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Standard purification protocol for Peptides from Natural Sources
Standard purification protocol for Protein fragments
Standard purification protocol for Synthetic Peptides
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Standard Purification Protocol for peptides from natural sources


Peptides are substantially more stable than proteins and thus tolerant to a much broader range of conditions, such as high concentrations of organic solvents, extremely acidic conditions like TFA or 70% formic acid and sometimes even boiling. Unlike most proteins, peptides are not normally inactivated by reversed phase chromatography.

RPC provides very high resolution and has therefore become the primary purification technique for peptides.

Peptide extracts originating from natural sources are very complex and require multi-step purification protocols.

Furthermore peptides normally occur in very low concentrations in living matter and large quantities of starting material may have to be dealt with to ensure that the purification results in enough material of the intended purity.

The Standard Purification Protocol for peptides from natural sources follows the Capture Intermediate purification Polishing strategy combining IEX, RPC and SEC in a way that utilises their independent selectivities for maximum overall resolution.

STEP ONE
STEP TWO
STEP THREE
STEP FOUR
IEX
Neutral RPC
on
RESOURCE RPC
Acidic RPC
on
Sephasil C8
SEC

Step one, the capture step, serves to concentrate the sample while reducing the protein background.

Step two, the first intermediate purification step, serves to remove the remaining protein background and to separate the peptides present in the sample. Polymer-based RPC is chosen for this step to allow cleaning the column with alkali to remove any irreversibly adsorbed proteins.

! Cleaning with alkali cannot be applied to silica-based RPC
media, since the silica will dissolve under alkaline conditions.

Step three, the second intermediate purification step, provides further separation of the peptides utilising the altered selectivity provided by the change in running pH.

Step four, the polishing step, serves to remove any remaining contaminants and aberrant forms of the target peptide like polymers, fragments etc. and to condition the target peptide for the intended further use.

Prerequisites

1. Analytical tools
  • A detection method specific for the target peptide.This is necessary in order to follow the progress of the purification and to evaluate optimisation measures.
  • A detection method to measure total protein content. This is necessary in order to calculate the relative content (purity) of the target peptide and to evaluate optimisation measures.

2. Knowledge of the sample stability.

This is necessary in order to choose running conditions compatible with solubility and biological activity of the target peptide and will be dealt with under initial tests below.

! It is of great help to know the isoelectric point (pI), the size and the hydrophobic character of the target peptide when designing the individual steps of the purification protocol.

! Peptides expose all or most of the constituent amino acid side chains and the chromatographic behaviour of a peptide will thus more or less be governed by the sum of the properties of the side chains. Physical properties of peptides with known structure can therefor be calculated to an extent meaningful to purification.

3. Sample condition

The starting material should be free of particulate matter, ionic detergents, and high Mw nucleic acids or columns may be blocked or clogged. If the starting material contains proteases these should be inactivated or recovery may suffer.

Initial tests
Before applying the standard purification protocol, tests are performed to select a suitable ion exchanger (AIEX or CIEX) for step one and to establish that the target peptide is fully soluble under the conditions used for RPC.

1. Choice of ion exchanger type and conditions.
HiTrap IEX Test Kit is used to select the appropriate ion exchanger type and to test the suitability of standard IEX conditions.

! In order to obtain representative results the column must not be overloaded, still enough of the target peptide has to be applied to enable its safe monitoring in the eluate.
As a rule of thumb the HiTrap IEX Kit columns accept loads between 5 and 10 mg of total protein.

Buffers
Prepare the following buffers:
  • HiTrap Q:
    - Buffer A: 20 mM TRIS-HCl, pH 8.0
    - Buffer B: 1.0 M NaCl in 20 mM TRIS-HCl, pH 8.0
  • HiTrap SP:
    - Buffer A: 50 mM acetate, pH 4.5
    - Buffer B: 1.0 M NaCl in 50 mM acetate, pH 4.5
Each experiment will require approximately 30ml of buffer A and 20 ml of buffer B.

Sample preparation
  • Adjust sample conditions to match those of Buffer A for HiTrap Q and for HiTrap SP respectively.
  • Filter or spin the sample to remove any residual particulate matter.

Experiments
  • Run HiTrap Q and HiTrap SP under standard conditions:
    - Flow rate: 1ml/min
    - Buffers as above
    - Gradient: AB in 20 column volumes.
    - Fraction size: 1ml.

Evaluation
  • Determine the total protein content and the target peptide content in all fractions.
  • Select the column and conditions that provides the following:
    - The target peptide eluting as a reasonably symmetrical peak within the gradient.
    - A recovery of the target peptide > 90%.
    - The highest relative target peptide content (target peptide content/total protein content).
If peak symmetry or recovery is unsatisfactory consult Trouble-shooting.
If the target peptide does not elute within the gradient or if purity is judged unsatisfactory perform pH scouting.

