The Rockefeller University Proteomics Resource Center

The Edman Sequencing Service

Table of Contents     

  • See our latest Poster Presentation from the ABRF meeting (3/14/00) (Requires Adobe Acrobat Reader).

 

Introduction

The Edman Sequencing Service offers two services for protein characterization and identification: 1) N-terminal sequence analysis using automated Edman degradation chemistry and 2)  internal protein sequence analysis by Edman degradation chemistry after in-gel or PVDF bound protein digestions followed by HPLC separation of the resulting peptides (1-3). The following information is provided in order to answer basic questions regarding these services. However, we strongly encourage you to come in, call (1-212-327-8869), or email Joseph Fernandez to discuss your sample with us, as every situation is unique.

N-terminal Sequence Analysis

N-terminal sequence analysis of a protein or peptide consists of repetitive cycles of the Edman chemistry followed by PTH analysis using microbore HPLC. One cycle of the Edman chemistry and the resulting PTH chromatogram represents identification of one amino acid. Each cycle of the chemistry takes 30-50 minutes depending on the instrument used. If the protein has a blocked N-terminus, no data can be obtained. It has been estimated that 40 - 70% of all naturally occurring proteins are N-terminally blocked.

Chemistry: Automated Edman degradation chemistry consists of three steps, a) coupling of phenylisothiocyanate (PITC) with the alpha-amino group of the protein/peptide at pH 9-10 to form a phenylthiocarbamyl (PTC) group, b) cleavage by anhydrous acid (trifluoroacetic acid (TFA) to generate an anilinothiazolinone (ATZ) amino acid and c) conversion of the ATZ derivative to the more stable phenylthiohydantoin (PTH) derivative. Solvent extractions between the above steps wash out by-products and excess reagents. Finally, the PTH amino acids are analyzed by microbore HPLC. It should be noted that cysteines are destroyed by the chemistry and therefore cannot be identified unless reduction and alkylation is performed. We routinely monitor for modified the cysteine derivatives carboxyamidomethylcysteine (CAMC), which is generated during sample preparation and propionamide cysteine (PAMC), which is formed through a side reaction of free acrylamide with cysteine during electrophoresis. Post-translational modifications can present their own individual problems.

Instrumentation: The Service has two automated protein sequencers. One Applied Bioystems (ABI) 494 gas-phase/pulsed-liquid Procise-HT sequencer is equipped with a 190C PTH analyzer, and the other ABI 494 pulsed-liquid Procise-HS sequencer is equipped with a capillary 140B PTH analyzer.  The ABI 494HT is used for moderate levels (1-10 pmol) of HPLC purified peptides as well as low-level PVDF-bound proteins. The ABI 494 HS is used only for very low-level HPLC purified peptides (0.1-1 pmol). All instruments analyze peptides suspended on a glass fiber disc using a polymer (polybrene) to retain samples through hydrophobic interactions. 

Yields and expectations: The performance of each instrument and individual sample is characterized by initial and repetitive yields as well as carryover. The initial yield (typically 50-80% based on standard proteins) refers to the quantity of amino acid recovered in the first cycle of the Edman chemistry and is expressed as a percentage of the total sample analyzed. The repetitive yield, typically 90-99%, represents the recovery of the PTH amino acid after each cycle of the chemistry, and is dependent on the instrumentation as well as the individual characteristics of the sample. Carryover (or lag) is the amount of the previous amino acid present in the subsequent cycle.  The expected number of amino acids which can be determined for a particular protein or peptide sample is dependent on its quantity, purity, and size. For example, 10 pmol of a 100 kD protein (on PVDF or solution) with an initial yield of 50% would yield 15-40 residues of usable sequence data depending on its structure and purity, while 10 pmol of an HPLC purified peptide fragment 20 amino acids long with an initial yield of 50% could probably be completely sequenced.

Internal Edman Sequence Analysis

The Service performs enzymatic digestion of SDS-PAGE purified proteins for internal protein sequence analysis.  The peptide fragments produced are analyzed by Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) and purified by reversed-phase HPLC (0.3 mm ID or 1.0 mm ID).  The HPLC fractions are then screened by MALDI-TOF MS to screen for peptides suitable for Edman sequence analysis.

