Peptide Analysis Services

JPT is the expert not only for peptide synthesis of a large variety of peptide formats and peptide-based products, but also conducts a wide variety of peptide analysis services supporting many different applications.
We analyze all the peptides we produce and offer additional quality measurements and analytical services.

Chemical & Physical Analyses

Peptide Purity Determination

Peptide Purity is determined by UV absorption at 220nm using RP-HPLC (reversed phase high performance liquid chromatography). This technique is used to separate, identify, and quantify components in a mixture, e.g. a target peptide and potential impurities.

  • Peptide sample in solution is pumped through a adsorbent column
  • Sample components interact differently with the adsorbent
  • Different flow rates lead to separation of components
  • UV detector generates a signal for each component at the end of the column
  • Allows for quantitative analysis of components

Material requisition: > 150 µg/sample

Time needed: 1-2 days

Peptide Molecular Weight Determination

Relative molecular mass or molecular weight of a molecule is calculated by the relative atomic masses of each constituent element. Together with HPLC purity, confirmation of the molecular weight of a synthetic peptide provides preliminary evidence on the success of peptide synthesis. In mass spectrometry, the molecular mass of a larger molecule such as a peptide is usually calculated based on the average molecular mass which is the average of the isotopic masses weighted by the isotopic abundances.

We determine the molecular weight of peptides by different mass spectrometric approaches. We use different ionizations (e.g. ESI and MALDI) as well as various detection methods such as TOF, ion trap or quadrupole, depending on the properties and associated detectability of a peptide.

Material requisition: > 150 µg/sample

Time needed: 1-2 days

Peptide Sequencing

The amino acid sequence represents the order of amino acids as they occur in a polypeptide chain and is critical for the biological function of a peptide. Whereas Edman degradation has been used for a long time, tandem mass spectrometry is now the method of choice for confirmation of peptide amino acid sequences. By using tandem mass spectrometry JPT is able to directly sequence peptides up to a chain length of 25 amino acids. For longer peptides we are applying peptide specific enzymatic degradation followed by subsequent sequencing of the resulting fragments.

Material requisition: 1mg

Time needed: 2 days

Peptide & Building Block Structure Confirmation

NMR (nuclear magnetic resonance) is a classical analytical technique used for structure determination and confirmation in organic synthesis, e.g. of peptides or building blocks for peptide synthesis. The principle is based on the different behavior of atoms (e.g. 1H, 13C) in a strong static and oscillating magnetic field depending on their chemical environment.

NMR is not frequently used for peptide analysis because NMR spectra of peptides are usually very complex with many signals overlapping. However, NMR is perfectly suited for the precise structural confirmation of very small peptides and building blocks, which is a synthesis service that JPT also offers.

Material requisition: 2-20 mg

Time needed: 2-3 days

Peptide Solubility Testing

Peptide solubility is a crucial factor for in vivo bioavailability and in vitro assay robustness. Lack of full solubility at the desired effective concentration will lead to decreased activity and false negative results. Turbidimetric Solubility Assay (TSA) allows a rapid determination of solubility using small peptide amounts. Depending on applications of the target peptides, different organic (DMSO, DMF, acetonitrile etc.) and inorganic (water, aqueous buffers like PBS) solvents can be selected for solubility measurements by TSA.

  • Known amounts of lyophilized peptides are dissolved in selected solvents
  • Special conditions (e.g. ultrasound sonication; vortexing; heating treatment) can be applied to improve peptide solubility
  • If a peptide is not soluble, the solution will be turbid and produce light scattering
  • The sample is measured at 850 nm to measure any light scattering
  • A plot of turbidity vs. peptide concentration indicates the maximum concentration dissolved, which is solubility

Material requisition: 1 mg per test condition

Time needed: 2 days

Peptide Stability Testing

Peptides have an inherent and sequence specific stability profile which depends on the properties of the individual peptides and storage conditions. Degradation of peptides during storage can be attributed to a variety of chemical reactions such as oxidation, hydrolysis, structural rearrangements and more. Despite this knowledge, stability of a given peptide is hard to predict. Stability testing is therefore advisable for certain applications and performed to determine the short and long term stability of peptides.

