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 Edman N-terminal Sequencing

 Professional Protein Sequencing Service

1. N-Terminal Sequencing (N-TS)

1.1 Equipment

1.2 N-Terminal Sequencing Principle

COUPLING
WASHING
CLEAVAGE
EXTRACTION
CONVERSION
RESOLUTION
ANALYSIS
REPEAT
RESULT EXAMPLE

1.3 Our Recommendation for Sample Preparation

1.3.1 Reagents May Potentially Interfere With Edman Degradation
1.3.2 Sample Purity
1.3.3 Amount
1.3.4 Approximate Molecular Weight
1.3.5 Modification
1.3.6 Sample Type or Dye
1.3.7 Sample Losses
1.3.8 Tips for SDS Gel Electrophoresis Prior to Sequencing
1.3.9 PVDF Types for Transferring Sequencing Samples from SDS Gels
1.3.10 Details for Sample Shipping to Creative Proteomics

1.4 Our Recommendation for Protein Blotting

1.4.1 Semidry PVDF Blotting Protocol
1.4.2 Protein Staining of PVDF Membranes
Ponceau Red Staining
Sulforhodamine Staining
CBB R250 Staining

1.5 Application Field of N-Terminal Sequencing

 1. N-Terminal Sequencing (N-TS)

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 N-terminal Sequencing (also called Edman sequencing) uses a chemical process based on the technique developed by Pehr Edman in the 1950's.

N-terminal sequencing is a method to excise amino acid one by one from N-terminal of protein or peptide to be segregated by HPLC (reversed phase HPLC), then to detect by UV to determine amino acid sequence by the time the peak is gained (Chromatogram).

Pure proteins (>90%) usually generate easily interpreted data, but insufficiently purified protein mixtures may also provide useful data.

Long sequences of 50 amino acids or more are possible with this technique.

 1.1 Equipment

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AB Procise 49X cLC Protein Sequencing System

It is a fully automated sequencer which employs Edman degradation, PTH derivatization of amino acids and HPLC separation followed by UV detection. The system is qualified as per vendor’s OQ/PV protocol which ensures the optimum performance.

 1.2 N-Terminal Sequencing Principle

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 COUPLING

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In the first step the free N-terminal amino acid is converted by PITC(phenylisothiocyanate) to its phenylthiocarbamyl (PTC) derivative under mild alkaline conditions (provided by n-methylpiperidine/methanol/water).

 WASHING

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The PTC-protein is washed with ethyl acetate and n-heptane to remove excess PITC.

 CLEAVAGE

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The PTC protein is then cleaved with neat trifluoroacetic acid, resulting in the formation of an intermediate anilinothiazolinone derivative (ATZ-amino acid), leaving the new amino terminus of the protein for the next degradation cycle.

EXTRACTION

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Following cleavage the ATZ amino acid is extracted with N-butyl chloride.  

CONVERSION

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The intermediate is converted to the more stable phenylthiohydantoin derivative (PTH-amino acid) with 25% TFA/water.

 

RESOLUTION 

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The PTH-amino acid together with several by-products formed during the Edman degradation chemistry is injected onto a C-18. PTH-amino acid is subsequently separated by HPLC. The retention time of the PTH amino acid is compared with the retention times of a standard mixture of 19 PTH-amino acids for identification. This standard chromatogram provides standard retention times of the amino acids for comparison with each Edman degradation cycle chromatogram.  

ANALYSIS  

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The HPLC chromatograms are collected using a computer data system for analysis. To determine the amino acid the chromatogram from the residue of interest is compared with the chromatogram from the previous residue by overlaying one on top of the other.

REPEAT

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This procedure is repeated for identification of the next amino acid again and again until the original peptide sequence is deconvoluted. 

RESULT EXAMPLE

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N-terminal Sequence: NH2NSXXECPXSH (X represents some kind of amino acid, confidential)

 1.3 Our Recommendation for Sample Preparation

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The following may be helpful in the preparation of samples for submission.

 1.3.1 Reagents May Potentially Interfere With Edman Degradation

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The sample should be in a volatile solvent such as H2O, acetic acid, acetonitrile, propanol, formic acid, TFA, TEA, or a volatile buffer such as ammonium bicarbonate. Detergents are particularly problematic in sequencing and should be avoided if possible. Small quantities of detergent may be used (i.e., 0.02% Tween 20, 0.01% Brij 35, 0.1% SDS, and 0.05% Triton X 100). All reagents used to resolve and solubilize proteins should be of the highest quality. Reagents that contain primary amines (Tris, glycine and ethanolamine) should be avoided. In addition, it is important to note that dialysis membranes are often a source of interfering contaminants. If dialysis is necessary in the purification process, only high quality, thoroughly cleaned membranes (e.g., Spectropor) should be used.
When samples are transferred to PVDF membranes many of these difficult reagents can be washed away with water and 50% methanol.
  

