Polypeptide Information

Peptide synthesis

1.The basic principles of peptide synthesis.

Solid-phase peptide synthesis is a major breakthrough in the field of peptide synthesis chemistry. Its main feature is the ability to perform the synthesis process continuously without the need to purify intermediate products, thus laying the foundation for the automation of peptide synthesis. Currently, automated peptide synthesis is predominantly carried out using solid-phase synthesis. The basic process is as follows: Based on Fmoc chemistry, the carboxyl group of the C-terminal amino acid of the target peptide to be synthesized is covalently linked to an insoluble polymer resin. The amino group of this amino acid serves as the starting point for peptide synthesis, and it reacts with the activated carboxyl group of other amino acids to form peptide bonds. This process is repeated iteratively to obtain the desired peptide. Depending on the amino acid composition of the peptide, different post-synthesis treatments and purification methods may be employed.

2.How is the quality of custom peptide services controlled?

peptide.ltd strictly follows the ISO quality management system for peptide production. Each peptide is assigned a unique identifier, and three HPLC and MS tests are conducted on crude products, purified collection fractions, and final lyophilized products to ensure the accuracy and quality of the products.

3.What is the production capacity and delivery time for peptide.ltd's peptides?

The company has expanded its frontline personnel engaged in peptide synthesis to 350 people, with an additional efficient liquid chromatography purification team of over 250 people. The company’s monthly output reaches 10,000 purified peptides. Regular peptides can be delivered within one week, and for the majority of cases, delivery can be completed within two weeks. Quantities range from milligrams to kilograms.

4.What methods are commonly used for peptide production?

Linear peptide chains are typically synthesized using the Fmoc solid-phase synthesis method, where amino acids are sequentially connected from the C-terminus to the N-terminus. Initially, the first amino acid is attached to an insoluble resin support via an acid-labile linker. After removing the Fmoc protecting group with piperidine, the second Fmoc-protected amino acid is added using methods such as pre-activation or “one-pot” coupling. Once the desired peptide sequence is completed, the peptide chain is cleaved from the resin using TFA, yielding a crude product.

5.How do you choose the purity of a peptide for different experiments?

The purity of a peptide is an important criterion, and the choice of purity depends on the purpose of the experiment. For less sensitive screening experiments, crude or >75% purity is recommended. For immunological studies, >85% purity is advised. For research involving receptor-ligand interactions, biological assays, or cellular studies, >95% purity is recommended. For structural studies, >98% purity is suggested.

6.What is the net content (peptide content) of a peptide, and what is its significance?

The weight of a dry peptide includes not only the peptide itself but also other components such as water, absorbed solvents, coordinating ions, and salts. The net content of a peptide refers to the weight percentage of the peptide in the total weight. The value of this percentage can vary widely, ranging from 50% to 90%, depending on the purity, sequence, synthesis, and purification methods. It is important not to confuse the net content of a peptide with its purity; they are two distinct concepts. Purity is typically determined by HPLC, representing the percentage of correctly sequenced components in the peptide sample. On the other hand, the net content of a peptide refers to the percentage of peptide material relative to non-peptide material in the sample. The net content of a peptide is usually determined by amino acid composition analysis or UV spectrophotometry. This information is particularly important in experiments where the concentration of the peptide is crucial for calculations.

7.If my peptide purity is 95%, what are the remaining 5% composed of?

Peptide purity is usually determined by HPLC using a standard gradient of 1% per minute. During the synthesis process, the efficiency of crosslinking between amino acids is not always 100%, resulting in a series of impurities with missing amino acids. Most of these impurities with missing amino acids are removed during the purification process, but a small number of them may exhibit similar chromatographic behavior to the target peptide. These impurities with missing amino acids in the peptide sample constitute the remaining few percentage points.

8.What impurities are present in non-HPLC purified peptides?

In crude and desalted-grade peptides, both peptide and non-peptide impurities can be present. Examples of non-peptide impurities include incomplete-length peptides and raw materials used in peptide post-treatment, such as DTT and TFA.

9.What is the appropriate length for a peptide?

When considering peptide synthesis, factors such as peptide length, charge, and hydrophobicity need to be taken into account. As the length of a peptide increases, the purity and yield of crude products decrease, and the difficulty of purification and the likelihood of synthesis failure increase. Although the sequence of the functional region of a peptide cannot be changed, sometimes it is necessary to add auxiliary amino acids upstream or downstream of the functional region to improve the solubility and hydrophobicity of the peptide for successful synthesis. If a peptide is too short, synthesis can also be problematic, mainly due to difficulties in the post-treatment process. Peptides with fewer than five amino acid residues generally require hydrophobic amino acids to facilitate post-treatment. Peptides with less than 15 amino acid residues can generally achieve satisfactory yields and success rates.

10.How can peptide solubility be determined from the peptide sequence?

(1) If the peptide contains a high proportion of highly hydrophobic amino acids such as Leu, Val, Ile, Met, Phe, and Trp, the peptide may have difficulty or may be impossible to dissolve in aqueous solutions. Both purification and synthesis of peptides containing these amino acids may encounter issues. (2) In general, if the proportion of hydrophobic amino acids is less than 50% and there are no consecutive runs of five hydrophobic amino acids, and the proportion of charged amino acids (positive charges from K, R, H, N-terminus, and negative charges from D, E, C-terminus) reaches 20%, the solubility of the peptide can be improved by introducing polar amino acids at the N- or C-terminus of the peptide.

11.Why are peptides containing Cys, Met, or Trp difficult to synthesize?

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.

12.Why do some peptides have lower synthesis yields or purities?

Peptide synthesis differs significantly from primer synthesis, as the inability to synthesize primers is rare, while the inability to synthesize peptides is more common. When amino acids such as Val, Ile, Tyr, Phe, Trp, Leu, Gln, and Thr are adjacent to each other or repeated in the peptide chain, the peptide chain cannot fully extend and dissolve during the synthesis process, leading to reduced synthesis efficiency. Several scenarios can result in lower synthesis efficiency and product purity, such as repeated Pro, Ser-Ser, repeated Asp, and four consecutive Gly residues.

13.How are peptides purified?

