Peptide synthesis: Purification and product analysis
Our instrumentation allows for single batch synthesis of up to 576 small scale peptides in one operation, as well as individual peptides in multigram amounts. For purification, our HPLC capabilities enable us to offer options specific to particular applications, ranging from un-purified peptides to purities of 80-98%. All purified peptides from AltaBioscience undergo reverse phase HPLC. Synthesis and subsequent purification can cover a range of applications including:
- 1mg to multigram amounts
- Range of purification options
- Cyclic, phosphorylated, fluorescent, isotopic labels, fatty acid incorporation
- Peptide Antigen Synthesis and Design Service
- Episcan scale; large numbers of small scale peptides suitable for HTS
- Histone peptides and microarrays
- Custom microarray service
Methodology & Peptide Purity
We use solid phase synthesis using Fmoc chemistry with the synthesis starting at the C-terminal end of the peptide and ending at the N-terminus.
When the peptide chain is complete, it is cleaved from the solid-phase resin with acid, a process that also removes the amino acid side chain protections. After removal of the acid, the peptide is ready for purification as required, then quality control (QC) by HPLC and mass spectrometry before being freeze dried, packaged and dispatched.
We offer a range of purity options from unpuriﬁed peptides through to peptide purities of 80-95% and 95-98%. The purity of our puriﬁed peptides is determined by reverse phase HPLC using either C-18 RP Vydac/Ace or Chromolith columns.
|Unpurified peptides||For large numbers of screening grade peptides.|
|95-98%||NMR studies, X-ray crystallography analysis and for peptides used as enzyme substrates or for mass spec standards.|
Impurities arise from non-target components such as truncated sequences of the target peptide, the peptide with protecting groups still intact, or material used in the cleavage process. Reverse phase chromatography will remove all the reagents used in the cleavage process. For analysis of HPLC output, a wavelength of 215nm is used as this is the optimum for the detection of the peptide bond and hence detects all peptide species present. The purity value obtained by this method does not include any water and triﬂuoroacetate salt which will be present in the dried material. All puriﬁed peptides are supplied with HPLC and MS traces. At AltaBioscience we make extensive use of capping during synthesis, so deletion peptides are very rare.
We provide various peptide salt forms. Peptides are typically supplied with triﬂuoroacetate as the counter-ion but we can offer both acetate or chloride salt forms on request. Peptide purified by HPLC with acetonitrile gradients and triﬂuoroacetic acid (TFA), exist as their TFA salts but this can be toxic to cell cultures. We can convert the TFA salts to another form to overcome this.
Additional information about your synthetic peptide provides assurance in the quality of the product synthesised. There are a number of methodologies available to give added quality assurance.
- High Performance Liquid Chromatography (HPLC)
HPLC is the primary method of analysing peptide purity. This is performed typically on a C18 reverse phase column, using an acetonitrile water gradient with TFA as the acidic species.
- Matrix Assisted Laser Desorption and Ionisation (MALDI-TOF)
A ‘matrix assisted laser desorption and ionisation – time of ﬂight’ 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 synthesised.
- Amino Acid analysis
All dried peptides contain a variable amount of water plus a ﬁxed amountof the peptide counter-ion, usually TFA. Quantitative amino acid analysis is the only method which enables the net peptide content to be determined. The peptide is acid hydrolysed to its amino acids and these are quantiﬁed after separation by ion exchange chromatography and detection with ninhydrin. 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%.
- N-terminal Sequencing
Amino N-terminal Edman sequencing can be used to conﬁrm the correct sequence of amino acids. Additional information about your synthetic peptide provides assurance in the quality of the product synthesised. There are a number of methodologies available to give added quality assurance.
Author: Sat Sandhu, Principal Peptide Chemist.