Digestion of PVDF samples: PVDF-bound proteins are digested with an enzyme of choice (trypsin, endoproteinase Lys-C, endoproteinase Glu-C, clostripain, endoproteinase Asp-N, thermolysin) in the presence of a non-ionic detergent such as hydrogenated Triton X-100 (1-3). Before the enzyme is added, cysteines are reduced with DTT and alkylated with iodoacetamide to generate carboxyamidomethyl cysteine which can be identified during N-terminal sequence analysis. PVDF-bound proteins are suitable for both N-terminal and internal Edman sequencing analysis.  Also, PVDF-bound samples can only be purified on a 1 mm x 250 mm HPLC column.   Please call to discuss your sample before submitting it for analysis.

Digestion of in-gel samples:  Proteins in an SDS-PAGE minigel can be reliably digested in-gel only with trypsin (3).    MALDI-TOF MS of the digestion mixture (5% of the total) is performed and the remainder is purified by reversed-phase HPLC.  Please note, N-terminal Edman sequencing of an undigested protein is not possible with proteins in a gel.  Please call to discuss your sample before submitting it for analysis.

Microbore HPLC purification: The Protein/DNA Technology Center has one microbore Hewlett-Packard 1090 HPLC and one Applied Biosystems Model 173 Capillary HPLC for analysis and purification of peptide mixtures using reversed-phase chromatography (C18 column). In-gel samples will be purified using a 0.3 mm ID x 150 mm column using the capillary HPLC while PVDF samples will be purified using a 1.0 mm ID x 250 mm ID column.  Several samples can be compared to each other by comparison of the HPLC chromatograms ("HPLC Peptide Maps"). Peptides collected from the HP-1090 HPLC are stored in approximately 200 fractions. Fractions will be temporarily stored for one year but final storage is the responsibility of the investigator. Peptides purified using the ABI Capillary HPLC will be collected into a 96 well microtiter plate.

MALDI-TOF MS of HPLC fractions: MALDI-TOF MS analysis is used for selecting HPLC fractions for N-terminal analysis. The selection criteria are: a)  the apparent purity of the HPLC fraction (apparent because MALDI-TOF MS is not uniformly quantitative) and b) the mass and thus the estimated length of the peptide.  The peptide mass information is useful for confirming the Edman sequencing amino acid assignments, and also in the possible detection of post-translational modifications.

The choice between PVDF and gel samples:  PVDF-bound proteins can be analyzed by N-terminal sequence analysis as well as used for internal sequence analysis.  However, the detergents used for releasing digested peptides from the membrane can interfere with MALDI-TOF MS analysis.  Proteins in a gel can only be analysed by  internal sequencing analysis, but very accurate peptides masses can be obtained that provide additional information that can be useful in both amino acid assignment and database searching.   Also, in-gel digests are suitable for purification on the higher sensitivity HPLC system.  We recommend that investigators contact us in order to determine the best approach to their specific problems. 

 

Sample Submittal

Note: Please contact the Edman Sequencing Service before final preparation of the sample.

Amount required:

  1. N-terminal Edman sequencing: 5-50 pmol (data has been obtained with as little as 1 pmol of purified protein).
  2. Internal Edman sequencing analysis: 5-100 pm (1-5 µg depending on sample molecular weight).

Note: Each internal sequence sample must have a suitable blank (negative control).

Sample conditions :

  • N-terminal sequence analysis :
    • PVDF: stained as described below, submitted as a dry membrane.
    • Solution: volume less than 100 microliter, volatile buffer with very little salt content.
  • Internal sequence analysis:
    • PVDF : stained as described below, dry, maximum practical limitation for sample amount should be approximately 20 lanes on a minigel. Best results are obtained when the sample is concentrated to the fewest number of lanes as possible without overloading.
    • Gel: gel samples must be limited to 1-3 lanes from a mini-gel (1 mm thick max) and can only be stained with 0.5% Coomassie blue G-250 as described below. 
    • Solution: please contact the facility before submitting sample.
    • Nitrocellulose: is not recommended because of lower recovery of peptides.
    • Note: Please submit all samples in 1.5 ml polypropylene microcentrifuge tubes for efficient handling. Thank you.