These reagents should be avoided:
A. non-volatile buffers
B. non-volatile amines
C. free amino acids
D. detergents and salts

What Buffer the Sample Is in, If Any? Please let us know.

 1.3.2 Sample Purity

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The sample should be as pure as possible (at least 75-80% purity) and contain only the protein or peptide. Purity below this point makes it difficult to identify which residue resides with which protein. A HPLC clean up of your sample is recommended when purity below this point, please contact us. As a general rule we suggest that soluble proteins for sequencing be more than 80% pure.

 1.3.3 Amount 

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A minimum of 10-100 picomoles (10 picomoles of a 50 KDa protein equals 500ng) of material is requested, although a lower amount is acceptable. Sequence can be obtained from less material (we have obtained sequence from samples with a starting amount of 10 picomoles), however, for lesser amounts, yield and quality of sequence decreases as sample starting material decreases. It is suggested that 3-5x as much sample as needed is prepared.  

More material will allow us to ensure that sequence interpretations are correct and to determine if the amino-terminal is blocked and no sequence is obtained. 

Sample amount can be estimated from Coomassie Blue or silver-stained gels or determined by a number of protein quantization assays.  

 1.3.4. Approximate Molecular Weight

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Long sequences of 50 amino acids or more are possible with this technique.    

 1.3.5 Modification

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  N-Terminally Blocked

 

Blocked N-termini provide the single largest impediment to protein sequence analysis. Proteins and peptides which are N-terminally blocked do not have a free N-terminal amino group. Therefore these proteins cannot be sequenced. An estimated 50-80% of all proteins naturally have chemically modified N-termini. More than 50% of all eukaryote proteins are blocked. Many proteins may be blocked at the N-terminus either by natural processes or by modification during manipulation and purification. Modifications include:

A. formyl groups
B. acetyl groups
C. pyroglutamic acid residues

Many proteins may be blocked at the N-terminus by modification during manipulation and purification via the following:


A. handling steps at elevated pH (>9.0)
B. inferior grade reagents and water (use the highest quality available)
C. due to an exposure to elevated temperatures if a glutamine is the N-terminal residue
D. protease inhibitors may react with amino groups
E. use of formic acid (e.g. in CNBr cleavage) may formylate the N-terminus- use 50-70% TFA as an alternative
F. not deionizing reagents such as urea - urea can be deionized on an ion exchange resin (e.g. Amberlite) prior to use  

If Your Sample Is N-Terminally Blocked:

Depending upon the nature of the modification, some N-terminal blocking groups can be removed with mild acid treatment. Failing this "quick fix" the standard solution is to carry out chemical or enzymatic cleavages followed by HPLC purification. On request we can perform deblocking procedures but we need a significant higher amount of protein and the deblocking does not work always, because mostly the type of blockage is unknown.

We will discuss options with you if N-terminal blockage is suspected.

Cysteine Containing and Modifying Procedure Used

Cysteine without special modification cannot be detected by N-terminal sequencing. Cys should be modified before sample submission if you wish to identify Cys.

Is the Protein Glycosylated and to What Extent If Known?

Edman sequencing steps without the detection of a PTH amino acid, reduced peak intensity or altered retention times can be caused by glycosilation, phosphorylation or other modifications. These amino acids cannot be sequence often.
Some of these modified amino acids can be identified using "Identification of Modified PTH-Amino Acids in Protein Sequence Analysis" by Mark W. Crankshaw and Gregory A Grant.

 1.3.6 Sample Type or Dye

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The samples can either be lyophilized, in solution or blotted on to PVDF membrane.

A. In 30-150 microliters of volatile solvents. The sample has to be shipped in frozen state!
B. Pure lyophilized peptide/protein is also acceptable.
C. On a PVDF membrane (blotted from SDS-PAGE gels).The sample should be as concentrated as possible on the PVDF membrane (e.g. 1 µg/lane, PVDF membrane size: max. 3x6 mm, smaller and more concentrated sample are preferred.). Several bands can be used. The bands should be stained with Coomassie Blue, Ponceau S, Amido Black or Sulforhodamine. 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.

 1.3.7 Sample Losses

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Sample loss of small amounts of protein is often caused by adsorption to purification apparatus. To avoid this problem try eliminating as many handling steps as possible.

A. Avoid dialysis by applying sample from gel filtration or ion exchange chromatography directly to a reverse phase HPLC separation set up.
B. Use polyethylene or polypropylene micro centrifuge tubes keeping protein mass to plastic area as high as possible.
C. Avoid drying or lyophilizing samples completely.