Peptides are typically purified using reverse-phase columns (such as C8, C18, etc.) with detection at 214nm. The buffer system usually consists of a solvent containing TFA at pH 2.0. Buffer A contains 0.1% TFA in ddH2O, and Buffer B contains 1% TFA/ACN at pH 2.0. The peptide is dissolved in Buffer A before purification. If it doesn’t dissolve well, it can be dissolved in Buffer B and then diluted with Buffer A. In the case of highly hydrophobic peptides, a small amount of formic acid or acetic acid may also be added. HPLC analysis of crude peptide products typically shows a main peak for peptides that are not too long (below 15 amino acids). For longer peptides with more than 20 amino acid residues, if there is no clear main peak, HPLC combined with mass spectrometry is used to determine the molecular weight and identify the desired peptide peak.

14.What is the suitable length for peptides used in immunization?

Generally, peptides used in immunization are around 10-15 amino acids in length. Longer peptides may provide better immunization effects, but the synthesis cost also increases. MAP (multiple antigen peptide) peptides are preferably longer than 15 amino acids for better effectiveness. Furthermore, peptides shorter than 10 amino acids usually exhibit poorer immunization effects.

15.If our synthesized peptide has poor solubility, does that mean there is a problem with the peptide?

It is challenging to accurately predict the solubility of a peptide and determine the appropriate solvent. Assuming there is a problem with the peptide synthesis solely based on poor solubility is not correct.

16.In what state are peptides?

The peptides we provide are in powder form, typically white in color. The composition may vary, resulting in differences in the color of peptide powder, such as a yellow-green color for peptides with FITC modification.

17.How to dissolve peptides?

Dissolving peptides is a complex process, and it is generally difficult to immediately determine the appropriate solvent. It is usually recommended to conduct preliminary tests and avoid complete dissolution until the suitable solvent is determined. The following methods can help you select the appropriate solvent: (1) Determine the charge of the peptide. Assign a charge of -1 to acidic amino acids such as Asp (D), Glu (E), and C-terminal COOH; assign a charge of +1 to basic amino acids such as Lys (K), Arg (R), His (H), and N-terminal NH2; and assign a charge of 0 to other amino acids. Calculate the net charge. (2) If the net charge is >0, the peptide is basic and can be dissolved in water. If it does not dissolve or has low solubility, add acetic acid (at least 10% concentration). If the peptide still does not dissolve, add a small amount of TFA (25 μL) to dissolve it, and then dilute it with 500 μL of water. (3) If the net charge is <0, the peptide is acidic and can be dissolved in water. If it does not dissolve or has low solubility, add ammonia solution (25 μL) to dissolve it, and then dilute it with 500 μL of water. (4) If the net charge is 0, the peptide is neutral and generally requires organic solvents such as acetonitrile, methanol, isopropanol, or DMSO for dissolution. Some suggest the use of urea to dissolve highly hydrophobic peptides.

18.How to store synthesized peptides?

For long-term storage, peptides should be stored in a light-protected container at -20°C. For short-term storage, they can be stored at 4°C. They can be transported at room temperature for a short period. Peptides are stable at -20°C, especially when freeze-dried and stored in a desiccator. Freeze-dried peptides can be stored at room temperature before being exposed to air. This reduces the impact of humidity. If freeze-drying is not possible, it is best to store them in small working aliquots. For peptides containing Cys, Met, or Trp, it is essential to include a reducing buffer to prevent oxidation, as these peptides are prone to air oxidation. Before sealing the vial, slowly pass nitrogen or argon gas over the peptide to reduce oxidation. Peptides containing Gln or Asn are also prone to degradation. Compared to simple peptides without these amino acids, their shelf life is limited.

19.How should the ends of peptides be treated? Should they be left free or protected?

Peptides are used to mimic proteins, and to mimic the behavior of proteins, we need to synthesize peptides with similar structures and charges. When a peptide segment is “cut out” from a protein, the charge of its ends will differ from the native protein. We need to modify the synthesis strategy to make them consistent. In general: If the sequence originates from the C-terminal of the protein, the N-terminus is protected through acetylation. If the sequence originates from the N-terminal of the protein, the C-terminus is protected through amidation. If the sequence originates from the middle part of the protein, both ends are protected through acetylation and amidation.

20.What is Electrospray Ionization (ESI) Mass Spectrometry?

ESI generates multiply charged ions of proteins or peptides, allowing the analysis of proteins with relative molecular weights up to 1×105. The resolution ranges from 1500 to 2000 amu, and the accuracy is approximately 0.01%. ESI is more suitable for online analysis of proteins with large relative molecular weights, and it requires nebulization or organic solvents to sensitize the samples.

21.What is Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS)?

MALDI-TOF is a precise method currently used for determining the molecular weight of proteins, particularly suitable for the relative molecular weight determination of mixed protein and peptide substances. It exhibits high sensitivity and resolution. It is an essential tool in proteomics research. When combined with liquid chromatography, it can efficiently identify peptide substances. Moreover, when various principles of mass spectrometry techniques are combined, it not only provides information on the relative molecular weight of peptides but also allows the determination of their sequence structure. This technique will play a decisive role in future proteomics research.

22.What is High-Performance Liquid Chromatography (HPLC)? What is Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC)?

The advent of HPLC has provided a favorable method for the separation of peptide substances. HPLC applications in proteins and peptides, compared to other compounds, can achieve separation goals in a short period under appropriate chromatographic conditions. More importantly, HPLC can produce bioactive peptides on a preparative scale. Therefore, numerous researchers have conducted extensive work in finding optimal conditions for the separation and preparation of peptide substances. Preserving peptide activity, selecting stationary phase materials, types of eluents, and analytical determination are all areas of current research.

Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC): Relationship between results and retention values: When using RP-HPLC to separate peptides, the retention of peptides with different structures on the column needs to be determined. In order to obtain a series of retention factors, Wilce et al. analyzed the retention properties and structures of 2106 peptides using multilinear regression. They established the relationship between the composition of different amino acids and the retention factors. In peptides composed of 2 to 20 amino acids, polar amino acid residues can reduce the retention time on the column. In peptides composed of 10 to 60 amino acids, an increased proportion of nonpolar amino acids can reduce the retention time. However, in small peptides containing 5 to 25 amino acids, an increased proportion of nonpolar amino acids can extend the retention time on the column. Additionally, several studies have reported the influence of peptide chain length, amino acid composition, temperature, and other conditions on the retention, and optimal conditions for the separation and extraction of each peptide have been obtained through computer analysis and processing.