Sample Preparation

When preparing samples for microsequencing, it is best to limit sample manipulations. When dealing with solution samples, never bring the sample to complete dryness. Based on extensive studies, we have found that SDS-PAGE or 2D IEF-SDS-PAGE (or optionally transferred to PVDF) is the best method for recovery of low quantities of protein. However, several other methods are listed below:

  • DESALTING: the best method for desalting is SDS-PAGE . Samples can be desalted by reversed-phase HPLC, but the recoveries are variable.
  • ELECTROELUTION: if proteins have been prepared by electroelution, they should be precipitated (TCA, ethanol) following electroelution in order to remove detergents and salts. Alternatively reversed-phase HPLC can be used but the recovery is variable.

Proteins submitted for internal sequence analysis are routinely reduced and carboxyamidomethylated prior to digestion.

Proteins transferred to PVDF: Samples can be separated by 1D (Laemmli SDS-PAGE) or 2D (O'Farrell) gels followed by electrophoretic transfer to PVDF. Enough protein should be loaded in a lane so that each band or spot contains 1-5 µg of sample. Lower amounts can be pooled for analysis after blotting. Artificial blockage of the N-terminus during sample preparation or electrophoresis has been reported to be a factor in reducing the amount of material that can be sequenced following electrophoresis (14). However, if fresh reagents of the highest quality are used, it should not present a problem as determined by the center. PVDF membranes should have a high protein binding capacity (0.1-0.22 µm pore size) so as to retain the most protein. Suggested suppliers are ProBlott (Applied Biosystems), Westran (BioRad), Novex, and Immobilon Psq (Millipore). Immobilon P (Millipore) retains less protein but can still be used. Even under the best circumstances there is always sample loss during the transfer step: therefore, if very little material is available, the sample should be submitted as a gel band to minimize sample loss.

Proteins in a gel:  Coomassie Blue stained gel samples from standard SDS-PAGE Laemmli electrophoresis can be accepted for internal sequence analysis (the gel is not stable to the Edman chemistry).    Because of the three dimensions of the gel, only up to 3 lanes of a 1 mm thick gel can be analyzed, more gel may decrease yields. 

Suggested method for electrotransfer of 1D or 2D gels (modified from 7-8)

Note: Please do not use a semi-dry blotting apparatus for protein transfer as this produces poor protein recovery on PVDF. A full immersion tank will produce much better results.  ALWAYS wear gloves as human keratin can be a major contaminant.

  1. Cut the PVDF to the same size as the gel. Prewet the PVDF by immersion (30 sec) in 100% methanol, followed by equilibration in transfer buffer (15 min).
  2. TRANSFER BUFFERS:
    1. CAPS: 10 mM CAPS, 10% methanol, pH 11.0. Degas buffer before use.
    2. TRIS-GLYCINE: 25 mM Tris, 192 mM Glycine, 20% methanol, pH 8.3.
      CAPS buffer is preferred for N-terminal sequence analysis; the background is significantly lower and there is no glycine contamination. If Tris-glycine is used, the membrane should be washed thoroughly with ddH2O following transfer and staining.
  3. Sandwich gel and PVDF membrane between Whatman 3MM paper, and assemble into blotting cassette (we use a Hoefer system; BioRad and others are also fine).
  4. The time and amperage of transfer should be optimized for each protein. A 20 kD protein can transfer in 30 min from a 1.0 mm gel at 0.5 amp (constant). Larger proteins require 1 or more hours. Some suggested conditions at constant current are
    • 500 mA for 30 minutes
    • 250 mA for 2 hours
    • 60 mA overnight
  5. After transfer is over, stain in one of the following depending on the application:   Note: always use a clean or preferably new Petri dish for staining.  DO NOT use trays previously used for western blots as this can introduce contaminating proteins.  
    • Staining procedure/recommendations: The following procedures are recommended before submission of PVDF samples to the Protein/DNA Technology Center.
    • Ponceau S. : (modified ref 10) 0.1% Ponceau S. in 1% acetic acid, 5 min. Destain : 5% acetic acid, followed by several ddH2O washes.
      Note: Ponceau S. supplied as a TCA solution (Sigma) does produce as good a result as using the dry reagent (Sigma) and solubilizing in 1% acetic acid.
    • Amido Black : 0.1% amido black in 40% methanol/10% acetic acid, 5 min.
      Destain : 1-3 times with 40% methanol/10% acetic acid (5 min each), followed by several washes with ddH2O.
    • India Ink : Modified from (9)
      1. equilibrate PVDF in 50 mM Tris-HCl, 150 mM NaCl, pH 4.9 for 5 min.
      2. Transfer PVDF to 50 mM Tris-HCl, 150 mM NaCl, 0.2% Triton X-100, 0.1% Pelican India Ink; 1 hour.
      3. Destain with buffer solution without Triton X-100 or India ink.
      4. Wash 2X with ddH2O.
    • Coomassie blue : 0.5-0.1% Coomassie blue in 50% methanol/10% acetic acid, 5 min.   Use 0.5% Coomassie blue for gels.
      Destain: 1-3 times with 50% methanol/10% acetic acid (5 min), followed by several washes with ddH2O. Coomassie blue can be very dirty and if used Coomassie G-250 is preferred. We prefer that one of the previous three stains be used first and Coomassie blue used as a last resort. One exception is that gel samples should be stained with Coomassie Blue G-250.

