Frequently Asked Questions (FAQ)

Peptide purity is the term used to describe the percentage of the peptide with the target sequence among the total quantity of material. Because peptide bond formation is not 100% efficient during peptide synthesis, not all polypeptide chains are made of the target sequence. For example, some chains might not be complete, or amino acids might not bind appropriately. These deleted or incorrect sequences form a certain percentage of peptides in most peptide mixtures. We analyze and purify crude peptides using reverse-phase HPLC, and then analyze the resulting material using MS to achieve the desired target sequence purity.

After your peptide has been purified and lyophilized, the white peptide powder will contain some non-peptide components such as water, salts, absorbed solvents, and counter ions. The peptide content describes the actual percentage weight of the peptide in your final product. This number varies but is commonly 50–90% depending on the purity, sequence, and methods used for synthesis and purification. When calculating the concentration of peptide solutions for biological assays or other experiments, the peptide content must be accounted for. The actual peptide concentration can be determined by subtracting the non-peptide weight from the total weight, which allows you to determine what volume of solvent to use. For example, if you were using 1 mg of the final product to make a 1-mg/ml peptide solution with a content of 80%, you would use 800-μl of solvent rather than 1000 μl.

It is important to note that peptide content and peptide purity are two distinct measurements. Purity is determined using HPLC and revealed the presence or absence of contaminating peptides with the incorrect sequences. In contrast, the net peptide content provides only information regarding the percent of total peptide vs. total non-peptide components: it does not consider the presence of multiple peptides. The net peptide content can be determined accurately by performing amino acid analysis or UV spectrophotometry.

It is difficult to determine the actual concentration of a peptide based on the weight of the lyophilized peptide. Lyophilized peptides might contain 10–70% water and salts by weight. Generally, hydrophobic peptides contain less bound water and salts than hydrophilic peptides.

If the peptide has a chromophore in its sequence (W or Y), the peptide concentration can be determined conveniently using the extinction coefficient of these residues as follows:

1. Molar extinction coefficients of chromophoric residues at 280 nm at neutral pH using a 1-cm cell:
   • Tryptophan 5560 AU/mmole/ml
   • Tyrosine 1200 AU/mmole/ml
2. The molar extinction coefficient of chromophoric residues is measured at 280 nm at neutral pH using a 1-cm cell. That of W is 5560 AU/mmole/ml, whereas that of Y is 1200 AU/mmole/ml
3. The extinction coefficient of the chromophores in a peptide sequence is generally additive; therefore, the overall molar extinction coefficient of the peptide depends on the type and number of chromophoric residues in the sequence.
4. When performing the calculations, the mg peptide per ml = (A280 x DF x MW) / e, where A280 = the actual absorbance of the solution at 280 nm in a 1-cm cell, DF = dilution factor, MW = molecular weight of the peptide, and e = molar extinction coefficient of each chromophore at 280 nm.
5. Hypothetical example: A 50× diluted solution of the peptide with the sequence GRKKR RQRRR PPQQW DCDLY RPYEK T (MW = 3418) would measure 0.5 AU at 280 nm in a 1-cm cell. The concentration of the original peptide in the stock peptide solution would be calculated as follows:

mg peptide/ml = (0.5AU x 50 x 3418 mg/mmole) / [(1 x 5560) + (2 x 1200)] AU/mmole/ml = 10.7

6. Cautions:

• Any absorbance calculation assumes that a peptide is unfolded and that the chromophores are exposed. This is generally an acceptable assumption for short, soluble peptides. If there are doubts about the solubility or folding of a given peptide, it is advisable to measure denaturing conditions (e.g., in the presence of 6 M GdnHCl or 8 M urea). It is important to remember that these peptide solutions will not be functional until the denaturants have been removed.
• If the sequence does not have W or Y amino acid, the peptide concentration can only be determined using amino acid analysis.