QYAOBIO Resource

Common FAQ, Blog, Publication, White paper in peptide synthesis

Home » Resource

Common FAQ

Custom peptide synthesis FAQ

Peptide name, sequence lot number
Peptide molecular formula
Theoretical molecular mass
Molecular mass by mass spectrometry
Purity by HPLC
Appearance and solubility test

With very few exceptions, peptides in Qyaobio are synthesized by chemical methods. There are three major synthesis methods of peptides: including solid phase peptide synthesis (SPPS), liquid phase peptide synthesis (LPPS), hybrid phase peptide synthesis (HPPS).

The peptide purity requirement is determine by the final applications.

Crude: Non-sensitive screening assays.
>70%: Non-sensitive screening assays, exploratory or relative MS assays.
>80%: ELISPOT assays, polyclonal antibody production, Non quantitative binding assays.
>85%: Immunological applications, polyclonal antibody production and non-sensitive screening.
>90%: SAR studies, receptor-ligand assays, bioassays.
>95%: In vitro bioassays such as ELISA, enzymology, Quantitative Mass Spectrometry SRM/MRM assays, monoclonal antibodies, quantitative receptor-ligated assays, enzyme kinetics, cell/tissue culture, biological activity.
>98%: Structural studies such as crystallography, NMR, X-ray or sensitive bioassays.

As most peptides contain the basic functionalities in sequences, such as the guanidino group of Arg, the ε-amino group of Lys, the imidazole moiety of His, the free N-terminus. All these basic moieties can form salt with acids. Except specific mentions, our peptides are provided as trifluoroacetate salts. Other salt forms are generated by an additional ion-exchange step, As the peptide will react to trifluoroacetic acid during the cleavage from the carrier resin.

Acidic peptides can form salts with bases, includes peptides with a large proportion of Asp, Glu, phospho, or sulfotyrosine. However, long peptides do not form stoichiometiric salts with acids or bases like low molecules.

As all research peptides are synthesized in TFA salt form. For cell-based assays or animal researches, we should remove TFA salt by switching it to HCl or acetate salt form with TFA<1%, in order to avoid the abnormal responses. Furthermore, higher purity>98% will get the optimal results depending on the budget.

In our normal peptide synthesis, Fmoc and Boc chemistry can both produce excellent peptides.

In hydrophobic peptide synthesis with high aggregation susceptibility, Boc chemistry provides a few advantages.

Removing Boc group in acid conditions will protonate the exposed amine terminal of peptides, this reduces the hydrogen bonding participation ,and increases its coupling availability.
Neutralization of activated amino acid will produce higher yields and purity peptides in difficult sequences.

Fmoc chemistry is more suitable for synthesis of larger peptides and small proteins with protected peptide fragments. Special resins are available for cleavage under mild conditions, in order to leave all other protecting groups intact.

The common method of solubility testing is based on charge determination. For small peptides up to 5 amino-acids, distilled water is the first option. Refer to the following guideline for other synthetic peptides.

Attribute -1 to every acidic residue(Asp, Glu) and the terminal carboxylic acid. Then assign -1 to each basic residues (Arg, Lys, His) and the terminal amine. Sum up both values to determine the overall charge of synthetic peptides.
Once the overall charge value is positive, try the distilled water at first. Then acidify the solution with the acetic acid (10 to 30%), when the peptides don’t dissolve. Add TFA if acetic acid does not allow peptide dilution to sufficient concentration.
If the overall charge value is negative, and peptides don’t contain cysteine, try to dissolve peptides in water. Then add ammonium hydroxide to achieve required concentration, once the peptides don’t dissolve.
In case the overall charge is zero, organic solvents like methanol, ethanol, isopropanol or acetonitrile, can be applied to dissolve peptides. According final application, the small amount of DMSO diluted with water can be employed. Furthermore, there are specific peptides are sensitive to oxidation, which contain cysteine, methionine or tryptophan residues, replace DMSO by DMF in these case.

Qyaobio synthesize peptides routinely between 20 and 90 amino acids. Although, we have extensive experience in peptide manufacturing from 40 AA to 100 AA. It is more difficult to synthesize peptide longer than 90 amino acids with high purity. In addition, Qyaobio is able to synthesize peptides up to 160 AA.

Chemically synthesized peptides have a free amine at the N-terminal and free acid at the C-terminal by default. We recommend the modifications for intra-cellular, in-vivo and in-vitro assays and researches. The modifications can increase the metabolic stability of peptides, and resistance to enzymatic degradation by exopeptidases, aminopeptidases, and synthetases.

Most lyophilized peptides are stable at room temperature for 2–3 weeks. Temperature at -20℃ is applied for long-time storage of lyophilized peptides. Furthermore, repeated freeze-thaw cycles must be avoided, and allow reaching room temperature before opening. The shelf life of peptide solutions is limited, it should be used as soon as possible once prepared.

There are several factors affect the peptide synthesis and purification, including amino acid succession, overall composition, peptide length, hydrophobic stretches.

Highly hydrophobic peptides are difficult to purify and solublize in aqueous solutions. These peptides normally involve residues like Leu, Val, Ile, Try, Phe and Met. Based on experience, we normally recommend one charged residue for every five amino acids.
Peptides with multiple Cys, Met or Try residues are difficult to achieve high purity, as these residues are prone to oxidation or hydrogen bonding. Normally, these peptides will result in slight delay, in reason of repeated synthesis and purification attempts.
Several Ser or Asp residues in one row will give rise to lower purity of peptides. Multiple Pro in sequence will create cis-trans isomerisation, this results in low apparent purity.
Peptides with the N-terminal Asn or Gln are difficult to achieve high quality, we recommend to remove, add or substitute with another amino acid.

