Peptide synthesis technology with great potential

Introduction

Peptide synthesis is a solid phase synthesis process generally from the n-terminal (amino terminal) to the c-terminal (carboxyl terminal) synthesis. In the past it was carried out in solution called liquid phase synthesis.  in 1963, Merrifield proposed solid-phase peptide synthesis (SPPS). The process of synthesis was convenient, rapid so that it became the preferred method and brought about a revolution of the peptides in organic synthesis. Afterwards through continuous improvement and efforts, today solid-phase method has become a common technique in peptide and protein synthesis while showing unparalleled advantages compared with the classic liquid phase synthesis. For example, greatly reducing the difficulty of purifying product in each step.

The synthesis of cyclic peptides

Development

Nowadays, according to different requirements of customers, such as sequence, purity, molecular weight, etc., polypeptides can be processed and synthesized to meet these specific needs. This is custom peptide synthesis which is a type of peptide synthesis service.

Customized synthesized peptide is crucial to a great number of fieldsso let’s have a look .

Application Fields

Peptide synthesis service and custom peptide synthesis service mainly include these three aspects :

  1. Peptide Synthesis
  2. Peptide Modifications
  3. Peptide Library Services

In fact, peptide synthesis service is much closer to our real-life situation than we imagined. Here are some examples:

Cancer immunotherapy: Cancer immunotherapy uses the body’s own immune system to attack cancer cells. Peptide-based vaccines use tumor-associated antigens that associate with T cells to target cancer.

Self-assembling peptide: Self-assembling peptides are short, synthetic peptides characterized by amphipathic sequences. These peptides are able to spontaneously self-assemble in aqueous solution to form highly organized structures such as hydrogels.

Peptide Venom Peptides: Bioactive peptides are the most dominant component of animal venoms. Venom peptides can vary in length and complexity, thus their synthesis requires a combination of chemical and recombinant synthesis.

Peptide Natriuretic Peptides: Functioning in the induction of natriuresis, (the excretion of large amounts of sodium in the urine), natriuretic peptides have been touted as useful biomarkers for the purpose of personalized heart failure treatments and are involved in maintaining heart pressure and volume, as well as regulating cardiovascular remodeling pathways.

Neurodegenerative Disease Peptides: A pathological hallmark of neurodegenerative diseases is the abnormal aggregation of extracellular or intracellular peptides and proteins such as beta amyloid, tau, prions, and polyQ-hHtt. Custom peptides can be used to study aggregation, and develop aggregative inhibitors and vaccines.

Antimicrobial Peptides: The prevalence of antibiotic-resistant microbes continues to hinder the treatment of a variety of diseases. Custom peptides can be used to develop intelligently designed antimicrobial drugs

Milk-derived peptide therapies: Nutrition aside, milk has gained recognition as a rich source of potentially therapeutic proteins and peptides that have a variety of functions, such as antimicrobial, antiviral, antioxidant, immunostimulant, and anti-hypertension activities.

Summary

The development of semi-automatic and automatic peptide synthesizer has brought the technology of peptide and protein synthesis to a peak. So far, people have achieved the synthesis of linear polypeptides composed of hundreds of amino acids (or more) and the yield is quite high. As the exploration of the life phenomenon and protein function becomes deeper and deeper, with the growing demand for a variety of functional materials, people pay more attention to special shapes and features of peptides such as cyclic peptide, peptide, peptide sample library, etc.

An introduction to Glycomics

Glycomics is the comprehensive study of glycomes (the entire complement of sugars, whether free or present in more complex molecules of an organism), including genetic, physiologic, pathologic, and other aspects. Glycomics “is the systematic study of all glycan structures of a given cell type or organism” and is a subset of glycobiology. The term glycomics is derived from the chemical prefix for sweetness or a sugar, “glyco-”, and was formed to follow the omics naming convention established by genomics (which deals with genes) and proteomics (which deals with quantitative proteomics).

Challenges

The complexity of sugars: regarding their structures, they are not linear instead they are highly branched. Moreover, glycans can be modified (modified sugars), this increases its complexity.

Complex biosynthetic pathways for glycans.

Usually glycans are found either bound to protein (glycoprotein) or conjugated with lipids (glycolipids).

Unlike genomes, glycans are highly dynamic.

This area of research has to deal with an inherent level of complexity not seen in other areas of applied biology. 68 building blocks (molecules for DNA, RNA and proteins; categories for lipids; types of sugar linkages for saccharides) provide the structural basis for the molecular choreography that constitutes the entire life of a cell. DNA and RNA have four building blocks each (the nucleosides or nucleotides). Lipids are divided into eight categories based on ketoacyl and isoprene. Proteins have 20 (the amino acids). Saccharides have 32 types of sugar linkages. While these building blocks can be attached only linearly for proteins and genes, they can be arranged in a branched array for saccharides, further increasing the degree of complexity.

