AltaBioscience

An Introduction to Synthetic Peptides

Page 57

Introduction to Synthetic Peptides

How they are made

Alta Bioscience uses solid phase synthesis to make all of its peptides. Here, the C-terminal amino acid

is anchored to polystyrene based resins and the peptide is grown amino acid by amino acid towards

the amino terminal. When the peptide chain is complete, it is cleaved off the resin with acid, a process

that removes the amino acid side chain protection at the same time. After removal of the acid, the

peptide is ready for QC by HPLC and mass spectrometry. After satisfactory QC, the peptides are

purifi ed by preparative reverse phase HPLC, then freeze dried, packaged and dispatched.

Aspects of purity

Peptide purity

The purity of all our purifi ed peptides is

determined by reverse phase HPLC. A

wavelength of 215nm is used for the analysis

as this is the optimum for the detection of the

peptide bond and hence detects all peptide

species present.

It should be noted that the purity value obtained

by this method does not include the presence of

any water and trifl uoroacetate salt which will be

present in the dried material. Unless specifi ed

in the order, all Alta Bioscience peptides are

supplied with trifl uoroacetate as the counter-ion,

acetate or chloride can be supplied on request.

Reverse phase chromatography will remove all

the reagents used in the cleavage process. All

Alta Bioscience peptides that are supplied to a

specifi ed purity will have been through a clean-

up process, even if the crude material exceeds

the requested purity. The laboratory makes

extensive use of capping during synthesis, so

deletion peptides are very rare. However some

truncated material and peptide with modifi ed side

chains could be present.

Net peptide content

All dried peptides will contain a variable

amount of water plus a fi xed amount of the

peptide counter-ion, usually trifl uoroacetic acid.

Quantitative amino acid analysis is the only

method which enables the net peptide content to

be determined. Here, the amount of each amino

acid is measured after total acid hydrolysis, the

sum total of which gives the amount of peptide

in the product. Typical values for net peptide

content range from 70% – 90% but in extreme

cases can be as low as 20%.

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An Introduction to Synthetic Peptides

synthesised.

3. Amino acid analysis

This technique is primarily used to measure the

net peptide content of a product. The peptide

is acid hydrolysed to its amino acids and these

are quantifi ed after separation by ion exchange

chromatography and detection with ninhydrin.

4. N-terminal sequencing

Amino terminal Edman sequencing can be used

to confi rm that the sequence of the amino acids

is correct.

Design and structure of peptides

By convention, peptides are written left to right

with the N-terminus at the left and the C-terminus

at the right. Care must be taken when specifying

modifi cations. An example of a typical modifi ed

sequence is shown below.

acetyl- KLPSSRY pS AGHLLD -amide

PhosphoSerine is spelled out as pS with spaces

before and after. The words acetyl and amide

are separated from the peptide by spaces and

hyphens. (Don’t forget that both amide and

acetyl spell out a real peptide sequence).

Amino acid classifi cation

The following table gives a general classifi cation

of the amino acids

Acidic, polar

Asp, Glu

Basic, polar

His, Lys, Arg

Polar uncharged

Asn, Cys, Gly, Gln,
Pro, Ser, Thr, Tyr

Nonpolar and
hydrophobic

Ala, Ile, Leu, Met,
Phe, Trp, Val

Solubility

Solubility, or primarily the lack of it, is the cause

of the majority of problems when working with

peptides. In general, peptides with a large

proportion of nonpolar amino acids will be

Levels of purity

Three levels of purity are offered, >95%, >90%,

>80%, in addition to unpurifi ed material. The

higher the purity, the higher the cost of the

fi nished product. In general, the >95% purity

is only needed when the peptide is to be used

as an enzyme substrate or in NMR and X-ray

crystallography analysis. It is not necessary to

specify high purity for peptides that are to be

used to raise antibodies.

If a peptide is requested to a set purity, Alta

Bioscience will put it through a purifi cation

process, even though the crude material passes

the HPLC purity specifi cation. All purifi ed

peptides are supplied with HPLC and MS traces.

Salt form of peptides

As peptides are usually purifi ed by HPLC with

acetonitrile gradients and trifl uoroacetic acid,

(TFA), as moderator, they exist as their TFA

salts. For most purposes this is not a problem

but when adding peptides to cell cultures, the

TFA can sometimes be toxic. This problem

can be avoided by specifying peptides in either

acetate or chloride salt forms.

The analysis of peptides

Alta Bioscience has the capability to analyse its

product by a wide range of methods.

1. HPLC

High Performance Liquid Chromatography,

HPLC, is the primary method of analysing

peptide purity. Performed typically on a C18

reverse phase column, 4.6mm x 250mm with

300Å pore size silica, using an acetonitrile water

gradient with TFA, as the acidic species.