 1.3.8 Tips for SDS Gel Electrophoresis Prior to Sequencing

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A. Use ultra-pure reagents.
B. Make up stock acrylamide solution fresh every 2 to 3 weeks.
C. De-ionize the acrylamide stock solution with ion exchange resin and store it in a brown glass container in a cool place.
D. Polymerize gel overnight to minimize any amino-reactive free acrylamide.
E. Pre-run the gel (5-10 minutes) before loading your sample.
F. Denature sample for 0.5-1hr at 65oC - avoid boiling sample.

 1.3.9 PVDF Types for Transferring Sequencing Samples from SDS Gels

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A. Weak binding PVDF-suitable for proteins from 5kD up to <100kD
Immobilon-P membranes/Millipore Corp.
B. Strong binders-more suitable for small polypeptides (<5kD)
Perkin Elmer/Applied Biosystems' "ProBlott"
BioRad's "Transblot"
Millipore's "Immobilon P (SQ)"
C. Nitrocellulose is not compatible with the Edman chemistry.

 1.3.10 Details for Sample Shipping to Creative Proteomics

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A. Please, fill the Edman protein sequencing form with information about the samples (e.g. sample name, sample type, MW, protein amount, staining method) and the requested number of N-terminal sequencing steps. Sign the form!
B. Send us an email before shipping the samples
C. The samples should be in an Eppendorf reaction tube sealed by Parafilm. Protein samples on PVDF membrane can either be shipped as small and dry membrane slides in reaction tubes or as a dried PVDF membrane in plastic foil with enclosed description of the protein bands which should be analyzed. We cut the marked protein band for analysis.
D. Liquid samples should be sent in frozen state.
E. Lyophilized sample and samples blotted on PVDF membrane can typically shipped at room temperature.
F. Please, send your samples in a padded envelope or in a box together with the Edman protein sequencing form.  

 1.4 Our Recommendation for Protein Blotting

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 1.4.1 Semidry PVDF Blotting Protocol

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We recommend the following protocol for semidry blotting on PVDF membrane:
- PVDF membrane: Immobilon P membrane or comparable
- Blot buffer: 50mM sodium borate, pH 9.0 / 20% methanol (HPLC quality)
(0.1% SDS can be added to blotting buffer if protein above 40kDa should be sequenced)
- Blotting condition: 1mA/cm1 PVDF membrane for 2-3 hours at 4°C

 1.4.2 Protein Staining of PVDF Membranes

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 Ponceau Red Staining

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Staining solution (0.5% Ponceau S, 1% acetic acid in Milli-Q water):
0.25g Ponceau S
0.5mL acetic acid
Add 50mL Milli-Q water

Procedure:
1. Wash the PVDF blot membrane 2x 3 minutes with plenty Milli-Q water.
2. Stain the PVDF membrane with Ponceau S staining solution for 1-3 minutes.
3. Destain the PVDF blot membrane under visual control with Milli-Q water until protein bands are well visible.
4. Dry the PVDF membrane.

 Sulforhodamine Staining

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Staining solution (0.005% sulforhodamine, 0.2% acetic acid, 30% methanol in Milli-Q water):
150mL methanol
1mL acetic acid
25mg sulforhodamine
Add 500mL Milli-Q water

Procedure:
1. Wash the PVDF blot membrane 2x 10 minutes with plenty Milli-Q water.
2. Dry the PVDF membrane at room temperature!
3. Stain the PVDF membrane in Sulforhodamine staining solution for 1-2 minutes.
4. Wash the PVDF membrane with Milli-Q water shortly.
5. Dry the PVDF membrane.

 CBB R250 Staining

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Staining solution (0.1% CBB R250, 10% acetic acid, 40% methanol in Milli-Q water): 0.1g CBB R250
40mL methanol
10mL acetic acid
Add 100mL in Milli-Q water

Destaining solution (10% acetic acid, 40% methanol in Milli-Q water):
40mL methanol
10mL acetic acid
Add 100mL Milli-Q water

Procedure:

1. Stain the PVDF blot membrane for 5 minutes in CBB R250 staining solution
2. Destain the PVDF blot membrane for 3 x 5 minutes with destaining solution under visual control until protein bands are well visible.
3. Dry the PVDF membrane.

 1.5 Application Field of N-Terminal Sequencing

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Improvements in this field have allowed minute quantities of purified protein / peptides to be easily sequenced. Protein Sequencing can now be routinely used as an analytical tool in life science research

A. Determination the N-terminal amino acids of a protein
B. Determination the N-terminal amino acids of a peptide
C. Check of the correct translation of a recombinant protein
D. Check of the sequence and purity of a recombinant protein
E. Check of the sequence and purity of a synthetic peptide
F. Determination of complete protein sequence in combination with additional methods
G. Quality control N-terminal sequencing is most commonly used to identify unknown proteins, confirm protein identity and quality (often for quality control of recombinant proteins), and identify protein N-terminus and cleavage sites
H. Homology based protein identification.

We have extensive experiences in this technology and will work with you from any steps in the workflow. Please contact us for a free project consultation.

 

     

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