands marked or the bands may be cut out and submitted. |
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Purity |
Liquid samples should contain only one protein or peptide. Buffers, SDS, salts, amino acids, primary amines, and other contaminants must be removed from your sample. These contaminants affect Edman degradation reaction on the instrument, contaminate the instrument or affect PTH amino acid detection. Samples submitted on PVDF (blotted from SDS-PAGE gels) should have well separated bands to minimize contamination. |
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Cysteine Modification |
Since unmodified Cys residues cannot be detected, Cys should be modified before sample submission if you wish to identify Cys. Procedures for modification can be obtained from the Protein Facility. |
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N-terminal blockage |
If the amino terminus is blocked, the protein or peptide cannot be sequenced using Edman degradation. We perform de-blocking procedures and will discuss options with you if N-terminal blockage is suspected |
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Glycosylation and other Modifications |
Glycosylated amino acids and phosphorylated amino acids may result in blank cycles, reduced peaks or altered retention times. |
- Sample Preparation:
- Most investigators will run their sample on an SDS-PAGE gel and then electroblot to PVDF, stain with Coomassie and cut out the band of interest. You must use PVDF, not nitrocellulose. Multiple lanes from the blot can be loaded onto the sequencer.
- If you can visualize the bands on the PVDF membrane with Coomasssie there should be sufficient material for sequencing. You may either send the whole membrane with the bands clearly marked and we will cut them out in our lab or you can excise the bands of interest and send them in a micro-centrifuge tube.
- You will need to either use pre-cast gels or let the gel polymerize overnight before running your sample. This will help eliminate any N-terminal blockage due to unpolymerized acrylamide in the gel. Be sure to use high-grade methanol as well.
- Samples can also be sent in liquid form if they are free of salts, buffers, amines, etc and contain a single protein.
- Liquid samples containing buffers or other contaminants can be cleaned up using an ABI ProSorb device (we charge $14.00 per sample).
- Sample Shipping:
Download the submission form and fill it out completely. Include the submission form with the samples when they are submitted. Samples will not be processed without a submission form or if no payment method is indicated. Samples may be submitted to the address below. - Turnaround time:
Usually 1-2 days. - Protein/Peptide Sequencing Submission Form (PDF)
