Peptide Chemistry

The Peptide Synthesis Laboratory provides peptides and peptide arrays to research scientists at the Francis Crick Institute. We work with most Crick groups, providing peptides, which range from alpha factor to highly modified and long peptides (> 60 amino acids) for protein interaction and structural studies.

Custom peptides

We can make peptides of many lengths and modifications including biotin addition, dye labels, phosphorylation, methylation acetylation, hydroxylation, and branched peptides, cyclic peptides and peptides linked by disulphide bridges. We can aid in design of peptides and conjugation and immunisation strategies of peptides for antibody generation. All peptides are analysed by high-pressure liquid chromatography and mass spectroscopy. Peptides are purified where appropriate using a range of reverse phase chromatographic methods.

Peptide arrays

Many protein-protein interactions can be studied using a synthetic peptide as one of the partners. We make peptide arrays on cellulose membranes, which can be probed by an interacting protein to allow the study of molecular recognition events at the cellular level. Typical experiments include scanning a protein across its length by making 16-25mers shifted by one amino acid, then looking for the site of interaction with its protein partner. Once a sequence is identified then amino acids can be switched to every other common amino acid in a mutagenesis experiment.

Stock peptides

We hold stocks of peptides, which are commonly used to synchronise yeast cells, elute proteins from affinity matrices and for immunological studies. Peptides are generally supplied same day or next day depending on location of researcher. Amount is as required by the experimenter.

Project peptides

We synthesise more unusual peptide-based reagents that require significant solution phase or manual synthesis. We have made for example, cysteine farnesylated peptides, peptides that are modified with ATP analogues, peptides which crosslink for mass spectroscopy based structural studies and peptides that contain a hydrocarbon bridge to constrain structure.

 

Projects

Synthesis of Factor VIII 1694-1708 sY (NH2-EDFDIsYDEDENQSPR-COOH)

This year we have made sulphotyrosine-containing peptides. Sulphation is a post-translational modification that is involved in protein-protein recognition and plays important roles in numerous physiological and pathological processes.

Studies of tyrosine sulphation have been hindered by the difficulty of introducing sulphate groups at specific positions of peptides and proteins. This is because tyrosine sulphate esters are rapidly degraded in acid and fragmented during mass spectrometry, making their synthesis and characterisation highly problematic. Recently it has been shown that protecting the sulphate group with particular protecting groups stabilises it during the TFA cleavage. Examples of such groups are trichloroethyl (TCE), dichlorovinyl (DCV) and neopentyl (np). Of these, the np group appears to be the most convenient as it is stable to piperidine and TFA, but can be removed after cleavage with sodium azide or ammonium acetate.

Synthesis was performed using Fmoc-Tyr(SO3nP)OH and double coupling Ile and Ser. During the synthesis HOBt in piperidine was used in order to avoid aspartimide formation. The peptide was cleaved using a solution of TFA/H2O/TIS 95%-2.5%-2.5% for 1h. To deprotect the neopentyl group from the tyrosine sulphate the peptide was dissolved in a minimal amount of DMSO. The solution was diluted to 100 ml with 2M NH4OAc and incubated at 37°C for 6h. Afterwards the solution was lyophilised and the peptide was desalted. The characterisation of the peptide by LC-MS was performed using negative ion mode. The sulphate-containing peptide was purified by RP-HPLC using 0.1 M ammonium acetate / 1% ACN as Buffer A and 90% ACN / 10% H2O as Buffer B using a gradient from 0% to 45% Buffer B over 40 min. The choice of this buffer is due to the fact that standard TFA-containing buffers can cause some loss of the sulphate group. The purified peptide was lyophilised three times in order to fully remove ammonium acetate. The characterisation by LC-MS showed the desired peptide (calculated mass: 1951.91; found mass: 1950.54).

Synthesis of lipidated peptides

The peptide chemistry lab has expertise in the synthesis of lipdated peptides. Isoprenyl groups, fatty acids, and unsaturated fatty acids have been attached to amino groups, cysteine side chains and serine side chains. Synthesis of geranylgeranylated peptides has also required synthesis of the geranylgeranylating reagent.

Isoprenyl groups are acid sensitive and therefore this modification has to take place once the peptide is cleaved from the resin. Fatty acids and unsaturated fatty acids are base sensitive therefore peptide modification is performed after chain assembly but before removal of peptide from the solid support.

Nicola O'Reilly

Nicola O’Reilly (Lead) 

nicola.oreilly@crick.ac.uk
+44 (0) 20 7269 3318