23.Why is gradient elution method used in reverse-phase HPLC?

Currently, reverse-phase HPLC is the most commonly used method for studying substances related to peptide synthesis. However, due to the significant differences in the properties of related substances present in peptide synthesis, it is often difficult to fully detect various organic impurities in the product using a simple isocratic elution method.

24.How are monoisotopic peaks and average isotopic peaks determined in a mass spectrum?

In low-resolution mass spectra, the peaks displayed are generally average isotopic peaks. However, commonly used mass spectrometry methods (such as MALDI-TOF) have high resolution, and small molecules (such as peptides) typically have 4-5 isotopic peaks, with the lowest molecular weight peak being the monoisotopic peak. When detecting large molecules (such as intact proteins) using mass spectrometry, there are many isotopic peaks that are difficult to distinguish on the spectrum. In such cases, using average isotopic peaks is more meaningful.

25.Are the differences between isotopic peaks always 1 Da?

The mass (m) of isotopic peptide fragments generally differs by 1 Da. However, because the peaks detected in mass spectrometry are the mass-to-charge ratio (m/z) of the peptide fragment, not the actual mass, the difference between them is less than 1 when the peptide fragment carries multiple charges. If the detected peptide fragment carries one charge, the difference between isotopic peaks is 1; if it carries two charges, the difference is 0.5; if it carries three charges, the difference is 0.33, and so on. Therefore, from the spacing between isotopic peaks, one can infer the charge state (z) of the peptide fragment and deduce the monoisotopic mass of the peptide fragment [(m/z)*z].

26.Is the monoisotopic peak always the highest peak?

The monoisotopic peak is always the lowest mass-to-charge ratio (m/z) peak among a group of peaks, but it is not necessarily the highest peak. When the molecular weight of the peptide fragment is small, it contains fewer total atoms, resulting in a lower probability of having isotopic atoms in the entire peptide fragment. In this case, the monoisotopic peak is often the highest peak. However, as the molecular weight of the peptide fragment increases, the probability of having isotopic atoms also increases. In such cases, isotopic peaks at +1 Da or even +2 Da can become the highest peaks. Statistical results show that when the molecular weight of the peptide fragment exceeds 2500 Da, the highest peak is often not the monoisotopic peak.

27.Besides HPLC, what other methods are used for peptide analysis?

Peptide analysis can also involve methods such as moisture determination, ion chromatography, elemental analysis, amino acid composition analysis, circular dichroism, NMR, IR, UV, endotoxin analysis, microbiological analysis, and others.

28.What is the commonly used counterion for peptides?

For peptides in the form of lyophilized powder, trifluoroacetate (TFA) salt is commonly used. If the peptides are intended for cell or animal experiments, it is recommended to convert them into acetate or hydrochloride salts.

29.What can isotopically labeled peptides provide?

We can provide peptides with stable isotope modifications, primarily using N15, C13, and D labeling. Since stable isotope amino acid raw materials are relatively expensive, it is recommended to choose simple amino acids for labeling, such as Gly, Val, Phe, Leu, Ala, etc.

30.What factors affect peptide stability?

Factors that affect the stability of synthesized peptides include deamidation, oxidation, hydrolysis, disulfide bond mispairing, racemization, β-elimination, aggregation, etc. Research shows that the most common degradation products in synthetic peptides are deamidation products, oxidation products, and hydrolysis products. Among the various amino acids that make up peptides, asparagine and glutamine are prone to deamidation reactions (especially at elevated pH and high temperature conditions); methionine, cysteine, histidine, tryptophan, and tyrosine are most susceptible to oxidation and are also sensitive to light exposure; aspartic acid residues participate in peptide bond formation and are prone to cleavage, especially Asp-Pro and Asp-Gly peptide bonds. Since a peptide molecule typically contains various unstable amino acid residues or peptide bonds, the possible degradation mechanisms and products of synthesized peptides are quite complex. Peptide aggregation is mainly driven by hydrophobic interactions, although it is currently challenging to accurately predict which peptides are prone to aggregation. However, at least for some medium to long peptides, it is necessary to study the potential presence of aggregates.

31.How do you select resin for Fmoc/tBu solid-phase peptide synthesis (SPPS)?

If synthesizing a peptide with a carboxylic acid C-terminus, Wang Resin is chosen. If synthesizing a peptide with an amide C-terminus, Rink Amide AM Resin or Rink Amide MBHA Resin can be selected. If synthesizing fully protected peptides, 2-Cl Trt Resin can be chosen.

32.What are the parameters of resin and what do they represent?

The parameters of resin typically include loading, bead size, and specifications such as 1%DVB. Loading: It is measured in mmol/g, indicating how many mmol of functional groups are present per gram of resin. Bead size: Commonly expressed as 100-200 mesh, the higher the value, the finer the beads. 1%DVB: It represents the proportion of cross-linking agent divinylbenzene in the copolymer of styrene and divinylbenzene.

33.What are the methods for calculating the loading of Fmoc-protected amino acid resin?

There are usually two methods for calculating the loading of Fmoc-protected amino acid resin. One method is the weight gain method, where the increased weight is divided by the added molecular weight and then divided by the total weight of the resin. The other method is through UV analysis. Currently, the loading values provided by our company are determined using UV analysis.

34.What are the commonly used fluorescent modifications?

 Dye Ex(nm) Em(nm)
 FITC 488 518
 5-FAM 490 520
 5-TAMRA 544 572
 TRITC 547 572
 Mca 322 390
 Amc 351 430
 AFC 380 500
 NBD 466 539
 Dnp 350 
 Texas Red 589 615
 Dabcyl 426 
 Glu(EDANS) 335 493

35.What is FRET detection?