Normally, the 10-25 residue peptides are recommended for antibody production. The longer peptides have more epitopes, but this also results in higher chance of forming in-native secondary structures with high stability. In addition, the shorter peptides less than 10 AA are not good enough unless valid reasons, such as potential sequence homology with the related protein family members.

The HPLC date refers to the information of peptide purity, it provides the percentage of target peptide in peptide material only, NOT the entire lyophilized powder.

The net peptides content refers to the percentage of peptide material in relation to the entire lyophilized powder. The typical net peptide content is 60-90%. Once for more accuracy, the net peptide content is achieved by Amino Acid Analysis (AAA), or Elemental Analysis (EA).

Generally, Qyaobio provides different types of cyclic reactions in peptides synthesis. In according, we have different analysis techniques to confirm the cyclizations.

Peptide cyclization of end to end, end to side, side to end are normally confirmed by both molecular shifts in Maldi-Tof mass spectrum and retention time alternative in analytical HPLC spectrum.

The dissulfide bridge cyclization of peptides are confirmed by HPLC before and after the cyclization step, the shift in retention time of the analytical HPLC spectrum can confirms the cyclic peptides.

For hydrocarbon-stapled peptides with alkene, the staple is confirmed by both the shift in molecular mass in mass spectrum, and retention time changes in analytic HPLC spectrum.

Peptide information

Professional Peptide Blog

Synthesis of Multi Antigenic Peptides

Synthesis of Multi Antigenic Peptides Introduction Multiple antigen peptides (MAPs) are a kind of polymeric peptide macromolecules bearing radial branches. It is a highly effective method for producing high-titer anti-peptide antibodies and synthetic peptide vaccines. MAP is essentially composed of three structural elements: (1) an amino acid, like glycine or f-alanine, that is…

Antigenic Peptides Design

Antigenic Peptides Design Introduction Antigenic peptides are synthesized via short amino acid sequences derived from carefully selected native target proteins. They can be used as immunogens to produce custom antibodies and are a popular option for using full-length proteins as immunogens. Their application has grown significantly in recent years. Advantages of Using…

Chemical Synthesis of Unnatural Amino Acids

Chemical Synthesis of Unnatural Amino Acids Introduction Unnatural amino acids (UAAs) not exist in natural polypeptide chains, so they are considered non-proteinogenic amino acids. They are an important component in the manufacture of many drugs and expand the range of protein functions via integrating them into proteins. Based on its structure may…

RGD Peptide

RGD Peptide Introduction RGD peptide is a synthetic peptide with the sequence GRGDSPK, which binds to integrins (receptor family of extracellular matrix proteins), thereby playing an important role in cell adhesion, migration, growth and differentiation. The RGD-sequence was originally discovered as the minimal binding epitope of fibronectin for cell attachment and…

Stapled Peptides for Drug Improvement

Stapled Peptides for Drug Improvement Introduction Stapled peptides are the new technology to bypass the intrinsic problem of peptide-drugs, this new strategy will increase the cellular uptake. Stapled peptides is consisting of peptide chains with α-helical structure by the external brace. The cross-link is achieved by the linkage of side chains…

Optimize Peptoid Synthesis

Optimize Peptoid Synthesis Introduction Peptoids (N-substituted glycines) are the peptidomimetic molecules for different applications. The automated solid-phase synthesis is the most common approach for selected synthetic peptoids, QYAOBIO can synthesize polypeptoids with various modifications for selected synthetic targets. Meanwhile, we also apply new solution-phase synthesis to overcome the limitations in…

Chemo Enzymatic Peptide Synthesis

Chemo Enzymatic Peptide Synthesis Introduction Chemo enzymatic peptide synthesis (CEPS) is a cutting-edge technology with unique versatile engineered enzymes. It is difficult to synthesize long and complex peptides by traditional SPPS (Solid Phase Peptide Synthesis). Moreover, the chain length increases will result in high impurity levels. Furthermore, peptide recombinant expression,…

Lipopeptides and Lipoglycopeptides

Lipopeptides and Lipoglycopeptides Introduction Lipopeptides and lipoglycopeptides are antibiotic classes with activity against gram-positive bacteria on the bacteria cell wall. Lipopeptides can create the ion-conducting channel, then cause potassium efflux and membrane depolarization, and disrupt the cell membrane finally. In order to inhibit protein synthesis and eventuate bacterial cell death…

Amide Bond Formation in Cyclization

Amide Bond Formation in Cyclization Traditional Amide Cyclization Many naturally active peptides are cyclized head-to-tail, these peptides are more resistant to hydrolysis since the absence of N- and C-terminus. Traditional coupling chemistry is used to cyclize the liner peptide precursor by amide bond formation. However, conventional head-to-tail amide formation is…

C-C Bond Cyclic Peptide Formation

C-C Bond Cyclic Peptide Formation C-C Single Bond Formation Traditional Cross Coupling Cross coupling corm the new carbon-carbon bond by transition metal catalysis. In most reactions, the transition metal like Pd inserts into a carbon-halide bond, then the subsequent trans-metallation of an organo-metallic compound induces two organic fragments bound to…

Email us

PEPTIDE@QYAOBIO.COM

Call Us

+86(021)-50795728
+86(027)-60707970