Add to this the complexity of the numerous crosslinking protein involved, not only as carriers of carbohydrate, the glycoproteins, but proteins specifically involved in binding and reacting with carbohydrate:

Carbohydrate-specific enzymes for synthesis, modulation, and degradation

Lectins, protein glycosylation of all sorts

Receptors, circulating or membrane-bound carbohydrate-binding receptors

Importance

Glycoproteins found on the cell surface play a critical role in bacterial and viral recognition.

They are involved in cellular signaling pathways and modulate cell function.

They are important in innate immunity.

They determine cancer development.

They orchestrate the cellular fate, inhibit proliferation, regulate circulation and invasion.

They affect the stability and folding of proteins.

They affect the pathway and fate of glycoproteins.

There are many glycan-specific diseases, often hereditary diseases.

There are important medical applications of aspects of glycomics:

Lectins fractionate cells to avoid graft-versus-host disease in hematopoietic stem cell transplantation.

Activation and expansion of cytolytic CD8 T cells in cancer treatment.

Glycomics is particularly important in microbiology because glycans play diverse roles in bacterial physiology. Research in bacterial glycomics could lead to the development of:

l Novel drugs

l Glycosylation Analysis

l Glycoconjugate vaccines

Tools used

The following are examples of the commonly used techniques in glycan analysis

High-resolution mass spectrometry (MS) and high-performance liquid chromatography (HPLC)

The most commonly applied methods are MS and HPLC, in which the glycan part is cleaved either enzymatically or chemically from the target and subjected to analysis. In case of glycolipids, they can be analyzed directly without separation of the lipid component.

N-glycans from glycoproteins are analyzed routinely by high-performance-liquid-chromatography (reversed phase, normal phase and ion exchange HPLC) after tagging the reducing end of the sugars with a fluorescent compound (reductive labeling). A large variety of different labels were introduced in the recent years, where 2-aminobenzamide (AB), anthranilic acid (AA), 2-aminopyridin (PA), 2-aminoacridone (AMAC) and 3-(acetylamino)-6-aminoacridine (AA-Ac) are just a few of them.

O-glycans are usually analysed without any tags, due to the chemical release conditions preventing them to be labeled.

Fractionated glycans from high-performance liquid chromatography (HPLC) instruments can be further analyzed by MALDI-TOF-MS(MS) to get further informations about structure and purity. Sometimes glycan pools are analyzed directly by mass spectrometry without prefractionation, although a discrimination between isobaric glycan structures is more challenging or even not always possible. Anyway, direct MALDI-TOF-MS analysis can lead to a fast and straightforward illustration of the glycan pool.

In recent years, high performance liquid chromatography online coupled to mass spectrometry became very popular. By choosing porous graphitic carbon as a stationary phase for liquid chromatography, even non derivatized glycans can be analyzed. Detection is here done by mass spectrometry, but in instead of MALDI-MS, electrospray ionisation (ESI) is more frequently used.

Multiple Reaction Monitoring (MRM)

Although MRM has been used extensively in metabolomics and proteomics, its high sensitivity and linear response over a wide dynamic range make it especially suited for glycan biomarker research and discovery. MRM is performed on a triple quadrupole (QqQ) instrument, which is set to detect a predetermined precursor ion in the first quadrupole, a fragmented in the collision quadrupole, and a predetermined fragment ion in the third quadrupole. It is a non-scanning technique, wherein each transition is detected individually and the detection of multiple transitions occurs concurrently in duty cycles. This technique is being used to characterize the immune glycome.

Coagulation Factor VIII & Its Test Method

Factor VIII is a variety of protein components involved in the blood coagulation process. Its physiological role is to activate when blood vessels bleed, stick to platelets and plug leaks in blood vessels. This process is called coagulation.

Structure and properties

In hemophilia, 80% of patients have hemophilia A, and this type of patients mainly lacks factor Ⅷ, which is owned by normal people.

The FⅧ gene is located at the end of the long arm of the X chromosome (Xq28), is 186 kb in length, and consists of 26 exons, of which exon 14 is 3.1 kb, which is one of the largest human exons found. FⅧ mRNA is 9kb long and encodes a precursor polypeptide consisting of 2351 amino acids. After removing the N-terminal 19-signal peptides, the mature egg is composed of 2332 amino acids. Amino acid sequence analysis showed that the FⅧ protein was composed of three A domains, one B domain and two C domains. The sequence of each region is A1-A2-B-A3-C1-C2. FⅧ, which is separated and purified from plasma and from the supernatant of recombinant cell culture, is composed of two peptide chains, and the heavy chain is A1-A2-B. Or A1-A2, with a molecular weight ranging from 90 to 200kDa; the light chain consists of A3-C1-C2, with a molecular weight of 80kDa, and the two peptide chains are connected by Ca2 +. Studies have shown that the B domain is not necessary for the coagulation activity of FⅧ.

