Frequently asked questions

A concentration of ≥ 2mg/mL is recommended.

See the product data sheet for recommended buffers.  It is important to avoid buffer additive reducing agents (e.g. DTT), protein stabilizers (e.g. BSA) or sodium azide. Additionally, the following buffers should be avoided: tris, glycine, ammonium salts and buffers containing free amines.

The free dyes are removed during the purification step via the spin column that is provided with the kit. 

On average, we observe 70-80% yields.

Yes, but it is important to use a membrane molecular weight cut off that is small enough to prevent protein from being lost through the membrane pores.

Dyes in AnaTag kits have a succinimidyl ester reactive group which can bind to any free amine available on your protein.  Hence the dye can bind to any Lysine residues and protein N-terminus.

Lyophilized peptides
For the long-term storage of peptides, we recommend that peptides be stored as a solid powder.  Lyophilized peptides can be stored at temperatures of -20 °C or lower with little degradation.  AnaSpec’s catalog peptides are checked every 2 years from the date of QC release to ensure that these peptides are within the required purity specification.  AnaSpec recommends that customers also re-check their custom peptides every 2 years from the date of QC release.

Peptides in solution

Peptides in solution are much less stable and are susceptible to degradation.  Solutions of water used to dissolve peptides should be sterile and purified.

In solution, some amount of the peptide may degrade depending on the amino acids present in the sequence.  A few examples of which are shown below:
- Peptides containing methionine, cysteine, or tryptophan residues should have limited storage time in solution due to oxidation. These peptides should be dissolved in oxygen-free solvents.
- Glutamine and asparagine can deamidate to Glu and Asp, respectively.
- Cysteines can undergo oxidative cyclization to form Cys-Cys disulfide bridges (intra or inter disulfide bridges can form).
- Charged residues (Asp, Glu, Lys, Arg, His) are hygroscopic (take up water from moisture in the air) and will easily form a viscous clear oil.  This physical change may not affect the properties of the peptide.  It is recommended that such peptides be first lyophilized to remove trace amounts of water, degassed with N₂ to ensure that there is no water vapor in the vial, and quickly capped.

To prevent any damage caused by repeated freeze-thaw cycles, we recommend that the user only dissolves the amount of peptide needed for the immediate experiment.  Any excess solutions should be stored at ≥ -20 ^oC until needed.

Avoid moisture
As moisture will greatly reduce the long-term stability of peptides, we recommend that the peptide should be allowed to equilibrate to room temperature in a desiccator before opening the vial.  Once the peptide has been dispensed, any remaining peptide in the tube should be gently purged with anhydrous nitrogen, the container recapped, sealed with parafilm, and stored at -20 °C.

Peptide solubility characteristics vary strongly from one peptide to another.  Residues such as Ala, Cys, Ile, Leu, Met, Phe, and Val will increase the chance of the peptide being hydrophobic and dissolving in aqueous solutions.  AnaSpec catalog peptides are tested for their solubility in certain solvents.  This information is located on every QC Datasheet that is shipped along with the peptide.  AnaSpec recommends that customers adhere to the below guideline for their custom peptides.

Solubility properties

Peptide solubility is highly dependent on the amino acid sequence. Hydrophobic peptides (high propensity of A, F, G, V, L, I, M, W, P) in nature, will require an organic solvent to dissolve. Acidic peptides (high propensity of D, E in the peptide sequence) require a basic aqueous buffer to dissolve, while basic peptides (high propensity of K, H, and R) require an acidic aqueous buffer to dissolve.

Selection of solvent

Taking into consideration the limitations of your assay, we recommend that the following guideline be used to determine the best solvent to dissolve your peptide 

Hydrophobic peptides

To reconstitute a hydrophobic peptide, add 100 µL of DMSO and sonicate until a homogenous solution forms. Next, add your buffer of choice to form a 1 mg/mL solution (a higher concentration of peptide will require a greater amount of DMSO).

Hydrophilic (acidic) peptide

To reconstitute an acidic peptide, add 100 µL of 1% NH₄OH to 1 mg of the peptide and vortex.  After the formation of a clear solution, add your buffer of choice to form a 1 mg / mL solution. 

Hydrophilic (basic) peptide

To reconstitute a basic peptide, add distilled water to the 1 mg of peptide and vortex.   

Use and storage
Reconstituted peptides can be stored frozen at -20°C for a short time, but it is advisable to prepare multiple aliquots to avoid multiple freeze-thaw cycles. We recommend that all aliquoted solutions be lyophilized if the peptide is going to be stored for extended periods at -20 ^oC.

Peptides with a propensity to aggregate
For peptides that tend to aggregate due to the presence of multiple Cysteines, we recommend that these peptides be dissolved in degassed solutions and/or acidic conditions.

Additionally, some types of peptides have a propensity to form secondary and tertiary structures once dissolved.  We recommend that these peptides be first dissolved in solvents such as HFIP (hexafluoroisopropanol) and then evaporated using a stream of nitrogen.  The HFIP helps to break up the hydrogen bonding network that aids in forming the secondary and tertiary structures.