Fluorescence resonance energy transfer (FRET) is a detection method that utilizes the principle of energy transfer between fluorescent molecules. In this technique, a fluorescent donor molecule (e.g., EDANS) and a fluorescent acceptor molecule (e.g., DABCYL) are attached to the ends of a synthetic peptide substrate for proteases. When the donor molecule is excited with light of a specific wavelength (e.g., 355nm), it emits fluorescence at a maximum wavelength of 490nm. The acceptor molecule, on the other hand, can absorb fluorescence and has a maximum absorption wavelength of 490nm.

When the peptide substrate is intact, the emitted fluorescence from the donor molecule is absorbed by the acceptor molecule, resulting in no detectable fluorescence. However, when the peptide substrate is cleaved by the protease, the donor molecule separates from the acceptor molecule, reducing or eliminating the quenching effect, and fluorescence is detected.

36.What are some commonly used combinations of fluorescent and quencher groups?

Some commonly used combinations of fluorescent and quencher groups include:

  • Abz/Tyr(3-NO2)
  • Mca/Dnp

37.When fluorescent modifications are made at the C-terminus, what are the available methods?

Fluorescent dye compounds usually have a carboxylic acid or NHS-ester group, so they require the peptide to have amino functional groups for conjugation. In the sequence, the modification can be made on the side-chain amino group of Lys. For C-terminal modification, Lys can be added or connected using ethylenediamine (ED: NH2CH2CH2NH2). Examples include Lys(Biotin), ED-Biotin, EDDnp, etc.

38.Can you provide an introduction to Maps-modified peptides?

Maps (Multiple Antigen Peptides) is a branched peptide in which the C-terminus of a linear peptide is connected to two, four, or more Lys residues, forming a branching structure. This increases the overall size of the molecule. When synthesizing Maps-modified peptides, due to the non-uniformity of coupling, the peptide product may have similar properties to non-target peptides, making it difficult to purify by HPLC and challenging to identify by mass spectrometry. Amino acid composition analysis is recommended.

39.What are the functions of peptide PEGylation?

PEGylation of peptides refers to the attachment of polyethylene glycol (PEG), which is a polymer with functional groups, to modify peptides. It is mainly used in protein drug modification to increase the half-life in the body, reduce immunogenicity, and enhance water solubility of the drug. In recent years, PEGylation has been widely applied in pharmaceutical research and development, playing an important role in drug delivery systems and sustained release of drugs.

40.What are the ways of peptide PEG modification?

The commonly used modification groups for PEG modification agents can be summarized as follows: Amino (-Amine) -NH2, aminoethyl -CH2-NH2, maleimide -Mal, carboxyl -COOH, thiol (-Thiol) -SH, succinimidyl carbonate -SC, succinimidyl acetate -SCM, succinimidyl propionate -SPA, succinimide-NHS, propionic acid -CH2CH2COOH, aldehyde -CHO, acrylate -Acrylate, acrylate -AC, azide -Azide, biotin -Biotin, fluorescein -Fluorescein, glutaric anhydride -GA, hydrazide -Hydrazide, alkyne -Alkyne, etc.

Common classifications of PEG modification agents: (1) Linear PEG modification agents: mPEG (2) Dual functional group modification agents: HCOO-PEG-COOH, NH2-PEG-NH2, OH-PEG-COOH, OH-PEG-NH2, HCL·NH2-PEG-COOH, MAL-PEG-NHS, (3) According to the molecular weight, there are various options such as 2000, 3000, 5000, 10000, 20000, etc.

Therefore, when performing PEGylation modification, it is necessary to determine the type of PEG, functional group requirements, and the range of molecular weight.

41.What is small molecule PEG?

Small molecule PEG refers to PEG with relatively low molecular weight. For example, mini-PEG is a compound with a clear molecular weight and an amino group at one end and a carboxylic acid group at the other end. Some commonly used compounds include: AEA (5-amino-3-oxapentanoic acid) AEEA (8-Amino-3,6-Dioxaoctanoic Acid) – 9 atoms (mini-PEG) TTDS (1,13-diamino-4,7,10-trioxatridecan-succinamic acid) Ebes (8-amino-3,6-dioxa-octyl)succinamic acid (PEG2-Suc-OH) AEEEA (11-Amino-3,6,9-Trioxaundecanoic Acid) – 11 atoms (mini-PEG3) dPEG(4) (15-amino-4,7,10,13-tetraoxa-pentadecanoic acid) dPEG(8) (alpha-amino-omega-carboxy octa(ethylene glycol)) dPEG(12) (alpha-amino-omega-carboxy dodeca(ethylene glycol))

42.Can pseudoproline dipeptides help with the synthesis of difficult peptides?

Yes, pseudoproline dipeptides can aid in peptide synthesis by reducing the generation of alpha-turns and beta-folded spatial structures in peptide coupling. This helps maintain the linearity of the peptide and improves the crude purity of the peptide. The structure of pseudoproline is simultaneously removed during peptide cleavage using TFA, resulting in a normal peptide. The main series of pseudoproline dipeptides include Fmoc-AA-Ser(Psi(Me,Me)pro)-OH and Fmoc-AA-Thr(Psi(Me,Me)pro)-OH, where AA represents the amino acid. If there are side chains, they are protected as well. For example: Fmoc-Ser(tBu)-Ser(Psi(Me,Me)pro)-OH and Fmoc-Asp(OtBu)-Thr(Psi(Me,Me)pro)-OH.

43.What are some common peptides used as epitope tags in antibodies?


44.What is TAT peptide?

The TAT peptide sequence is Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg (RKKRRORRR), which can facilitate the translocation of peptides across cell membranes.


45.What kind of peptides can be quantified using UV analysis?

Peptides containing Trp or Tyr residues can be quantified using UV analysis. The peptide content can be calculated based on the molar extinction coefficients: Tryptophan: 5560 AU/mmole/ml Tyrosine: 1200 AU/mmole/ml (at 280 nm, at neutral pH, using a 1-cm cell) The molar extinction coefficient of a peptide can be calculated by summing the coefficients for each Trp or Tyr residue. Then, the peptide concentration in mg/mL can be calculated using the formula: mg peptide per mL = (A280 x DF x MW) / e A280: Absorbance value at 280 nm (1-cm cell) DF: Dilution factor MW: Peptide molecular weight e: Molar extinction coefficient of the peptide

46.What cyclic peptides does peptide.ltd offer?