Factor Ⅷ in normal human plasma is a glycoprotein with a molecular weight of up to 1 million to 2 million, which contains two components of low molecular weight and high molecular weight. Low molecular weight has coagulation activity (Ⅷ: C), and high molecular weight components have factor Ⅷ related antigen (ⅧR: Ag) and VW factor (ⅧR: VWF). The synthesis site of these three components controls the gene locus of synthesis And hereditary methods are different. Ⅷ: C is synthesized by liver and spleen or monocytes, and is genetically controlled by X chromosome. Ⅷ: C activity is reduced in hemophilia A, which is recessive inheritance of sex chromosome. ⅧR: Ag and ⅧR: VWF are synthesized by endothelial cells, megakaryocytes, and platelets, and are inherited by autosomes. The normal plasma VWF value is 10mg / L, and its activity is produced by a series of plasma multimers. The molecular weight of the multimer is about 400,000 to 20 million or more. It exists in plasma, platelets and subvascular endothelium, and the plasma VWF concentration decreases slightly Or the selective loss of high molecular weight polymers can reduce platelet adhesion. In VWD patients, these two components are lacking, or the molecular structure of these two components is abnormal, various physiological activities of factor Ⅷ polymers are abnormal, and Ⅷ: C function also has corresponding effects. ⅧR: VWF contains low, medium and high molecular weight polymers, which constitute different subtypes of VWD. The polymer of ⅧR: VWF can bind platelets to the lower layer of vascular endothelium by binding with special receptors of platelets, and maintain normal bleeding time. The effect of ristomycin and ⅧR: VWF on platelets is affected by platelets. Body related. The Ⅷ: C and ⅧR: VWF of patients with this disease also decreased to varying degrees. After the factor Ⅷ was purified, the patient’s bleeding time was shortened in a short time, while the Ⅷ: C increased after 6 to 24 hours. It is believed that normal ⅧR: VWF has the effect of stabilizing Ⅷ: C. When ⅧR: VWF is lacking, it can affect the activity of Ⅷ: C. Therefore, the disease may be due to a defect in the high molecular part of factor , or the entire factor Ⅷ complex. Caused by physical defects. VWD has also recently been found to be defective in fibrinolysis.

test methods

Human factor VIII-deficient plasma was used as the matrix plasma, and the test factor human factor VIII titer was measured by a one-stage method.

Reagent

(1) 9.5 g of 3.8% sodium citrate, dissolved in water and diluted to 250 ml.

(2) Imidazole buffer solution (pH 7.3) Take 0.68 g of imidazole and 1.17 g of sodium chloride, add water to dissolve it into 100 ml, add 42.2 ml of 0.1 mol / L hydrochloric acid solution, and dilute to 200 ml with water.

(3) Diluent: Take one volume of 3.8% sodium citrate, add 5 volumes of imidazole buffer, and add an appropriate amount of 20% human albumin to a final concentration of 1%.

(4) Activated partial thromboplastin (APTT) reagent

(5) Human coagulation factor viii antibody deficiency plasma is human plasma or artificial matrix plasma with human coagulation factor VIII content less than 1%

(6) 147 g of calcium chloride (CaCl2 · 2H2O) in 0.05 mol / L calcium chloride solution, dissolved in water and diluted to 1000 ml, formulated into a 1 mol / L calcium chloride stock solution, diluted 20 times with water before use, formulated 0.05mol / L calcium chloride solution.

Preparation of human coagulation factor VIII standard solution The human coagulation factor VIII-deficient plasma was used to dilute the standard to 1 IU of factor VIII per 1 ml, and the dilutions were diluted 10-fold, 20-fold, 40-fold, and 80-fold, respectively. use.

Preparation of test solution Use human factor Ⅷ deficient plasma to dilute the standard to 1 IU of coagulation factor 8 per 1ml, and then dilute with the diluent 10-fold and 20-fold or 40-fold, and place in an ice bath for use.

Assay

Take 0.1ml of activated thromboplastin reagent and incubate it in a 37 ° C water bath for a certain period of time (usually 4 minutes). Add coagulation factor Ⅷ deficient plasma 0.1ml and test solution 0.1ml, mix well, and incubate in a 37 ° C water bath for a certain period of time ( Usually 5min), add 0.1ml of 0.05mol / L calcium chloride solution that has been preheated to 37 ° C, and record the setting time.