2. MALDI-TOF

A ‘matrix assisted laser desorption and

ionisation – time of fl ight’ mass spectrometer is

used to determine the molecular weight of the

peptides. Highly accurate, fast and requiring

small amounts of sample, it is the ideal method

to ascertain that the target peptide has been

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An Introduction to Synthetic Peptides

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diffi cult to dissolve in aqueous solutions, the

more polar residues that are present, the easier

it will be to dissolve a peptide. Peptides that are

acidic, i.e. contain more acidic amino acids than

basic, will be more soluble at higher pH and visa

versa, peptides that are overall basic will be most

soluble at lower pH.

Length of peptides

Although Alta Bioscience has made some very

long peptides of over 80 amino acids, the solid

phase method essentially has a realistic upper

limit of about 50 amino acids. Above this length,

the high risk of failure tends to make a synthesis

fi nancially uneconomic. As the length increases,

so does the number of impurities that have to

be removed from the target sequence, thus the

absolute purity of the product will be lower. A

longer peptide will also have a higher chance of

containing a sequence region that is diffi cult to

make.

The ease of synthesis of any peptide is entirely

dependent on its sequence, a diffi cult sequence

can easily prevent even a short peptide of 10

amino acids being made. Alta Bioscience will

freely give as much help as possible concerning

the viability of a synthesis. Peptides that are

potentially diffi cult, could cost more to make than

easy ones.

Things to avoid

Some sequences can be particularly diffi cult

and if they can be avoided in some way, the

synthesis will be much easier or even made

possible.



N-terminal Gln should be avoided at

all costs. It is very unstable and rapidly

forms the cyclic pyroglutamic acid as shown

in the illustration. It is best to add either

pyroglutamic acid itself, or include an

acetyl group at the N-terminal Glutamine.

H

2

N

CH

C

CH

2

O

CH

2

C

NH

2

O

HN

CH

C

CH

2

NH

O

H

2

C

C

O

Peptide

Peptid

e

NH

3

+

Figure 1. Mechanism of pyroGlu formation



Peptides containing long strings of Valine

or Isoleucine are virtually impossible to

synthesise and work with.



A peptide with no charged or polar groups

may be very insoluble.



Multiple additions of phospho amino acids

can cause major synthesis and purifi cation

problems. The peptides can be made but

the costs rise steeply with each additional

phospho group.



If possible, it is best to avoid cysteine when

designing peptides for raising antibodies.

In proteins, cysteine usually exists as a

disulphide bridge so it would present a very

different shape if presented as the monomer,

as shown in fi gure 6.



These amino acids decrease solubility:-

Trp, Val, Ile, Phe

If in doubt please ask, we are happy to

give advice free of charge

Things to include if possible



Proline breaks up beta sheet formation

and although non-polar, helps to solubilise

peptides.



A spacer between a dye or tag and the

rest of the peptide sequence is usually

advantageous. A range of spacers can be

used. Ahx, amino hexanoic acid is a simple,

useful spacer. SGSG is a hydrophilic

sequence designed by Alta Bioscience for

use as a biotin spacer. A range of PEG

spacers are available with varying numbers

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An Introduction to Synthetic Peptides

of atoms.



It is always cheaper to put a dye or tag at the

N-terminus rather than the C-terminus.



These amino acids increase solubility:- Lys,

His, Arg, Asp, Glu, Ser, Thr.

Modifi cations and unnatural amino
acids

There is a huge number of modifi cations

possible, listed below are the more common

ones. The structures of many of these unusual

amino acids are shown in the accompanying

paper, ‘Table of the amino acids’.



Phosphorylated amino acids

Phosphorylated Ser ,Thr and Tyr can be placed

at any specifi ed site in a peptide. However,

multiple incorporations can cause synthesis and

purifi cation problems.



Terminus modifi cations

N-terminal acetyl and C-terminal amides remove

the charges at the ends of a peptide and make it

much more like the parent protein.



Methylation

Mono, di and tri methylated Lys, mono and

dimethyl Arg are found in histone proteins,

these methylated amino acids can be easily

incorporated at specifi c positions.



D amino acids

All the D amino acids can be added at any

position.



Analogues

Amino acids with longer or shorter versions of

the side chain length are available. For example,

homoserine and homoarginine are longer

variants of serine and arginine while ornithine

and diamino butyric acid are shorter analogues

of lysine. These are very useful in fi ne tuning the

shape of peptides.



Isotopes

Amino acids enriched with the stable isotopes

13C and 15N can be incorporated into peptides

for use in quantitative mass spectrometry. It

is advised to focus on the amino acids with

nonreactive side chains, such as Val and Phe.

The more complex amino acids tend to be

prohibitively expensive, if available at all.