! It is advantageous to have the target peptide eluting late in the gradient since this allows a larger part of the background to elute in the run-through fraction after adjusting the gradient accordingly.

2. Solubility in RPC eluents

Neutral RPC
  • Adjust pH to 7.0.
    If the sample does not already contain a buffer to control pH in the range of 7, make up to 10 mM with ammonium phosphate before pH adjustment.
  • Filter or spin the sample to remove any formed precipitate.
  • Determine the content of target peptide in the clarified solutions.
  • Make up to 60% with acetonitrile.
  • Filter or spin the sample to remove any formed precipitate.
  • Determine the content of target peptide in the clarified solutions.

Acidic RPC
  • Adjust pH to 2 by adding TFA (final concentration around 0.1%)
  • Filter or spin the sample to remove any formed precipitate.
  • Determine the content of target peptide in the clarified solutions.
  • Make up to 60% with acetonitrile.
  • Filter or spin the sample to remove any formed precipitate.
  • Determine the content of target peptide in the clarified solutions.

Evaluation
Target peptide recoveries < 90% indicate incompatibility with the conditions used.

Standard Protocol

STEP ONE, IEX

Column
  • Use the type of ion exchanger selected under initial tests.
  • Select a column to match the amount of sample.

Sample preparation
  • Adjust sample conditions to match those of eluent A.
  • Filter or spin the sample to remove any particulate matter.

Method
  • Use the default values recommended for the column chosen.

Eluents
  • Use the eluents selected under initial tests.

Evaluation
  • Determine the total protein content and the target peptide content in all fractions and calculate the relative target peptide content (target peptide content /total protein content).
  • Check purity of collected fractions by:
    - Chromatography on µRPC or Mini S.
    - Capillary electrophoresis.
    - MS.
  • Stop if purity is satisfactory
    or
  • Pool fractions containing target peptide and proceed to STEP TWO.

STEP TWO, Neutral RPC

Column
  • Select a RESOURCE RPC column to match the amount of sample in the pool from step one.

Sample preparation
  • Adjust sample conditions to match those of eluent A.
  • Filter or spin the sample to remove any particulate matter.

Method
  • Use the default values recommended for the column chosen.

Eluents
Prepare the following eluents:
    -Eluent A: 10 mM ammonium phosphate pH 7.0 in water.
    -Eluent B: 10 mM ammonium phosphate in water containing 60% acetonitrile (final conc.).

! If the target peptide is sensitive to oxidation, degas eluents thoroughly by helium sparging and cool them.

Evaluation
  • Determine the total protein content and the target peptide content in all fractions and calculate the relative target peptide content (target peptide content /total protein content).
  • If peak symmetry or recovery is unsatisfactory, consult Trouble Shooting.
  • If the target peptide does not elute within the gradient, adjust conditions as follows:
    - Increase pH to reduce retention.
    - Decrease pH to increase retention.
  • Check purity of collected fractions by:
    - Chromatography on µRPC or Mini S.
    - Capillary electrophoresis.
    - MS.
  • Stop if purity is satisfactory.
    or
  • Pool fractions containing target peptide and proceed to STEP THREE.

STEP THREE; Acidic RPC

Column
  • Select a Sephasil C8 column to match the amount of sample in the pool from STEP TWO.

Sample preparation
  • Adjust sample conditions to match those of eluent A.
  • Filter or spin the sample from STEP TWO to remove any particulate matter.


Method
  • Use the default values recommended for the column chosen.

Eluents
Prepare the following eluents:
    -Eluent A: 0.06% TFA in water.
    -Eluent B: 0.05% TFA in water containing 60% acetonitrile (final conc.).

Evaluation
  • Determine the total protein content and the target peptide content in all fractions and calculate the relative target peptide content (target peptide content /total protein content).
  • If peak symmetry or recovery is unsatisfactory, consult Trouble Shooting.
  • If the target peptide does not elute within the gradient, adjust conditions as follows:
    - Increase pH to reduce retention.
    - Decrease pH to increase retention.
  • Check purity of collected fractions by:
    - Chromatography on µRPC or Mini S.
    - Capillary electrophoresis.
    - MS.
  • Stop if purity is satisfactory.
    or
  • Pool fractions containing target peptide and proceed to STEP FOUR.

STEP FOUR, SEC

Sample preparation
  • Check that the sample is stable at the conditions chosen.

Column
  • Select a Superdex peptide column for target peptide Mr >3 000.
  • Select a Superdex 75 column for target peptide Mr >3 000.

Method
  • Use the default values for recommended for the column chosen.

