Custom AQUA™ Peptides Storage and Handling Guidelines
Custom AQUA Peptides
To meet the specific requirements of Protein– AQUA™ experimentation, Sigma® has developed a specialized custom peptide offering. Custom AQUA Peptides are synthesized with one labeled amino acid per peptide. The labeled amino acid contains 98 atom % 13C and 98 atom % 15N isotopic content. Each peptide is stringently tested to ensure high purity (HPLC), accurate molecular mass (LC-MS), and specific amino acid content (AAA). Custom AQUA Peptides are available in small package sizes (5 × 1 nmol) to enable convenient sample preparation and provide an appropriate peptide quantity.
The difference in the molecular mass of an AQUA peptide and its corresponding native peptide is key to differentiating the two via mass spectrometry. Due to the inherent isotopic mixtures in natural amino acids, it is important to clearly resolve all spectral peaks attributed to the native peptide from those attributed to the AQUA peptide. For this reason, a mass difference of 6 Daltons or greater is recommended. The stable isotope amino acids summarized below provide mass differences of 6 Daltons or greater and are available for incorporation into your Custom AQUA Peptide.
Storage and Handling of AQUA Peptides
AQUA Peptides are provided as 5 × 1 nmol packages. Each 1 nmol vial contains an AQUA peptide lyophilized as a thin film located at the bottom of the vial. In most cases, the AQUA peptide remains as a film. However, during transit, portions of the peptide may separate from the film and adhere to other surfaces within the vial, including the cap. It is imperative that the peptide be quantitatively solubilized in order to attain accurate analytical results.
Aliquots of peptide stock solutions and all working solutions should be stored at –20 °C and diluted as needed. Because stock solutions and subsequent working solutions are very dilute, the peptide may slowly deposit onto the surface of the vial over extended periods of time. Therefore, it is recommended that all peptide solutions stored for more than one day be vortexed prior to use. Working peptide solutions stored for more than one month should be discarded, as the integrity of the peptide and accuracy of the concentration may no longer be reliable.
1. Determine the Solubility Characteristics of your AQUA Peptide.
Complete solubilization is imperative for accurate Protein–AQUA™ quantitation. Peptide solubility is dependent on amino acid composition and sequence. The number and types of ionic charges in the peptide will determine its solubility in aqueous solutions. The more charged amino acids the peptide contains, the more soluble it is in aqueous solutions.
In general, peptides are more charged at pH 6-8 than at pH 2-5. At low pH, only the amino groups are ionized; whereas, at near neutral pH (6-8), both the carboxyl groups and the amino groups are ionized, resulting in negative and positive charges, respectively. However, due to the very low concentrations of the prepared solutions, AQUA Peptides generally dissolve at pH 2-3, which is often used for reversed phase HPLC solvents.
A peptide is classified as hydrophobic if 50% or more of the amino acids in the peptide sequence are hydrophobic, especially when the hydrophobic amino acids are clustered together. Hydrophobic amino acids include:
If your peptide is not hydrophobic, it can be considered hydrophilic. Using the peptide sequence of your AQUA Peptide, determine if it is a hydrophobic peptide or a hydrophilic peptide.
2. Dissolve your AQUA Peptide according to its Solubility Characteristics.
Hydrophilic Peptides: Dissolve the vial contents (1 nmol of AQUA Peptide) by adding 20 μl of 10% (v/v) aqueous formic acid solution. Vortex to fully dissolve the peptide.
Hydrophobic Peptides: Hydrophobic peptides are generally difficult to dissolve directly into aqueous media. Dissolve the contents of the vial (1 nmol of AQUA Peptide) by adding 20 μl of one of the following: neat acetic acid (HOAc), dimethyl sulfoxide (DMSO), acetonitrile (ACN), or dimethylformamide (DMF). Sonicate for one minute to ensure complete solubilization.
3. Prepare a Stock Solution
Add 180 μl of 0.1% formic acid in water and gently swirl to mix. This solution serves as a stock solution (5 pmols per μl). Working solutions can then be diluted from the stock depending on the application and sensitivity of the assay.