Cyclic peptides include disulfide bonds (S-S) formed between Cys-Cys, known as oxidized peptides. peptide.ltd can provide peptide synthesis with one pair of S-S bonds, two pairs of S-S bonds, or three pairs of S-S bonds. They can also assist in the formation of intermolecular oxidations, such as dimerization within the same peptide or oxidation between different peptides. Additionally, they offer cyclization through amide bonds, including head-to-tail cyclization and side-chain cyclization. They can also synthesize cyclic peptides with ester bonds and thioester bonds.

47.What modifications are available for Click Peptides?

The representative reaction of click chemistry is the copper-catalyzed azide-alkyne cycloaddition reaction. Click chemistry has made significant contributions to the field of chemical synthesis and has become one of the most useful and attractive synthetic concepts in areas such as drug development and biomedical materials. In the context of peptides, click chemistry involves incorporating azide or alkyne functional groups. Some commonly used compounds for click peptide synthesis include: Fmoc-4-azidophenylalanine (Fmoc-Phe(N3)-OH) Fmoc-Azidohomoalanine Fmoc-D-propargylglycine (Fmoc-D-Pra-OH) Fmoc-L-propargylglycine (Fmoc-L-Pra-OH) Fmoc-Lys(N3)-OH (Fmoc-azidolysine or Fmoc-lys(azide)) Azidoacetic acid 6-Azidohexanoic acid Propiolic acid

48.What are the amino acids with side chain methylation modifications?

The main amino acids with side chain methylation modifications are Lys(Me), Lys(Me2), Lys(Me3), Orn(Me), Orn(Me2), Orn(Me3), Arg(Me), Arg(Me2, Symmetrical), and Arg(Me2, Asymmetrical).

49.Where are C-terminal thioester peptides used?

After C-terminal thioester modification, peptides can be used in Native Chemical Ligation reactions. They undergo chemoselective transthioesterification, a sulfur transfer reaction, with another peptide having an N-terminal cysteine residue under neutral conditions, resulting in rearrangement to form an amide bond and connecting the two peptides. This method can be applied to connect peptide fragments in the synthesis of long peptides.

50.What factors should be considered when coupling peptides with other compounds or carriers?

When coupling peptides with drugs or specific functional compounds, or when attaching peptides to carriers, chemical bonds are used for the conjugation. Peptides typically have amino or carboxylic acid functional groups, which can be coupled with corresponding carboxylic acids or amines. Another option is to use thiol and maleimide chemistry for conjugation under pH 8 conditions. Peptides can be modified with functional groups such as Cys or Maleimide. A commonly used compound for Maleimide modification is 3-Maleimidopropionic Acid (CAS 7423-55-4).

51.How can peptide molecular weight be calculated?

Peptide molecular weight can be calculated using software such as Peptide Companion or by inputting the sequence into certain websites. Due to differences in calculation precision, there may be slight variations in the decimal portion of the molecular weight.

52.How can units be converted?

1 mg = 1000 mcg (or μg) 1 ml = 1000 μl 1 mole (mol) = 1000 millimoles (mmol) 1 millimole (mmol) = 1000 micromoles (μmol) 1 micromole (μmol) = 1000 nanomoles (nmol) 1 nanomole (nmol) = 1000 picomoles (pmol)

Peptide purification

53.What is the difference between peptide content and peptide purity?

In a peptide product, apart from the peptide itself, it also includes impurities such as water and organic salts introduced during the production process. Peptide purity refers to the content of the peptide itself and the impurities in the peptide product, excluding impurities such as water. On the other hand, peptide content refers to the net amount of the target peptide in the product, usually detected using methods such as elemental analysis or amino acid analysis. Therefore, even if a peptide has a purity of 99%, its content may be only around 70-80% due to the presence of impurities such as water and organic salts in the product.

54.What are the commonly used methods for peptide separation and purification?

The commonly used method for peptide separation and purification is reverse-phase high-performance liquid chromatography (RP-HPLC).

55.What is the commonly used mobile phase system for peptide separation and purification using RP-HPLC?

The commonly used mobile phase system is a mixture of water and acetonitrile, with trifluoroacetic acid (TFA) as the ion-pairing reagent. Peptides are separated by applying appropriate gradient elution based on their characteristics.

56.Why is trifluoroacetic acid added as an ion-pairing reagent in the mobile phase, and what other mobile phase systems or ion-pairing reagents can be used for peptide separation and purification?

The addition of trifluoroacetic acid helps to adjust the pH of the eluent and enhances the separation efficiency by interacting with peptides as an ion-pairing reagent, significantly improving peak shape. Other mobile phase systems or ion-pairing reagents that can be used for peptide separation and purification include acetic acid systems, phosphoric acid systems, hydrochloric acid systems, heptafluorobutyric acid, and others. By appropriately adjusting the pH, good separation results can be achieved.

57.How to dissolve peptides?

Most peptides can be dissolved in ultrapure water. For peptides that are difficult to dissolve, their amino acid sequence should be analyzed first. For acidic peptides, they can be dissolved in a small amount of alkaline solution (such as 0.1% ammonia water), followed by dilution to the desired concentration. For basic peptides, they can be dissolved in a small amount of acidic solution (such as acetic acid or trifluoroacetic acid), followed by dilution to the desired concentration. For hydrophobic peptides, strong polar organic solvents such as DMF, methanol, ethanol, isopropanol, and DMSO can be used for dissolution.

58.What are the commonly used chromatographic packing materials in reversed-phase liquid chromatography?

The most commonly used packing materials are C18, C8, and C4 reversed-phase silica columns.

59.How to choose the chromatographic column packing material for purification processes?

Different peptides have significant variations in physicochemical properties and hydrophobicity. In most cases, peptides with a molecular weight smaller than 4000 and hydrophilic peptides show the best separation efficiency on a C18 column. Peptides with a molecular weight larger than 5000 and extremely hydrophobic peptides exhibit the best separation efficiency on a C4 column. C8 columns, on the other hand, fall between C18 and C4 columns, and their performance is more similar to C18 columns. For peptides with specific selectivity, phenyl columns and polymer columns can also be chosen.

60.What are the principles for selecting polymer packing materials in the purification process?