Use 0.1ml human coagulation factor Ⅷ standard solution of different dilutions to replace the test solution, and perform the same operation.

The logarithm of the anti factor viii antibody titer (IU / ml) of the standard solution and the logarithm of its corresponding coagulation time (seconds) were subjected to linear regression processing to obtain a linear regression equation. Calculate the human factor VIII titer of the test solution and multiply it by the dilution factor to obtain the human factor VIII titer of the test solution (IU / ml).

The Largest Cancer Genome Study Announced Public I

A large-scale international cooperation project carried out comprehensive research on more than 2,600 tumor specimens from 38 cancer tissues, and provided a lot of new insights into the genetic basis of cancer.

Since the first human genome sequencing was completed in 2001, comprehensive genomic characterization of tumors has been a major goal for cancer researchers. Since then, sequencing technology and analysis tools have continued to advance, driving rapid development in the field.

Of the six papers published in Nature on February 6, 1-6, the Pan-Cancer Analysis of Whole Genomes (PCAWG) Alliance presents the most comprehensive and largest meta-analysis of cancer genomes to date. Previous analyses focused on protein-coding regions in the cancer genome, while the PCAWG analyzed the entire genome. Each article focuses on an important aspect of cancer genetics. Taken together, these six articles are of great significance for a comprehensive grasp of the genetic complexity of cancer.

Before describing each study in detail, it must be pointed out that the PCAWG relies on a large amount of data and a complex organizational framework. The entire project relies on an interdisciplinary team of scientists from four continents, involving a total of 744 academic institutions. To protect patient data while conducting distributed research, scientists must overcome technical, legal, and ethical obstacles. Researchers are divided into 16 working groups, each focusing on one aspect of cancer genomics, for example, some groups evaluate mutation repetition rates, and some groups infer tumor evolution.

The consortium conducted a comprehensive analysis of 38 different tumors, sequenced 2,658 whole cancer genomes, and also sequenced corresponding non-cancer cell samples from the same individual. In addition, the researchers also analyzed the sequence and abundance of 1,188 tumor transcriptomes, the RNA transcripts in tumors.

These collaborations involve extensive quality control, coordinated data processing, and large-scale, systematic experimental validation of different computational processes for detecting mutations. Multiple calculation algorithms and processes must be used and compared simultaneously. This requires hundreds of terabytes of data, distributed in multiple data centers, and may require millions of hours of processing time-and cloud computing greatly alleviates these problems. Obviously, the cooperation of the PCAWG Alliance as an excellent example proves the key role of cloud computing in promoting international cooperation and advancing the development of data-intensive fields.

The first paper 1 outlined the breadth and depth of the PCAWG dataset. The Alliance reports that, on average, each cancer genome carries 4-5 driver mutations, which provides cancer cells with a selection advantage. Of the tumors analyzed, only 5% did not find any driver mutations. In contrast, many tumors have chromosomal rearrangements (17.8% of tumors) and chromosome fragmentation (22.3% of tumors). The typical manifestations of these two genomic disasters can lead to major structural changes in the genome.

The other five papers are cut from different perspectives of the dataset. In the second paper, Rheinbay et al. identified driver genes in non-coding DNA. This work is very challenging because detecting mutations in non-coding regions is much more difficult than mutations in coding regions, let alone assessing their repetition rate. Nevertheless, the author systematically identified non-coding driver mutations by carefully modeling and eliminating artifacts.

The authors’ results question the previously reported non-coding driver mutations, such as the long non-coding RNAs NEAT1 and MALAT1. In addition, the authors also revealed new driver mutations, such as a mutation that repeatedly appears in the non-coding region of the key tumor suppressor gene TP53. They also found frequent mutations in the non-coding region of the telomerase gene TERT that cause abnormally high expression of telomerase (thus promoting uncontrolled division of tumor cells). This finding confirms the results of a previous pan-cancer study: higher telomerase mutation rates (12%) in advanced (metastatic) tumors. Although this study does not directly rule out the existence of other non-coding driver mutations, it can at least show that such mutations are not common.

In the third and fourth papers, Alexandrov et al. and Li et al. focused on genomic abnormalities known as “signature”. For example, defective DNA repair mechanisms or exposure to environmental mutagens, can lead to this characteristic DNA mutation. To further refine known mutation features and mine new features, a very large genomic data set is required. To their credit, Alexandrov et al. and Li et al. identified a total of 97 features. These newly discovered features include not only conventional single-nucleotide features, but also features related to polynucleotide variations and insertions or deletions of small DNA fragments.