The industry standard is to deliver peptides in a lyophilized form and to state the delivery amount as the weight of the lyophilized powder “Gross weight”.

Gross weight contains the peptide of interest (including impurities), water, and a strong acid or base used in the preparative HPLC purification of the peptide.  The majority of peptides are purified using trifluoroacetic acid (TFA).  Water and TFA are byproducts of the purification process and there will always be trace amounts that cannot be removed completely. 

TFA forms a salt with basic groups (Lysine, Arginine, Histidine, and free N-terminal amine) present in the peptide sequence.  Usually, the minimum number of TFA molecules associated with a peptide is directly proportional to the number of basic residues in a given peptide sequence. 

Therefore, the more basic residues present in a peptide, then the lower the actual amount of peptide present in the sample.  To take into consideration the amount of TFA or water present in the sample, we recommend that the peptide content be determined for every custom peptide.  The net peptide content (NPC) is the fraction of peptidic material present in the lyophilized material.  In combination with the peptide purity, it allows one to determine the exact amount of the peptide of interest.

NPC is traditionally measured 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 measure total peptidic content.

Quant Peptides

AnaSpec offers Quant-Peptides corresponding to two proprietary peptide quantitation methods (with and without Quant-Tag), offering net peptide content with better accuracy and reproducibility than AAA or CHN.

AnaSpec 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, AnaSpec employs Fmoc chemistry as it is more mild, flexible, and versatile compared with BOC chemistry.     

AnaSpec’s QC datasheet for catalog peptides includes:

• Testing attributes (appearance, % peak area by analytical HPLC, identity by MS, and solubility)
• Test method
• Acceptance criteria
• QC results

This information is listed on all of our QC datasheets and the raw data for each test can be provided upon request.

AnaSpec can provide additional testing attributes as a fee for service offering.       

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 volume
For 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 Mass
Mass = 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.

Peptides are shipped as lyophilized powder or oil at room temperature via an express courier. On request, we can also deliver your peptides in solution.

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".

The standard biotin modification does not include a spacer. Spacers (PEG, X, etc.) are available on request.  Please contact service@anaspec.com for more guidance on the type of spacers that we offer.

Generally, AnaSpec can perform seven types of cyclization reactions as shown below:

 

• Head-to-tail, side-chain-to-head, side-chain-to-tail, and side-chain-to-side-chain cyclization products are usually confirmed by both a shift in molecular mass of 18 mass units in the mass spectrum as well as a change in the retention time in the analytical HPLC spectrum.




• A disulfide bridge cyclization is typically confirmed by a mass shift of 2 mass units in the mass spectrum and a shift in the retention time of the analytical HPLC spectrum. Ellman’s testing may also be used to confirm absence of free thiol groups.




• A hydrocarbon-stapled peptide consists of alkene that has been formed using a ruthenium catalyst. The hydrocarbon-staple ensures that the peptide conforms to an α-helix motif, which has been shown to be useful for some therapeutic applications. This staple is confirmed by both a shift in the molecular mass (loss of 28 mass units) in the mass spectrum as well as a change in the retention time in the analytical HPLC spectrum.




• Thioether-bond (-CH2-S-CH2-) containing peptides are another type of cyclic peptide. This type of cyclization is confirmed by both a shift in the molecular mass in the mass spectrum as well as a change in the retention time in the analytical HPLC spectrum.

AnaSpec can synthesize ‘wobble’ or degenerate peptides. As there are known differences in the incorporation rates of different amino acids, we can compensate for these rates to produce roughly equimolar amounts of each ‘wobble’ peptide.

AnaSpec can synthesize peptides containing non-radioactive atoms from commercially available building blocks. This includes amino acids containing ¹³C and/or ¹⁵N for Nuclear Magnetic Resonance (NMR) or Mass Spectrometry applications, just to name a few.

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).

AnaSpec’s 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. 

These “pure” fractions are then pooled together and lyophilized.  The “pure” powder is then tested for both purity and identity to ensure that the correct peptide has been manufactured.

Finally, an independent QC group at AnaSpec again tests the peptide to ensure that all of the testing attributes meet the required specifications.

Mass spectrometry (MS) is an analytical tool used to determine the identity of molecules.  In a typical MS procedure, a sample is ionized by bombarding the sample with a beam of electrons.  The results are a plot of intensity as a function of the mass-to-charge ratio (m/z). These measurements can often be used to calculate the exact molecular weight of the sample components as well.

MALDI-TOF (Matrix-Assisted Laser Desorption Ionization - Time of Flight) Mass Spectrometry

MALDI-TOF is a technique that uses a pulsed laser that generates a laser energy absorbing matrix to create ions from large molecules with minimal fragmentation.  MALDI-TOF instruments are often equipped with a reflectron that functions to increase the time of flight between ions of different m/z, which yields an increase in resolution.

ESI (Electrospray Ionization) Mass Spectrometry

ESI a technique used in mass spectrometry to produce ions using an electrospray in which a high voltage is applied to a liquid to create an aerosol. It is especially useful in producing ions from macromolecules because it overcomes the propensity of these molecules to fragment when ionized.