Unnatural amino acids

Compounds such as phenylglycine, napthyl

alanine, nor leucine and beta alanine are readily

incorporated into peptides.



Spacers

These are used to pull dyes and tags away

from the active site of a peptide, some common

examples are shown here:-

Hydrophobic aminohexanoic

acid

Hydrophilic

SGSG a short peptide sequence

Hydrophilic

PEG, ranging from 9 to 88 atoms

Please let us know if you need

a compound that isn’t in the above

list of modifi cations



Biotin.

S

H

2

C

CH

CH

CH

C

NH

HN

O

H

2

C

C

H

2

H

2

C

C

H

2

C

OH

O

Binds irreversibly to streptavidin and is used

extensively in screening assays and to bind

peptide to substrates.



Desthiobiotin.

H

3

C

C

H

2

CH

HC

C

NH

HN

O

H

2

C

C

H

2

H

2

C

C

H

2

C

OH

O

Binds to streptavidin but can be displaced

by biotin. Useful when you need to get your

peptide out of a binding experiment.

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An Introduction to Synthetic Peptides

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Peptides with Dyes

A very wide range of dyes and tags are available,

a short list of the more common ones is shown

here. The accompanying paper, “Introduction to

dyes, labels and tags” describes these more fully.



FAM



Tamra



The DyLight™ range of dyes



Dansyl



NBD



Edans



Dabcyl



Mca

Cyclic peptides

Alta Bioscience can synthesise both cyclic and

cross linked peptides.

Cyclic disulphide

If a peptide is made with two cysteine residues,

careful oxidation in solution will result in a cyclic

compound, created as the cysteines bridge to

form their dimer, cystine. This reaction generally

proceeds smoothly with good yield and minimal

polymer formation. The bridge can be broken

under physiological conditions.

Figure 1. Diagram of a peptide with a disulphide

bridge.

Cross linked peptides

Many bioactive peptides contain several

disulphide bridges. Alta Bioscience has had

considerable success in the synthesis of these

complex compounds.

Cyclic with a peptide bond

Either the two ends of a peptide or specifi c –

CO

2

H and –NH

2

residues can be reacted

to form a peptide bond, resulting in a cyclic

compound. Care must be taken in the design of

the peptide for this method to work well.

Figure 2. Cyclised with a peptide bond

Cyclic thioethers

These are useful when designing peptide

libraries where the peptide needs to be

presented as a constrained shape. The

cyclisation process proceeeds smoothly, in

good yield. The thioether bond is stable under

physological conditions.

Figure 3. Diagram of a thioether cyclic peptide

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An Introduction to Synthetic Peptides

Peptides for raising antibodies

In general, synthetic peptides are too small to

elicit an antibody response, Alta Bioscience

uses two methods to convert its peptides into a

suitable form.

1 MAP peptides

MAP peptides are octomeric molecules with

the peptide chains

branching out from a central

poly-lysine core, as shown in fi gure 4. The eight

peptide chains increase the molecular weight

of the compound suffi ciently for it to be easily

recognised as an antigen. It provides an easy

and fl exible method for antibody production.

K

K

K

K

K

K

K

K

K

K

K

K

K

K

Figure 4. Diagram of an octomeric MAP peptide

It is also possible to make chimeric MAPs with

two different peptides sequences, each forming

four of the chains.

The MAP method however, isn’t suitable for

peptides which come from the C-terminus of a

protein, as that particular amino acid is the one

conjugated to the core peptide and thus not

exposed.

Dialysis through a 2-3kDa membrane is the only

purifi cation method which is required for these

molecules.

2 Peptide – protein conjugates

Here, a synthetic peptide with a free cysteine

residue, is covalently attached to the lysines in a

protein carrier molecule. The size of the protein

triggers the antibody system, which recognises

the attached peptides. The most popular carrier

protein is keyhole limpet heamocyanin, KLH,

which elicits a strong antibody response and

contains a very large number of lysine residues

which are used to attach the peptide antigen.

This particular approach can be used to attach

the peptide in any orientation, i.e. at either the N

or the C terminus. However, it is not suitable for

any peptide containing cysteine, as that amino

acid is added to the sequence to act as the linker

to the protein.

Figure 5. Diagram of a peptide-protein conjugate.

Antigen design considerations

In general, peptides for antibodies will be

hydrophilic and fl exible, coming from the exterior

of the parent protein. A hydophilicity plot will

indicate which parts of the protein are likely to

be on the outside of the structure. The Kyte-

Doolittle or the Hopp-Woods algorithms will be

very useful here.

Structure predictions can be done with Chou-

Fasman plots. The best source for the data

would be the European Bioinformatics Institute.

Cysteine should be avoided where possible.

