One of the most challenging aspects of working with synthetic peptides is determining the best solvent in which to dissolve the peptide. General guidelines for proper storage, handling and dissolving your synthetic peptide are provided below.
Storage of Lyophilized Peptides
Lyophilized peptides are shipped at ambient temperature and will remain stable for days to weeks. For long term storage, the material should be stored at –20 °C or colder and away from bright light.
Exposure to moisture will decrease long-term stability of lyophilized peptides. Before using the peptide, remove from cold storage and allow the peptide to equilibrate to room temperature before removing the lid of the container. This will reduce the uptake of moisture that is present in the surrounding atmosphere.
There is no universal solvent for solubilizing all lyophilized peptides, while also maintaining their integrity and compatibility in biological assays. Selecting the best solvent may be a result of a “trial-and-error” process. Wherever applicable, it is advisable to first try solvents that are relatively easy to remove by lyophilization, in case the initial solvent does not does not dissolve the peptide. Always test a portion of the peptide before dissolving the entire peptide sample. The steps below provide a general guide for peptide solubilization. For any solvent used, the tolerance of the solvent in your assay should be taken into consideration before attempting these strategies.
Determining Solubility Characteristics
Evaluating the amino acid composition of the peptide is a preliminary tool in understanding the solubility characteristics of your peptide. The number and types of ionic charges in the peptide determine solubility in aqueous solutions. In general, the more charged residues the peptide possesses, the more soluble it is in aqueous solutions. Please note that because peptides generally have more charges at pH 6–8 than at pH 2–6 they are more likely to be better dissolved at near neutral pH. Exceptions are for peptide sequences that are very hydrophobic and tend to aggregate. While hydrophobicity of a sequence can be the primary cause of aggregation, peptides will also aggregate or “gel” through extensive hydrogen bonding.
Use the following guidelines to determine if the peptide is basic, acidic or neutral:
Step 1. Assign a value of –1 to each acidic residue (D, E and C-terminal COOH).
Step 2. Assign a value of +1 to each basic residue (K, R and the N-terminal NH2).
Step 3. Assign a value of +1 to each H residue at pH <6 or zero at pH >6.
Step 4. Add up the total number of charges of the peptide at pH 7 (all D, E, K, R, C-terminal COOH and N-terminal NH2) to calculate the overall net charge of the peptide.
Dissolving Approach for Charged Peptides
Based on the above guidelines, determine the overall solubility type of the peptide using the following strategies:
Acidic Peptides (net charge is negative)
A peptide is considered acidic if the overall net charge of the peptide is negative. If the peptide is acidic, and/or if the total number of charges of the peptide at pH 7 is greater than 25% of the total number of residues, add a small amount of 0.1 M ammonium bicarbonate to dissolve the peptide and dilute with water to the desired concentration. Ensure the resulting pH of the peptide solution is approximately 7 and adjust the pH as needed.
Basic Peptides (net charge is positive)
A peptide is considered basic if the overall net charge of the peptide is positive. If the peptide is basic and the total number of charges of the peptide at pH 7 is between 10 and 25% of the total number of residues, add a small amount of 25% acetic acid to dissolve the peptide and dilute with water to the desired concentration.
Neutral Peptides (net charge is zero)
A peptide is considered neutral if the overall net charge of the peptide is zero. If the total number of charges is greater than 25% of the total number of residues, use the strategy described for acidic peptides.
If the total number of charges of the peptide is less than 10% of the total number of residues, the use of organic solvents is recommended.
Before trying stronger solvents, sonicate the peptide solution to confirm that the peptide is insoluble in the chosen solvent. Sonication enhances solubilization, breaking the solid peptide into smaller particles. If, after sonication, the solution has gelled, appears cloudy, or has visible particulates, the peptide has not dissolved completely but is suspended. At this point, a stronger solvent is necessary. If the peptide does not dissolve, lyophilize and remove the volatile buffer solution. Once the sample is dry, alternative solvents can be tested on the same sample.
Dissolving Approach for Hydrophobic/Uncharged Peptides
For peptide sequences containing greater than 50% hydrophobic residues, neutral peptides with less than 25% charged amino acids, and/or peptides that have less than 10% charged amino acids, the use of organic solvents is recommended. Acetonitrile (ACN), dimethylsulfoxide (DMSO) or dimethylformamide (DMF) are suggested solvents. Addition of chaotropic compounds such as guanidine hydrochloride or urea can facilitate in breaking up hydrophobic interactions or reduce the “gelling” of peptides by disrupting hydrogen bonding network. Consideration of the tolerance of your assay to these organic or chaotropic reagents should be maintained during the selection process. Peptide sequences containing Cys (C) and Met (M) are unstable in DMSO. It is important to dissolve the peptide completely in the initial solvent (such as acetic acid, acetonitrile, DMSO or DMF) because the rate of dissolution of the peptides into these solvents is usually higher than in a water/solvent mixture. If the water/solvent mixture is used first to dissolve the peptide, you may end up adding a much larger than necessary amount of non-aqueous solvent to your peptide sample. Sonication may also be necessary to facilitate complete dissolution of the peptide.
After the peptide is dissolved in the initial solvent, especially those dissolved in organic solvents, dilute the peptide by slowly adding (dropwise) the peptide solution into the buffered solution with gentle but constant agitation. This technique prevents localized concentration of the peptide in the aqueous solution, which can potentially result in precipitation of the peptide. The benefit of this strategy is that peptide precipitation can be visually monitored and acted upon accordingly.
Preparing a Working Stock Solution
Prepare a peptide stock solution at a higher concentration than required for the experimental assay by dissolving the peptide in sterile distilled water or sterile dilute acetic acid (0.1%), where applicable. The stock solution peptide can be further diluted with the assay buffer.
Caution: If the assay buffer, free of nonvolatile salts and/or organic solvent, is initially used to dissolve the peptide and fails to dissolve, recovery of the peptide may be a challenge. If the peptide does not dissolve in water or acetic acid, the peptide solution can be lyophilized back to its original state. Once the peptide is lyophilized, alternate solvents can be tried.