When purification needs to be performed under pH or temperature conditions higher than normal, polymer columns with an extremely wide pH range can be used. The advantage of using such columns is that they are not degraded under extreme pH conditions, allowing separation and purification using strong acids and bases as the mobile phase.

61.Factors affecting the results of peptide purity analysis

There are many factors that can affect the results of peptide purity analysis, including the mobile phase system, column type, column temperature, wavelength, and performance specifications of the chromatograph. Any variation in these factors can lead to errors in the results.

62.What are the causes of baseline drift often observed during peptide analysis? How to resolve it?

Gradient elution with fixed trifluoroacetic acid concentrations sometimes causes absorption baseline drift at the 210-220 nm detection range, which is a common cause of baseline drift in many reverse-phase separations. To reduce or eliminate baseline drift caused by the spectral absorption changes of trifluoroacetic acid, it is recommended to choose a detection wavelength close to 215 nm and compensate for the baseline drift by using 15% less trifluoroacetic acid in solvent B compared to solvent A. For example, if the trifluoroacetic acid concentration in solvent A is 0.1%, solvent B can be used with a concentration of 0.085%.

63.After separation and purification by reverse-phase chromatography, how can impurities such as TFA and acetonitrile in the mobile phase be removed from the product?

Most of the TFA and acetonitrile can be removed by freeze-drying, as long as they are within the allowable limits. This method is simple and effective. However, for drug peptides that require stricter TFA content requirements, freeze-drying alone may not completely meet the requirements, and specialized salt conversion or desalting methods are needed.

64.In what forms do peptides commonly exist as salts? How can salt conversion or desalting be performed?

Most peptides are purified in the presence of TFA, so peptides are predominantly found as TFA salts. Drug peptides may also exist as acetate salts or hydrochloride salts, while a few drug peptides may have other specific salt forms. Salt conversion methods mainly involve ion exchange and HPLC, while desalting can be performed using G25 (a type of dextran gel) column.

65.Does TFA only serve to regulate the pH in the buffer solution? The higher the concentration of TFA, the more significant the baseline drift. Does that mean a lower concentration of TFA is better in terms of buffer solution pH, as long as it is within the acceptable range?

(1) TFA acts as an ion-pairing reagent and is typically used at a concentration of 0.05-0.1%. Excessive concentration can acidify the solution and may affect the column’s lifespan if used for a prolonged period. (2) Additionally, TFA can inhibit the silanol groups on the silica surface, improving peak shape for basic compounds. Sometimes, if separation is not satisfactory with 0.1% TFA, increasing the concentration to 0.2% can be considered. However, it is important to rinse the chromatographic column promptly after use. (3) When running gradients, there may be baseline drift, but it does not significantly affect the preparative process.

66.Why is pretreatment necessary for peptide samples before loading onto a preparative column? What are the methods for pretreatment?

Preparative columns handle more sample material than analytical columns, making them more prone to contamination. Therefore, it is necessary to pretreat the peptide samples to effectively extend the lifespan of the preparative column. The main methods for pretreatment include extraction, filtration, centrifugation, precolumn application, and the use of online filters.

Antibody preparation

67.What antibody services does peptide.ltd provide?

peptide.ltd provides preparation services for monoclonal antibodies and polyclonal antibodies for both domestic and international clients. They offer antigen synthesis and expression services, antibody purification services, as well as antibody labeling and detection services, among others.

68.What animal sources are used for peptide.ltd's polyclonal antibodies?

We use experimental animals such as rabbits, mice, rats, chickens, guinea pigs, and sheep to produce antibodies.

69.What advantages does peptide.ltd have in antibody preparation compared to other antibody companies?

Advantages of our custom antibody services include:

Free antibody consultation service: We offer free assistance in selecting the most suitable antibody packages, analyzing proteins, and designing peptide antigens.

I. Extensive experience in antigen design: Through extensive exploration and integration of genomics, proteomics, and structural genomics, we have developed an effective antigen design method. Antibodies produced through immunization with peptide antigens ensure successful Western Blot experiments.

II. Rich experience in antibody production, ensuring high-quality antibodies for customers: We have successfully produced over 5,000 types of antibodies.

III. Regular communication with customers to provide updates on antibody projects.

IV. Highly competitive prices, guaranteeing the provision of high-quality services at the lowest market rates.

V. Strong support for post-sales technical services: We are the first in China to propose lifelong technical services for antibody customers.

70.Can peptide.ltd prepare antibodies against modified antigens?

Yes, we can. Currently, we are able to prepare antibodies against protein modifications such as phosphorylation, acetylation, and methylation (mono-, di-, and trimethylation), among others.

71.What guarantees are there for peptide.ltd's antibody preparation?

The final antibodies or antiserum must pass all validations required by the customer (ELISA, Western Blot, IHC, etc.). All antigen and antibody validation data are provided to the customer. If the requirements of the customer are not met, no fees will be charged.

72.How are antibodies from peptide.ltd stored?

For purified antibodies, we recommend aliquoting them into small portions and storing them at -80°C. They can also be aliquoted and stored at -20°C with the addition of 50% (v/v) glycerol.

73.How does peptide.ltd ship custom antibodies?

For domestic customers, we usually include ice packs and use express delivery for next-day arrival. We can also ship the antibodies lyophilized at room temperature or with ice packs, depending on the customer’s requirements. If dry ice transportation is needed, we may charge a fee accordingly.

74.What is the process for ordering custom antibodies from peptide.ltd?

Customers can directly contact our sales representatives, who will provide a quotation based on the customer’s customization requirements. Our antibody preparation technical experts can answer various customer inquiries and help them select the best solution. Once the customer agrees to our proposal and quotation, we proceed with the antibody preparation. We provide regular progress reports to the customer during the preparation period.

75.We have used your antibody custom service. Can we cite peptide.ltd in our published articles?

We highly welcome customers to cite peptide.ltd in their published articles. We hope that all customers achieve satisfactory research outcomes and publish high-quality research papers.

76.Can your company prepare antibodies for sandwich ELISA?

We have extensive experience in preparing antibodies for sandwich ELISA, and the detection sensitivity can reach the picogram level.

77.We have customized monoclonal antibodies. Can your company help us store the hybridoma cell line?