The following illustration in fi gure 6, shows that

a single cysteine would present a very different

shape to the immune system compared with the

disulphide bridged, cystine.

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An Introduction to Synthetic Peptides

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Cys S--S Cys

Cys SH

Figure 6. Differences in shape between the

disulphide bridge in a protein and a linear

peptide.

Peptides for micro arrays

Virtually any type of sequence can be printed

onto a micro array. To reduce steric hindrance

effects, it is helpful to specify a spacer such as

Ahx or a PEG between the peptide sequence

and any biotin which is used to anchor the

peptide onto the array slide. The biotin is

usually added at the N-terminus but there are no

synthesis diffi culties in having either a C-terminal

biotin or it anywhere along the peptide chain.

If a cysteine is being used as the linker amino

acid for binding to maleimide surfaces, then the

array peptide must not contain any sequence

cysteines. If two Cys residues were present,

there would be no control over which of them

would act as the linker.

It is advisable to specify the linker group to be

at the N-terminal of the peptide. The synthesis

proceeds C to N with capping, so only the full

length peptide would contain the linker. All failure

sequences would be washed away and take no

part in the binding.

Handling peptides

Storage

Alta Bioscience supplies all its peptides as freeze

dried materials and these can be regarded as

stable compounds for shipping purposes. For

long term storage however, it is recommended

to store them in a deep freeze at -20°C. When

taking them out of the freezer, it is important

to allow the bottles/vials to warm up to room

temperature before opening the container. This

is because peptides are often hygroscopic and it

avoids condensation of atmospheric water on the

peptide.

Peptides in solution can degrade, primarily due

to oxidation of Cys, Met and Trp residues but

they are also susceptible to attack by microbes,

so it is advised to store solutions at -20°C when

not in use. It is diffi cult to predict the storage

life of a peptide as it is highly dependent on its

amino acid content and sequence.

Dissolving peptides

This can be a very diffi cult operation.



Always try to use volatile materials such as

dilute acetic acid and ammonia solutions

when fi rst dissolving an unknown peptide. If

everything fails, the buffers can be removed

by lyophilisation and the dissolution attempted

again.



If the peptide is acidic, i.e. contains more

Asp and Glu residues than His, Lys or Arg,

then fi rst attempt to dissolve the peptide in

dilute ammonia solution, e.g. 0.5%

ammonium hydroxide. Do not use this

method if your peptide has disulphide

bridges, the high pH may cause them to

unfold.



If the peptide is basic, i.e. contains and

excess of His, Lys and Arg groups, then

try and dissolve the peptide in something like

10% acetic acid.



DMSO is a very good solvent and has the

advantage of being tolerated by cells, it is

AltaBioscience

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An Introduction to Synthetic Peptides

AltaBioscience is a leading manufacturing laboratory providing analysis and synthesis of DNA, proteins and other biochemical
molecules to clients world-wide. Founded in 1973 at the University of Birmingham, England, we offer a well established and
comprehensive range of synthetic, sequencing and analytical methodologies, which are available to academia and commercial
clients. The following internationally recognised accreditations position AltaBioscience amongst the few laboratories world-
wide working to such high standards. ISO 9001:2008 Quality management system for the laboratory as a whole, and ISO
17025:2005 Technical competence in amino acid analysis and protein sequencing.

This publication is one of a series presenting answers to questions frequently asked by established researchers, as well as
those new to their fi eld. Should you have a question which is not dealt with, or if you fi nd an item lacking clarity, we invite you to
bring it to our attention by sending an email to E: [e-mail]

AltaBioscience, Building Y10, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom

T: [phone] F: [phone] E: [e-mail] W: www.altabioscience.com

however diffi cult to remove by drying. Add a

small amount of a high purity grade DMSO

to the stock peptide solution until it dissolves.

Once dissolved, water or buffer solution can

be added very slowly to dilute the DMSO

content. Stop the water addition if the peptide

starts to precipitate out. DMSO isn’t suitable

for peptides containing single Cys as it

promotes disulphide bridge formation.



Gentle warming and sonication are

useful tactics in getting peptides to dissolve.



Peptides originating from the transmembrane

regions of proteins will certainly be diffi cult to

dissolve.

References

The original paper

1. Merrifi eld R. B. ‘Solid Phase Peptide

Synthesis’. J. Am. Chem. Soc. 85, 2149 (1963)

Recent reviews

2. Cheng W., White P. D. ‘Fmoc Solid Phase

Peptide Synthesis: A Practical Approach’ Oxford

University Press, 2000

3. Albericio F,. Kates S. A. ‘Solid-Phase

Synthesis: A Practical Guide’ CRC Press, 2000

© Copyright by AltaBioscience
August 2011
Reproduction forbidden without permission