Eluents
  • Select an eluent suitable for the further use or storage of the target peptide.

! The resolution of peptides in SEC is affected by the ACN concentration.
ACN concentrations higher or lower than 25 - 30% will cause varying degrees of excessive retardation.

Evaluation
  • If peak symmetry or recovery is unsatisfactory, consult Trouble Shooting.
  • Determine the total protein content and the target peptide content in all relevant fractions and pool accordingly.
  • Check purity of the pool by:
    - Chromatography on µRPC or Mini S.
    - Capillary electrophoresis.
    - MS.

    Trouble-shooting
    When a chromatogram shows unexpected deviations such as non-symmetrical or excessively broadened peaks, when recovery suffers, or when the system pressure deviates from normal, trouble-shooting has to be performed. The strategy outlined below starts with the perhaps most common and obvious source of malfunction, faulty eluents. Then make sure that the system performs normally to avoid the risk of misjudging column function tests. Finally, test sample behaviour.

    General trouble-shooting strategy

    I. Check the eluents
    - Prepare fresh solutions.
    Cause
    Results not OK
    II. Check solvent delivery
    - Run installation test.
    Results OK.
    Results not OK
    Refer to instrument manual.
    III. Check the column
    - Run a known sample.
    Results OK
    Results not OK.
    Column malfunction
    Refer to trouble-shootingguide below.
    Sample incompatible with experimental conditions
    Refer to trouble-shooting guide below.
    The following table lists symptoms of malfunction.
    The actions recommended are intended as "first aids".

    Trouble-shooting guide

    Symptom
    Cause
    Action
    I. Pressure
    - LowAir in the pump heads.
    Worn-out piston seals.
    Leaks.     		Purge the pump with MeOH.
    Replace piston seals.
    (Refer to instrument manual)
    - HighClogged tubing or column.Watch pressure while removing column and tubing in the "upstream" direction until theclogged part is identified.
    - IrregularSolvent delivery malfunction.Re-run with fresh eluentsPurge the pump with MeOHl.
    Replace piston seals.(Refer to instrument manual)
    II. Aberrant peak shapesAlways check column first by running a known sample.
    -Triangular with sharp frontOverload. Reduce sample load.
    -Excessively broadOverload.
    Column bed irregularities.
    Air in the column.    		Run column with 2 column volumes of MeOH to remove any trapped air.
    Perform recommended CIP.
    -TailingColumn bed irregularities.
    Air in the column.
    Non-specific adsorption.    		Run column with 2 column volumes of MeOH to remove any trapped air.
    Perform recommended CIP.
    Try additives like ACN or MeOH.
    -FrontingChannels or air in the column bed.Run column with 2 column volumes of MeOH to remove any trapped air.
    -Split peaksPartially blocked frit.Column bed irregularities.
    Air in the column.    		Run column "backwards" with 2 columnvolumes of MeOH to remove any particles inthe frit or trapped air.
    -IrregularSolvent delivery malfunction.
    Sample incompatible with experimentalconditions.    		Run Installation Test.
    Run sample stability test.
    Try additives like ACN or MeOH,chaotropic salts.
    Change conditions, type of column or technique.
    - Ghost peaks(Peaks appearing even inblank runs)Column regeneration incomplete.
    Quality of eluent chemicals and /orsolvent too low.
    Aged eluents.     		Increase eluting component concentration in eluent B.
    Use HPLC quality chemicals and solvents only.
    Unexpectedly low or missingPeaksLow recovery of activity.Non-specific adsorption.
    Sample incompatible with experimental conditions.    		Run sample stability test.
    Try additives like ACN or MeOH chaotropic salts.
    Try to block non-specific binding sites by injecting e.g. albumin.
    Change conditions, type of column or technique.
    Base line instability
    - IrregularAir in the detector cell.
    Detector problem.     		Check constrictor function.Run Installation Test.
    - DriftingDetector problem.
    Bleeding of precipitated ornon-specifically adsorbed material.
    Faulty or aged eluents.     		Run Installation Test.Perform specified CIP procedure.
    Re-run with fresh eluents.

    pH Scouting

    Sample preparation

    • Adjust sample conditions to match those of the appropriate eluent A.
    • Filter or spin the sample to remove any particulate matter.

    Method
    • For CIEX, decrease the running pH in steps of 0.5 -1.0 pH units from the target peptide pI to the lower limit of the sample stability window.
    • For AIEX, increase the running pH in steps of 0.5 -1.0 pH units from the target peptide pI to the upper limit of the sample stability window.
    • Use the values for gradient and flow rate recommended for the column chosen

    Eluents
    • Select buffers suitable for the pH range to be scouted from the buffer list.

    Evaluation
    • Select the running pH that provides the highest purity.