For the customization service of monoclonal antibodies, peptide.ltd not only provides customers with selected hybridoma cell lines but can also assist in storing these cell lines. Depending on the specific situation, we may not charge any fees or charge a fee accordingly.

78.Are there discounts for large-scale antibody preparation?

Please contact our sales department for more information.

79.Have customers who used your antibody services published high-quality papers?

In recent years, scientists and research institutions from around the world have adopted peptide.ltd’s peptide, antibody, and other custom services, resulting in a series of high-level research papers (published in journals such as Science, Nature Series, Molecular Cell, Neuron, PNAS, Cancer Research, JBC, etc.). The number of papers exceeds one thousand.

80.Should I choose monoclonal or polyclonal antibodies for customization?

The choice between monoclonal and polyclonal antibodies depends on the specific experiment. Please discuss with our antibody experts.

81.Should I select a peptide or protein as the antigen?

The choice between a peptide or protein antigen depends on the specific experiment where the antibody will be applied. For experiments where the protein is in its native state, the antibody generally recognizes conformational and linear epitopes of the protein. Examples of such experiments include co-immunoprecipitation (CO-IP), fluorescence-activated cell sorting (FACS), neutralizing/blocking assays, and ELISA for detecting native proteins. For experiments where the protein is denatured or partially denatured, most conformational epitopes are destroyed, and the antibody primarily recognizes linear epitopes. Western blotting, immunohistochemistry (IHC), immunocytochemistry (ICC), and immunofluorescence (IF) are examples of such experiments. Antibodies prepared using peptides as antigens recognize linear epitopes and can provide satisfactory results when the protein is denatured or partially denatured during the experiment. However, they may not guarantee recognition of conformational epitopes of the protein. Antibodies prepared using proteins as antigens, which can recognize both linear and conformational epitopes of the protein, can be applied to almost all experiments. Therefore, using a protein as an antigen is generally preferred. However, if the peptide antigen can meet the experimental requirements and obtaining the protein is challenging, preparing antibodies using peptides as antigens may be more suitable.

82.When should I consider designing peptides as antigens?

If the amino acid sequence is known but the antigen protein cannot be obtained due to gene cloning or expression issues, or if antibodies specific to certain specific epitopes of the antigen protein are required, peptides can be synthesized to serve as antigens. For example, if the goal is to produce antibodies targeting a specific region of a protein, such as studying the precursor of the N-terminal of a protein, a peptide antigen can be designed for the N-terminal. If the purpose of the antibodies is to recognize modified amino acids, such as phosphorylated serine, threonine, or tyrosine, or acetylated lysine, the peptide needs to be modified accordingly. In general, antiserum can recognize the peptide sequence used for immunization but may not recognize the folded structure of the natural protein. Whether antibodies against discontinuous epitopes can recognize the antigenic determinant cluster depends on whether the peptide used for antibody production contains secondary structures.

83.Why does your company provide custom antibody services despite the availability of many catalog antibodies?

There are several reasons for this:

  • New proteins are constantly being discovered, and customers require antibodies against these proteins.
  • Many catalog antibodies available for purchase do not meet the requirements of specific experiments or are suitable for different experimental techniques.
  • Catalog antibodies are often limited in quantity and can be expensive.

Amino acids

84.What are the methods of amino acid synthesis?

Amino acids can be synthesized through three main approaches: protein hydrolysis, organic synthesis, and fermentation.

85.What are D-, L-, and DL-forms of amino acids? What is meant by protected amino acids?

D- and L-forms refer to the configuration of amino acids. Based on their optical activity, amino acids can be classified as L-form (left-handed) or D-form (right-handed) (L and D are derived from Latin terms). Naturally occurring amino acids are predominantly in the L-form, except for Gly, which is achiral. DL-form refers to racemic amino acids, which contain both D- and L- carbon atoms. Typically, this distinction is made for the alpha carbon, so the term DL-form is not commonly used. Protected amino acids refer to derivatives of amino acids in which the functional groups of the amino acid are modified or protected to temporarily deactivate their reactivity. This can include protection of the amino group, carboxyl group, and side-chain functional groups.

86.What are the common protecting groups for carboxyl groups in amino acids?

Common protecting groups for carboxyl groups in amino acids include tert-butyl, methyl, ethyl, benzyl, allyl, pentafluorophenyl esters, p-methoxybenzyl ester, and others.

87.What are the common protecting groups for amino groups in amino acids?

Common protecting groups for amino groups in amino acids include tert-butoxycarbonyl, fluorenylmethoxycarbonyl, allyloxycarbonyl, trifluoroethyl carbamate, benzyl carbamate, ethoxycarbonyl, p-toluenesulfonyl, ortho-nitrobenzyl, and others.

88.What are the common classifications of amino acids?

There are 20 common amino acids, which can be classified into several categories based on their side chains: aliphatic amino acids (Ala, Gly, Val, Leu, Ile), aromatic amino acids (Phe, Tyr, Trp, His), amide or acidic side chain amino acids (Asp, Glu, Asn, Gln), basic side chain amino acids (Lys, Arg), sulfur-containing amino acids (Cys, Met), hydroxy-containing amino acids (Ser, Thr), and imino acids (Pro).

89.What are the purposes of protecting groups for amino acids?

The protection of amino acids plays a crucial role in peptide chemical synthesis as it directly determines the success of the synthesis. Since many of the common 20 amino acids contain reactive side chains, they need to be protected. The requirements for these protecting groups are that they should be stable during the synthesis process, have no side reactions, and be completely removable after synthesis quantitatively.

The amino acids that commonly require protection during synthesis include Cys, Asp, Glu, His, Lys, Asn, Gln, Arg, Ser, Thr, Trp, and Tyr.

The functional groups that require protection include hydroxyl, carboxyl, thiol, amino, amide, guanidine, indole, imidazole, etc. However, Trp can sometimes be left unprotected as indole is relatively stable. In certain circumstances, some amino acids can also be left unprotected, such as Asn, Gln, Thr, and Tyr.

90.What are the common methods for removing OC protecting groups?

There are three common methods: trifluoroacetic acid (TFA) method, which is commonly used, concentrated hydrochloric acid method, and silica gel catalysis method.