It is common to see Na (Sodium) and K (Potassium) adducts of the peptide in the mass spectrum.  Sodium and Potassium analytes originate from the water used to purify the peptide. Even distilled and deionized water has trace amounts of sodium and potassium ions, which can never be entirely removed. These analytes associate with the free carboxyl groups of the peptide and become easily ionized during the ionization step.

As a result, the mass spectrum of some peptides includes the molecular weight of either sodium and potassium adducts.  This is not an indication that the peptide is not pure, nor should it be confused with an incorrect molecular weight.

AnaSpec recommends that the N-terminal and C-terminal ends of a peptide, mimic the protein of interest.  Generally speaking, if the peptide sequence is from the middle of a protein sequence, then both ends of the peptide could be capped to avoid unwanted interactions. AnaSpec recommends that the N-terminal end be capped with an Acetyl group [Ac-NH₂] while the C-terminal end be capped with an Amide group [C(O)-NH₂].

For peptide sequences that represent the N-terminus, AnaSpec recommends keeping the N-terminus free (NH₂) while the C-terminus of the peptide should be amidated [C(O)-NH₂].

For peptide sequences that represent the C-terminus, AnaSpec recommends keeping the C-terminus free (COOH) while the N-terminus of the peptide should be acetylated [Ac-NH₂].

The above are suggestions only and any modifications or sequence specifications are based on your specific applications.

Maleimide-Thiol Linkage

The preferred method for linking a peptide to the carrier protein is through the side chain thiol (-SH) of a cysteine residue located at either end of the peptide.  Usually, the carrier protein is first modified using NBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester).  This reagent adds maleimide groups to the surface amines locate on the carrier protein.  This maleimide-adduct is then reacted with the thiol group from the peptide and forms a new Sulfur-Carbon bond.  The peptide is now conjugated to the surface of the protein via the maleimide linker.  

Glutaraldehyde Linkage

Glutaraldehyde cross-links primary amino groups on the peptide to those on the carrier.  This linkage is usually employed when there are no available Lys residues present in the peptide sequence.  Thus, the N-terminal amine is used as the main point of conjugation.

Carbodiimide Linkage

Carbodiimides couple free carboxyl and amine groups, whether C- or N-terminal or on side chains (i.e lysine, aspartic acid, or glutamic acid) from the peptide to the carrier protein via an amide bond. Amide bonds are extremely rigid and this type of linkage results in considerable steric hindrance as the peptide is tightly bound and close to the carrier protein.

Peptides containing one or more lysine, aspartic acid, or glutamic acid can couple to the carrier protein at various places in the peptide giving alternative conjugates.

MAPS (Multiple Antigenic Peptides)

In some applications (whereby a protein carrier is not necessary) multiple peptide strands coupled to a Lysine backbone may be of great use.  Typically 4, 8, or 16 copies of the peptide can be synthesized on a small multivalent core consisting of Lysine.  The peptide is synthesized by the usual solid-phase peptide synthesis and cleaved from the resin to give the crude MAP-4, -8, or -16. As the molecular weight of the peptide is now increased several-fold, it may serve as a standalone and not require the linkage to a carrier.

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)

When either Glutamine (Q) or Glutamic acid (E) is located at the N-terminus of a peptide sequence, a percentage of the Q or E may spontaneously cyclize to form Pyroglutamate (pGlu). This peptide can now be referred to as a pyroglutamyl peptide. Pyroglutamyl peptides are considered a normal subset of such peptides and are included as part of the peptide purity during HPLC analysis.
pGlu could affect a peptides activity or characteristics and hence have either a positive or negative effect on peptide application. In general, it is believed that the hydrophobic γ-lactam ring of pGlu may play a role in peptide stability against degradation by gastrointestinal proteases.
If only pGlu is desired at the peptide N-terminus AnaSpec also has the capability of replacing the Q or E with pGlu during synthesis. Note that in a peptide sequence, pGlu can also be referred to as Pyr.

During peptide manufacturing, ions in the synthesis and purification solutions, bind to the charged amino acid side chains of the peptide, to form salts. These ions are referred to as the peptide counterion and help to balance the peptides electrical charge.

Trifluoroacetic Acid (TFA) is frequently used in peptide synthesis and purification.

It forms a Trifluoroacetate salt with basic amino acids such as Lysine, Arginine, Histidine, and the free N-terminal amine. Hence Trifluoroacetate tends to be the default counterion.

The minimum number of TFA molecules associated with a peptide is directly proportional to the number of basic residues in a given peptide sequence. Therefore, the higher the number of basic residues present in a peptide, the lower the Net Peptide Content of the lyophilized powder. To take into consideration the amount of TFA or water present in the sample, we recommend that the peptide content be determined for every custom peptide.

Water and TFA are byproducts of the purification process. However, TFA can pose toxicity concerns for in-vivo and pre-clinical studies. Therefore, AnaSpec offers non-toxic Acetate, Chloride, or Ammonium salt exchange.