  • Peptide aliquots are stored under defined conditions (e.g. -20°C, -80°C, room temperature)
  • They are analyzed by HPLC-MS at certain time points (e.g. 3 months, 6 months, 12 months)
  • Acquired data demonstrate HPLC purity of peptides under the tested conditions in the recorded time frame
  • This information is useful to determine expiry dates and necessary storage conditions for peptides
  • In order to predict long term stability of peptides in a short time frame, a stress stability test at elevated temperatures (e.g. 40°C) can be performed

Material requisition: approx. 1 mg/sample

Time needed: depends on time points

Peptide Quantitation / Peptide Content Determination

Due to their chemical properties lyophilized peptides may still contain traces of moisture and counter ions on protonated amino functions (N-terminus, Arg, His, Lys, etc.). This is not considered an impurity, but reduces the actual peptide content by approximately 10 to 30%.

An accurate determination of the peptide content can be performed by quantitative amino acid analysis. The analysis includes an acidic hydrolysis of the peptide, transforming the sample into free amino acids. Then amino acid analysis and quantification of total peptide content are performed.

Material requisition: 1 mg/sample

Time needed: 2 weeks

Amino Acid Composition

The amino acid composition provides additional evidence on the assembly success for a given peptide. To confirm the results of peptide sequencing it is possible to perform a quantitative amino acid analysis. It provides quantitative information on amino acid ratios within a peptide and yields exact peptide content for accurate calculation of the peptide concentration.

  •  Acidic hydrolysis of the peptide, transforming the sample into free amino acids
  • Amino acids are quantified by ion exchange chromatography (or ion chromatography)
  • Ion chromatography separates ionizable molecules based on their total charge and enables separation of similar molecules that would otherwise be difficult to separate

Material requisition: 1 mg/sample

Time needed: 2 weeks

Residual Solvent Determination (e.g. DMF, Acetonitrile …)

Residual solvents from peptide synthesis and purification such as DMF, Acetonitrile, DMSO or NMP can be cytotoxic or lead to false results in biological applications.

Residual solvents can be determined by gas chromatography that is able to separate chemicals in a complex sample. The peptide sample is added into a column with a gas stream (carrier gas, mobile phase) which the different chemical components pass at different rates depending on their various properties and their interaction with the column filling (stationary phase). As the chemicals exit the column, they are detected and identified.

Material requisition: 1.5 mg/sample

Time needed: 2 weeks

Residual Water Determination

Due to their chemical properties lyophilized peptides may still contain traces of moisture. This is not considered an impurity, but reduces the actual peptide content by approximately 2 to 15%. In order to determine the exact composition of a lyophilized peptide probe the amount of residual water also needs to be analyzed.

Residual water in a peptide is determined by gas chromatography. The peptide sample is heated to desorb the water and then added into a column with a gas stream (carrier gas, mobile phase) which different chemical components pass at different rates depending on their various properties and their interaction with the column filling (stationary phase). As the water exits the column, it is detected and quantified.

Material requisition: 1.5 mg/sample

Time needed: 2 weeks

Residual Counter-Ion Determination (e.g. TFA)

Due to their chemical properties lyophilized peptides may still contain counter ions on protonated amino functions (N-terminus, Arg, His, Lys, etc.). These counter ions result from acids being used in the workup or purification of the peptides and are not considered an impurity. However, they reduce the actual peptide content by 5 to 25% depending on the peptide sequence. In order to determine the exact net-amount of a lyophilized peptide sample, the amount of residual counter-ions needs to be analyzed. Furthermore, when TFA (trifluoroacetic acid) is being used during peptide synthesis and purification, potential cytotoxicity needs to be considered for some applications. Then, TFA can be removed by anion exchange using chloride (HCL) or acetate (HOAc). To assess whether the anion exchange was successful it is possible to determine residual TFA amounts. Gas chromatography is being used to determine residual counter-ion amounts.

Material requisition: 1.5 mg/sample

Time needed: 2 weeks

Enantiomeric Integrity

The alpha carbon of naturally occuring alpha amino acids (all but glycine) is a chiral carbon atom. Therefore, each amino acid has two enantiomers, the L- and D-amino acid. L-amino acids represent the 20 natural proteinogenic amino acid, wheras D-amino acids are only rarely found in organisms.

We only use L-amino acids for peptide synthesis (unless you specifically order D-peptides). However, some amino acids, like histidine and cysteine, are prone to racemication (swapping from L to D) during chemical synthesis. The chirality affects the unique 3D structure of each peptide and protein. As the protein or peptide structure is responsible for the recognition by a receptor or an enzyme it is important to ensure enantiomeric integrity of synthetic peptides.