Suggested method for staining  gels with Coomassie Blue G-250

  1. Remove the gel from the electrophoresis chamber and place into enough 0.5% Coomassie blue G-250 (made in 50% methanol/10% acetic acid) to cover the gel.  We suggest using a new Petri dish or the top cover of a box of P-1000 pipette tips.  Stain for about 5 minutes.
  2. Discard stain (note for protein chemistry work please do not reuse Coomassie solutions) and rinse briefly with high purity water to remove most of the residual stain in the tray.
  3. Destain with 40% methanol/10% acetic acid, replacing the solution every 10-20 minutes until faint bands are observed.  Kimwipes rolled up into a ball can be added to speed up the destaining.
  4. Start destaining in high quality water (preferably Milli-Q water) until bands are very clean.  Usually we destain overnight in water with several pieces of Kimwipes present.  The gel will usually expand a little.  Bands can now be excised and submitted for analysis.

Protein gels can also be stained with Gel Code Blue (Pierce).  Please follow instructions from the manufacturer.

Examples

To see procedures on the operation of the Edman sequencing Service please follow the links below (requires Adobe Acrobat reader).

ABRF2000Poster.pdf

To see examples of actual data from a standard protein as well as what a written data report from the Center looks like click on the following links (requires Adobe Acrobat reader).

Sequence Report for protein sequence data (galseq.pdf)

BLAST search results for sequence data:  BLAST

Below are examples of  PVDF blots for assistance in protein quantity estimation PVDF Blot.

Staining of PVDF-bound standard proteins.   Standard proteins (indicated by arrows) used were human transferrin (75K), bovine serum albumin (68K), rabbit muscle actin (45K), soy bean trypsin inhibitor (21K), and horse heart myoglobin (14K).  Lanes from left to right were 1) Bio-Rad molecular weight markers, 2+3) 20 pmol each standard protein, 4+5) 10 pmol each standard protein, 6+7) 5 pmol each standard protein, 8+9) 2 pmol each standard protein, and 10) blank lane.   All staining conditions were as described above.  CBB is an abbreviation for Coomassie Brilliant Blue. 

Below is an example of a SDS-PAGE gel stained with Coomassie G-250.   Click on gel to see a larger picture of the gel.

gel.jpg (11028 bytes)

Internal Edman sequencing analysis requires a minimum of 1-2 pmol and preferably  at least 5 pmol.

Data Return and Acknowledgements

Data return: Unconfirmed, initial N-terminal sequence assignments and candidate proteins from an initial database search will be given over the phone, in person, by e-mail, or by FAX the day after a sample is run (usually 15-20 cycles). If the sample has a blocked N-terminus, the investigator will be charged for only the first 5 residues (the minimum charge). If no MALDI-TOF MS data is obtained or no significant peaks are observed in the HPLC chromatogram, the minimum charge is $250.  The finalized data report will be emailed to the investigator.

The Service will perform a data base search using the protein sequence data via the Blast/Fasta Server or NCBI. All investigators are strongly encouraged to perform their own data base search in addition to the Service's search; however, please follow our suggestions to avoid misinterpretation of the data.

In the case of internal Edman sequencing samples, if an initial database search using the MALDI-TOF MS data of the unfractionated digestion mixture yields any significant candidates for protein identity, these results will be discussed with the investigator prior to further analysis of the samples in order to determine the best course for further analysis.  Results from the HPLC separation of the peptides and MALDI-TOF MS analysis of the fractions will be discussed with the investigator in order to determine the best peptides to sequence. After each peptide is N-terminally sequenced, the investigator will be notified of the results before the next peptide is sequenced in order to determine if the investigator requires additional data.

