Peptide
Synthesis
By
specialising in the preparation of synthetic peptides for many years and remaining at the
forefront of synthetic developments, AFFINITI is well positioned to use the latest and most appropriate
methodology for the synthesis of a required peptide. The quality of a peptide is
affected by the skill and efficiency that goes into each of the constituent operations of
its preparation, namely synthesis, purification and analysis.
The
use of high quality reagents and purified solvents, the monitoring of synthetic steps,
etc. are all crucial to a quality synthesis. Whilst every effort is made to ensure
that individual syntheses proceed as efficiently as possible, the final products are
always subjected to a number of quality control procedures appropriate to the grade of
peptide being supplied.
Quantities routinely supplied are 5mg, 10mg,
25mg, 50mg and 100mg at one of two purity levels, with absolute values dependent upon the
number of amino acid residues, sequence complexity and overall amino acid composition.
Modified
and Complex Peptides
Peptides
are normally supplied with free N-terminal amino and C-terminal acid groups but
modifications, such as N-terminal acetylation or C-terminal amidation, are available at no
extra charge.
Modification
of peptides, be it:
by
the incorporation of D-amino acids, 'unnatural' amino acids (such as b-alanine,
diiodotyrosine, ornithine, hydroxyproline, b-naphthylalanine, etc.),
at
the termini with substituents such as DNP, FITC, biotin, long chain alkanes,
para-nitroanilides, AMC, etc.,
on
certain side chains as with the phosphorylation of serine, threonine or tyrosine, or
sulphation of tyrosine,
by
cyclisation via disulphide bond formation or by formation of cyclic amides
is routinely available upon request.
Phosphopeptides
There is an increasing requirement for peptides
that are specifically phosphorylated on serine, threonine or tyrosine residues.
Whilst technically more complex than the synthesis of non-phosphorylated peptides, A
FFINITI has
much expertise that enables it to offer such products on a custom basis.
Multiple Antigen Peptides
Multiple
antigen peptides (MAPs) represent a novel and efficient method for the production of
antisera to peptide immunogens which avoids completely the necessity for coupling peptides
to carrier proteins. Such an approach has the advantage of eliminating carrier
antibodies and circumventing problems that can occur during peptide-carrier protein
conjugation.
MAPs
are prepared by synthesising eight copies of the desired peptide on an inert branching
lysine core giving a molecule with a molecular weight in the region of 10-20kDa.
Although often difficult to prepare due to steric problems, AFFINITI has
many years' experience in their preparation and has developed techniques which overcome
the problems effectively. MAPs are characterised by amino acid analysis and are
normally used without extensive purification, as this has not been found to be necessary
for full functionality.
Peptide
Panels
For
those requiring small quantities of a large number of peptides that have not undergone
individual purification and analysis but yet are suitable for initial screening purposes,
AFFINITI has introduced its ‘3P (PACEmaker Panel Peptides)
Service’. Please contact AFFINITI Customer Service for full details and pricing.
Peptide
purification
In the majority of cases, peptides are purified
to the required specification by techniques including gel filtration, ion exchange, and
reversed-phase and ion exchange high performance liquid chromatography (hplc). High
purity peptides (>95%) are prepared by the most appropriate route for the structure and
quantity required and undergo rigorous purification prior to despatch. Lower grade
purity peptides are prepared by analogous methods to those of the higher purity products
but are subjected to slightly less rigorous purification procedures. Nevertheless,
the purity of these lower specification products is often in excess of 90%, although this
cannot be guaranteed.
Peptide
analysis
Peptide
purity is assessed by reversed phase, and additionally in some cases by cation exchange,
high performance liquid chromatography and is stated as the percentage of the total peak
area of the major peak. Additionally, all peptides are subjected to mass
spectrometric analysis as a routine part of AFFINITI’s service in order to ensure confidence in the
integrity of the product supplied. A copy of the analytical chromatogram and mass
spectroscopic data is included in the data sheet that accompanies each product.
Whilst
amino acid sequencing is not routinely carried out or, indeed, is deemed necessary it
should be noted that the organisation and protocols used for synthesis are such that
errors in sequence are extremely unlikely. Actual amino acid content (as may be
determined by amino acid analysis) will normally exceed 70%; however, no minimum
percentage of residual salts can be specified since counter ions to charged
functionalities within individual peptides make this highly sequence-dependent.
Amino acid analysis is not carried out as part of any routine service, save for MAP
characterisation, but is available upon request, at an extra charge, for both amino acid
ratio confirmation and net peptide content determination.
Some cysteine-containing
peptides are very sensitive to oxidation. Whilst all reasonable measures are taken
to maintain the reduced form, no guarantee can be given that cysteine-containing peptides
will remain in a reduced state upon subsequent storage and use. For
cysteine-containing peptides at the lower purity levels, purity determinations exclude
oligomeric materials.
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