91.What are the functions and sources of essential amino acids?

There are known to be twenty types of basic amino acids, among which eight amino acids, namely lysine, threonine, leucine, isoleucine, valine, methionine, tryptophan, and phenylalanine, cannot be synthesized by the human body and are therefore called essential amino acids. They need to be obtained from food sources.

Functions of the eight essential amino acids: Leucine:

  • Promotes sleep
  • Reduces sensitivity to pain
  • Alleviates migraines
  • Relieves restlessness and anxiety
  • Helps alleviate symptoms of chemical imbalances caused by alcohol and aids in controlling alcohol poisoning Food sources: Milk, fish, bananas, peanuts, and protein-rich foods


  • Reduces or prevents the occurrence of herpes simplex infections (fever blisters and cold sores)
  • Enhances focus and concentration
  • Enables the proper utilization of energy-producing fatty acids
  • Assists in resolving certain cases of infertility Food sources: Fish, legume products, skim milk, almonds, peanuts, pumpkin seeds, and sesame seeds


  • Reduces appetite
  • Increases libido
  • Improves memory and cognitive agility
  • Alleviates depressive moods Food sources: Bread, legume products, skim milk, almonds, peanuts, pumpkin seeds, and sesame seeds

Isoleucine and Valine:

  • Essential for the formation of hemoglobin
  • Regulates sugar and energy levels, aiding in improved physical performance
  • Assists in muscle tissue repair
  • Accelerates wound healing
  • Treats liver dysfunction
  • Increases blood glucose levels and stimulates growth hormone production Food sources: Eggs, soybeans, black wheat, whole grains, brown rice, fish, and dairy products


  • Indispensable for the absorption and utilization of proteins in the body
  • Prevents the accumulation of fat in the liver
  • Promotes antibody production and enhances the immune system Food sources: Meat and other protein-rich foods


  • Facilitates fat breakdown, preventing the occurrence of fatty liver, cardiovascular diseases, and kidney disorders
  • Eliminates harmful substances such as lead and other heavy metals
  • Prevents muscle weakness and fatigue
  • Treats rheumatic fever and toxemia during pregnancy
  • Acts as a beneficial antioxidant Food sources: Soybeans, other legumes, fish, garlic, meat, onions, and yogurt


  • Helps alleviate restlessness and anxiety
  • Promotes sleep
  • Helps control alcohol intoxication Food sources: Brown rice, meat, peanuts, soy protein

92.What is the color reaction between amino acids and ninhydrin?

What is the color reaction between amino acids and ninhydrin

α-Amino acids can react with ninhydrin to form a blue-violet color substance. This reaction is specific to α-amino acids and can be used to identify their presence. It is also commonly used for colorimetric determination and thin-layer chromatography analysis of α-amino acids.

93.What reactions involve the α-amino group in amino acids?

The α-amino group in amino acids participates in several reactions:

Reaction with nitrous acid, where the volume of nitrogen gas formed can be used to calculate the amino acid content.

Reaction with acylating reagents, which can be used for amino group protection.

Alkylation reaction, used to determine the N-terminal amino acid of a peptide chain.

Formation of Schiff’s base, which serves as an intermediate step in transamination reactions.

Deamination reaction, an important intermediate step in amino acid degradation reactions.

94.What is the definition of an amino acid and how are they classified?

Definition of an amino acid: A compound that contains one or more amino groups and carboxyl groups is called an amino acid.

Classification of amino acids: A. Based on the position of the amino and carboxyl groups, there are different types such as α-amino acids and β-amino acids. B. Based on their involvement in protein synthesis, amino acids can be classified into proteinogenic amino acids (the common twenty types) and non-proteinogenic amino acids (including but not limited to ornithine, citrulline, GABA, etc.). C. Based on whether they can be synthesized by the human body, amino acids can be categorized as essential amino acids and non-essential amino acids. There are eight essential amino acids that the human body requires: leucine, isoleucine, lysine, phenylalanine, methionine, threonine, tryptophan, and valine.

95.Sources and Manufacturing Methods of Amino Acids

The main sources and manufacturing methods of amino acids include protein hydrolysis, microbial fermentation, enzyme engineering technology, and chemical synthesis. Amino acids produced through chemical synthesis are generally in the DL form and need to be separated to obtain the corresponding D or L forms. The asymmetric synthesis of amino acids has seen rapid development in recent years.

96.Types of Organic Reactions Performed by peptide.ltd

peptide.ltd can perform addition reactions, substitution reactions, oxidation-reduction reactions, rearrangement reactions, ammonolysis reactions, hydrogenation reactions, alkylation reactions, esterification reactions, and hydrolysis reactions. The main products include amino acid protecting reagents, peptide coupling agents, linkers, amino acids and their derivatives, non-natural amino acids, protected amino acids, and amino alcohols.

97.Chirality Purity of Non-Natural Amino Acids from peptide.ltd

Non-natural amino acids from peptide.ltd are primarily obtained through enzymatic cleavage. Due to the specificity of enzymes, the chiral purity of the separated amino acids can reach over 99.5%.

98.Common Protecting Groups for Amino Acid Carboxyl Groups

a) Methyl or ethyl esters b) Benzyl esters or substituted benzyl esters c) tert-Butyl esters d) Allyl esters e) Hosu esters, onp esters, opfp esters f) Trimethylsilyl esters or triethylsilyl esters g) Amides or hydrazides

99.Common Protecting Groups for Amino Acid Amino Groups

  1. Boc or bpoc
  2. Fmoc
  3. Cbz or 2-clz
  4. Trt
  5. Pht
  6. Tfa
  7. Nps
  8. Ac or for
  9. Adoc

100.Applications of Pseudoproline Dipeptides

Pseudoproline dipeptides of the structure I can be used as reversible protecting groups for Ser, Thr, and Cys and have proven to be versatile tools in the field of peptide chemistry for overcoming certain inherent issues. The presence of ψPro in a peptide sequence disrupts the α-turn structure, which is considered a source of intermolecular aggregation. The increased solvation and coupling kinetics caused by the presence of pseudoproline in peptide assembly, such as Fmoc solid-phase peptide synthesis, make chain elongation easier, especially for peptides containing “difficult sequences.”