Acknowledgements:

When data obtained by the Service is used for in a publication, please include the following acknowledgement:

"Edman sequencing data was obtained at the Rockefeller University Protein/DNA Technology Center."

When publishing data obtained using the Internal Edman sequencing analysis service please use the following references in addition to the above acknowledgement

"Fernandez, J., Gharahdaghi, F., and Mische, S.M. (1998) Electrophoresis, 19, 1036-1045.

"Fernandez, J., Andrews, L., and Mische, S.M., (1994) Anal. Biochem. 218, 112-118."

Acknowledgement and referencing of the Protein/DNA Technology Center is greatly appreciated by the staff of the Center. Acknowledgements also aid the Center in obtaining highly competitive shared instrumentation grants and helps to establish other essential services at the facility.

References

  1. Fernandez, J., DeMott, M., Atherton, D., and Mische, S.M., (1992) Internal Protein Sequence Analysis: Enzymatic Digestion for less than 10 Micrograms of Protein Bound to Polyvinylidene Difluoride or Nitrocellulose Membranes. Anal. Biochem., 201, 255.
  2. Fernandez, J., Andrews, L., and Mische, S.M., (1994) An Improved Procedure for Enzymatic Digestion of Polyvinylidene Difluoride-Bound Proteins for Internal Sequence Analysis. Anal. Biochem., 218, 112.
  3. "Fernandez, J., Gharahdaghi, F., and Mische, S.M. (1998) Routine identifiaction od proteins from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels or polyvinyl difluoride membranes using matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS).  Electrophoresis, 19, 1036-1045.
  4. Atherton, D., Fernandez, J., DeMott, M., Andrews, L., and Mische, S.M., (1993) Routine Protein Sequence Analysis Below Ten Picomoles: One Sequencing Facilities Approach, in Techniques in Protein Chemistry IV (Angeletti, R.H., Ed.) Academic Press, San Diego, pp 409-418.
  5. Laemmli, U.K. (1970) Cleavage of Structural Proteins During the Assembly of the Head of Bacteriophage T4. Nature, 227, 680.
  6. O'Farrell, P.H., (1975) High Resolution Two-Dimensional Electrophoresis of Proteins. J. Biol. Chem., 250, 4007.
  7. Mozdzanowski, J., and Speicher D.W., (1992) Microsequence Analysis of Electroblotted Proteins 1. Comparison of Electroblotting Recoveries Using different Types of PVDF Membranes. Anal. Biochem., 207, 11-18.
  8. Matsudaira, P. (1987) Sequence from Picomole Quantities of Proteins electroblotted onto Polyvinylidene Difluoride Membranes. J. Biol. Chem., 262, 10035.
  9. Hughes, J., Mack, K., and Hamparian, V. (1988) India Ink Staining of Proteins on Nylon and Hydrophobic Membranes. Anal. Biochem., 173, 18.
  10. Salinovich, O., Montelaro, R.C (1986) Reversible Staining and Peptide Mapping of Proteins Transferred to Nitrocellulose after Separation by Sodium Dodecylsulfate-Polyacrylamide Gel Electrophoresis. Anal. Biochem., 156, 341.
  11. Schaffner, W., and Weissman, C. (1973) A Rapid, Sensitive and Specific Method for the Determination of Protein in Dilute Solution. Anal. Biochem., 56, 502.
  12. Simpson, R.J., Moritz, R.L., Nice, E.E., and Grego, B. (1987) A high-Performance Liquid Chromatography Procedure for Recovering Subnanomole Amounts of Protein from SDS Gel Electrophoresis for Gas-Phase Sequence Analysis. Eur. J. Biochem., 165, 21.
  13. Towbin, H., Staehelin, T., and Gordon, J. (1979) Electrophoretic Transfer of Proteins from Polyacrylamide Gels to Nitrocellulose Sheets: Procedure and Some Applications. PNAS, USA, 76, 4350.
  14. Moos, M., Nguyen, N.Y., and Liu, T.Y., (1988) Reproducible high yield sequencing of proteins electrophoretically separated and transferred to an inert support. J. Biol. Chem., 263, 6005.

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