To ensure enantiomery purity we hydrolyze the peptides and analyze the resulting amino acids by gas chromatography using a chiral column to separate amino acid enantiomers.

Material requisition and time needed: upon request


Biological Testing

Bacterial Endotoxin Testing

Endotoxins are lipopolysaccharides (LPS), large molecules consisting of a lipid and a polysaccharide, that are found in the outer membrane of Gram-negative bacteria. Endotoxins are released after destruction of the bacterial cell wall or secreted in the form of bacterial outer membrane vesicles. In contrast to bacteria, endotoxins are very heat stable even after sterilization. They induce strong responses from animal immune systems, e.g. inflammation. There are two standard tests for endotoxin testing, LAL Test and Haemotox rFC.

LAL (Limulus amebocyte lysate) is an extract of blood cells from the Atlantic horseshoe crab (limulus polyphemus). LAL reacts with the bacterial endotoxin lipopolysaccharides, which can be detected by gel-clot, turbidimetric, or chromogenic methods.

Haemotox rFC is a recombinant endotoxin receptor (factor C) that is alternatively used to limulus amebocyte lysate. The reaction is measured via highly sensitive fluorescence detection. This new test appears to be more accurate than the traditional LAL test.

Material requisition: 1.5 mg/sample

Time needed: 2 weeks

Peptide Sterility Testing

Sterility is important if peptides are intended for use in cellular assays or for development of cell & immunotherapies or vaccines. Although peptide synthesis is performed under low bioburden conditions we can not guarantee sterility without further testing. For sterility testing lyophilized peptides are dissolved in water and subsequently incubated in tryptic soy broth. Subsequent to incubation turbidity is determined as indicator for contaminating microorganisms (aerobic microorganisms, yeast & fungi).

Material requisition: 1.5 mg

Time needed: 2 weeks

Cell Toxicity Testing

When applying peptides in cellular assays or other biological systems, testing peptide samples for cell toxicity may be advisable as certain counter-ions or residual solvents are able to display cytotoxic properties. An in vitro toxicity test determines if the dissolved peptides show cytotoxic effects i.e. influence cellular metabolic activity. Therefore, the dye resazurin is used that is metabolized by living cells.

  • A T cell lymphoma cell line is treated with the dissolved peptides
  • After 24h the dye resazurin is added
  • Viable, metabolically active cells will reduce the dye to resorufin
  • Resofurin is photometrically quantified thus determining any cytotoxic effects of the peptides

Material requisition: 1 mg

Time needed: 2 days

Bioburden Determination

Bioburden is defined as the number of microorganisms living on a surface that has not been sterilized. The aim of bioburden testing (microbial limit testing) is to measure the total number of viable microorganisms (total microbial count) prior to sterilization. Products used in the pharmaceutical or medical field require control of microbial levels during processing and handling. We test for total aerobic microbial count (TAMC) and total combined yeast and mold count (TYMC).

Material requisition: 1 mg

Time needed: approx. 1 week


Bio- & Cheminformatics

Manufacturability Evaluation /Ranking

JPT has developed an algorithm called PepSelector that performs an evaluation of manufacturability and feasibility ranking of peptide sequences. PepSelector applyies a proprietary manufacturability algorithm on the basis of literature and experimental data from several tens of thousands of synthetic peptides. The algorithm considers amino acid coupling efficiencies, side reactions during the synthesis process, peptide stability, peptide solubility, correlation of physicochemical properties and secondary structure propensities with manufacturability, the ability to be purified and reconstituted in aqueous buffers.

The goal of applying this algorithm is to predict potential difficulties and rank a given set of peptides according to their overall manufacturability.

Although PepSelector does not provide a go/no go differentiation of peptides for production it enables selection of peptides with higher success probabilities against peptides with an increased risk to fail. As a consequence it is an efficient tool to help ranking peptides for the fast, efficient and successful synthesis in turnaround sensitive studies such as neo-epitope based individualized immunotherapies. In addition to manufacturability evaluation the program may provide access to potential optimization of sequences that can be performed by simple shortening, extension or frame-shifting the peptide sequences along their parental antigen sequences.

Time needed: 1-2 days


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