Question 1

How to store peptides?

Accepted Answer

Lyophilized Peptides For long-term storage of peptides, we recommend storing them as a solid powder. Lyophilized peptides can be stored at -20°C or lower with minimal degradation. Customers are advised to re-inspect customized peptides every 2 years from the date of quality control release. Peptides in solution Peptides in solution are less stable and prone to degradation. Aqueous solutions used to dissolve peptides should be sterile and pure. In solution, a certain amount of peptide may be degraded, depending on the amino acids present in the sequence. Some examples are listed below: – Peptides containing methionine, cysteine or tryptophan residues should have limited storage time in solution due to oxidation. These peptides should be dissolved in an oxygen-free solvent. – Glutamine and asparagine can be deamidated to glutamic acid and aspartic acid, respectively.

Question 2

How do you dissolve peptides?

Accepted Answer

The solubility characteristics of different peptides vary considerably. Residues such as alanine (Ala), cysteine (Cys), isoleucine (Ile), leucine (Leu), methionine (Met), phenylalanine (Phe), and valine (Val) add to the peptide’s hydrophobicity and solubility in aqueous solutions, and it is recommended that the customer follow these guidelines when customizing peptides. Solubility Properties The solubility of peptides is highly dependent on the amino acid sequence. Naturally hydrophobic peptides (A, F, G, V, L, I, M, W, P with higher propensity) require organic solvents for solubilization. Acidic peptides (higher propensity for D, E in the peptide sequence) require basic aqueous buffers for solubilization, while basic peptides (higher propensity for K, H, and R) require acidic aqueous buffers for solubilization.

Question 3

What is the difference between net vs gross peptide weight?

Accepted Answer

The industry standard is to deliver peptides in a lyophilized form as a Gross weight. Gross peptide weight is the total weight of all components present in the lyophilized powder. This includes the peptide of interest, any peptide impurities, water, residual solvents, and counterion. On the contrary Net peptide weight, calculated from the Net peptide content, is the weight of only the peptide component present in the lyophilized powder. This offers more accurate concentrations of your subsequent peptide solutions. Hence, we recommended net peptide weight amounts for Custom Peptide orders. Net Peptide Content measurement is performed by amino acid analysis (AAA; limited accuracy but requires a low material amount) or elemental analysis (CHN; requires milligrams of peptide but is more accurate). Both methods yield a percentage value (ie., 75% net peptide content).

Question 4

What type of chemistry do you use?

Accepted Answer

MOL Changes utilizes solid-phase Fmoc chemistry rather than BOC chemistry. Fmoc chemistry allows for the removal of protecting groups using mild conditions during peptide elongation, while BOC chemistry requires the use of a strong acid to remove the protecting groups during peptide elongation. Additionally, Fmoc chemistry utilizes trifluoroacetic acid (TFA) to remove the peptide from the resin, while BOC chemistry utilizes hydrofluoric acid (HF). HF is colorless, highly corrosive, and a contact poison. Therefore, MOL Changes employs Fmoc chemistry as it is more mild, flexible, and versatile compared with BOC chemistry.

Question 5

How do I calculate molarity of my peptide?

Accepted Answer

Molarity refers to the molar concentration of a solution, which is the number of moles of solute dissolved in 1 liter of solution, expressed as mol/L, or M. Molarity [M] = Mass / (Volume x Molar Mass); Mole = Concentration (g/L) x Volume (L)/MW (g/mol) Example:Given: 1mg of dry peptide powder with MW: 20KDa (Molar mass of peptide is 20 g/mol)To determine molarity with known mass and known volumeFor a 1ml solution of this peptide:Molarity = Mass (0.001g) / (volume (0.001L) x Molar Mass (MW 20,000) = 50μM To determine mass to achieve a certain molarity:If for your assay, you need 0.01mM working peptide solution in 1ml of water, then calculate mass required as follows: Mass = Molarity x Volume x Molar MassMass = 0.01mM x (1/1000 L) x 20,000g/mol = 0.0002g Hence, you will need 0.2mg/ml of peptide to have a working solution of 0.01mM.

Question 6

In what form are peptides delivered?

Accepted Answer

We deliver as lyophilized or raw powder and peptide solution.

Question 7

How do I know if my peptide will be soluble?

Accepted Answer

The solubility of a peptide in a given solvent is dependent on the amino acid sequence and often hard to predict. In the case of catalog peptides, the solubility information is listed on the QC datasheet. For custom peptides, please see the section entitled “How should I solubilize my peptide”.

Question 8

What is the difference between peptide content and peptide purity?

Accepted Answer

Peptide content is not an indication of the peptide purity as these are two separate measurements. Purity is determined by analytical HPLC and indicates the presence/absence of other peptidic contaminants (i.e. truncated peptides). Net peptide content determines the actual amount of the peptide (peptide of interest as well as the peptide contaminants) present in the sample. The net peptide content is traditionally measured by Amino acid analysis (AAA; limited accuracy but requires a low amount of material) or elemental analysis (CHN; requires milligrams of peptide but is more accurate).

Question 9

How do you generally purify your peptides?

Accepted Answer

MOL Changes peptides are generally purified by reverse-phase preparative HPLC, using 2 buffers containing 0.1% trifluoroacetic acid (TFA). The first buffer (Buffer A) is composed of 0.1 % TFA in deionized water and the second buffer (Buffer B) is composed of 0.1 % TFA in acetonitrile (ACN). Generally, crude peptides are first dissolved in a solution of either Buffer A, some amount of Buffer B, or an organic polar solvent such as DMSO. If an organic polar solvent or Buffer B was used, this clear solution will first be diluted with Buffer A. Next, the peptidic solution is filtered and loaded onto the preparative HPLC system. Utilizing an optimized method gradient, fractions are then collected based on the absorbance readings. Each fraction is then analyzed via an analytical HPLC to ensure that the purity of each fraction meets the required purity specification.

Question 10

How do I write my peptide sequence termini?

Accepted Answer

The international nomenclature used for the sequence termini is as follows: – N-terminus: H means free amine (NH2-), Ac means acetyl [CH3C(O)-NH-], Pyr means pyroglutamic acid– C-terminus: OH means free acid (-COOH), NH2 means amide [-CONH2] – Modifications on the side chain of amino acids are depicted in the parenthesis after the corresponding amino acid. For example; phosphorylated serine = S(PO3H2) or epsilon-N-acetylated lysine = K(Ac)

Question 11

What is a peptide counterion?

Accepted Answer

During peptide production, ions in the synthesis and purification solutions bind to the charged amino acid side chains of the peptide, forming salts. These ions are known as peptide counterions and help balance the peptide’s charge. Trifluoroacetic acid (TFA) is commonly used in peptide synthesis and purification. It forms trifluoroacetate salts with basic amino acids (e.g., lysine, arginine, histidine) and the free N-terminal amine. Therefore, trifluoroacetate is typically the default counterion. The minimum number of TFA molecules bound to a peptide is proportional to the number of basic residues in a given peptide sequence. Thus, the more basic residues present in a peptide, the lower the net peptide content of the lyophilized powder. To account for the TFA or water content present in the sample, we recommend performing a peptide content assay for every custom peptide.

Question 12

hy are peptides containing Cys, Met, or Trp difficult to synthesize?

Accepted Answer

Peptides containing Cys, Met, or Trp are challenging to synthesize and obtain high-purity products. This is mainly due to the instability and susceptibility to oxidation of these functional groups. Special attention is required for the use and storage of these peptides to avoid repeated opening of the vials. Did we miss your question? Drop us a line and we willget back within 24 hours.

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