Not only that, Li and colleagues were among the first research teams to discover reproducible features involving structural variants (SVs), where structural variants refer to the rearrangement of large genomes. Due to the diversity and complexity of structural variation, identifying such features is much more difficult than identifying mutation features.

By grouping the mutations, the researchers identified a total of 16 structural mutation characteristics, revealing other relationships such as the inferred mechanism link between the two structural mutations, deletion and inversion (the last characteristic involves the reversal of the direction of the DNA fragment). The researchers also provided new insights into the role of these 16 features play in cancer. Analysis has shown that mutations in specific DNA repair genes are linked to some cancer characteristics. For example, the consortium found that mutations in the gene CDK12 are related to tandem repeats in DNA; truncated variants of the DNA repair enzyme MBD4 appear simultaneously with mutational features involving the DNA sequence CpG site. Taken together, these newly discovered features lay the foundation for our understanding of the mechanisms of cancer development and the role of mutagenic exposure in this process.

To be continued in Part II…

 

An Overview of Anthocyanin

Anthocyanins, also known as anthocyanins, are a class of water-soluble natural pigments that are widely present in plants in nature. They are colored aglycones obtained through hydrolysis of anthocyanins. Most of the main coloring substances in fruits, vegetables and flowers are related to it. Under different pH conditions of plant cell vacuoles, anthocyanins make the petals appear colorful. It is known that there are more than 20 anthocyanins, and there are 6 important foods, namely geranium pigment, cornflower pigment, delphinium pigment, peony pigment, morning glory pigment and mallow pigment. Anthocyanins in the natural state exist in the form of glycosides, called anthocyanins, and few free anthocyanins exist. Anthocyanins are mainly used in food coloring, and can also be used in dyes, medicine, cosmetics and so on.

Main source

Anthocyanins are widely found in flowering plants (angiosperms), and the content of anthocyanins in plants varies greatly with variety, season, climate, and maturity. According to preliminary statistics: Anthocyanins are contained in 73 families and 27 genera, such as purple sweet potato, grape, blood orange, red cabbage, blueberry, eggplant, cherry, redberry, strawberry, mulberry, hawthorn, morning glory Flowers and other tissues have a certain content.

The earliest and most abundant anthocyanin was grape skin red pigment extracted from red grape dregs. It was launched in Italy in 1879. The pigment can be extracted from the waste of wineries-grape dregs. Elderberry contains a large amount of anthocyanins, and they are cyanidin, with a fresh weight of 200 ~ 1000 mg per 100 grams. In addition, anthocyanins are also widely present in food crops such as barley, sorghum and legumes. Studies have found that grape seed and pine bark extracts have the highest anthocyanin content.

Extraction Method

Solvent extraction

Solvent extraction is a common method for extracting anthocyanins. The solvents used are mostly methanol, ethanol, acetone, water, or mixed solvents. In order to prevent the degradation of non-acylated anthocyanins during extraction, a certain concentration of hydrochloric acid or formic acid is often added to the extraction solvent, but these acids will cause partial or complete hydrolysis of the acylated anthocyanins during evaporation and concentration. In addition, for samples that may contain fat-soluble components in the extract, organic solvents such as n-hexane, petroleum ether, ether, etc. are required for extraction. The traditional solvent extraction method has a long extraction time, low production efficiency, and hot solvents easily cause degradation of anthocyanins and reduction of physiological activity.

The traditional method of extracting anthocyanins abroad is to use low temperature (4 ~ 8 ℃) or normal temperature (25 ℃) in the dark for 16 ~ 20 h, or use 01 5% and 1% trifluoride. Extraction of acetic acid in methanol for 24 h at 4 ℃. Considering the toxicity of residual methanol in food, 1% HCl ethanol solution can also be used instead of methanol solution. In addition, in order to avoid the hydrolysis of acylated anthocyanins, weak acids such as tartaric acid and citric acid can be selected instead of hydrochloric acid. In China, hot solvent (50 ~ 70 ℃) is usually used for extraction for 1 ~ 2 hours. The solvent can choose alcohol solution or acidified aqueous solution with different concentrations.

Pressurized solvent extraction

Pressurized solvent extraction, also known as pressurized liquid extraction and rapid solvent extraction, is to increase the boiling point of the solvent by external pressure, and then increase the solubility of the substance in the solvent and the extraction efficiency.

The extraction of functional ingredients from food by PSE technology is mainly focused on the research of flavonoids, phenols and other antioxidant active ingredients. This technology has also been reported in the extraction of anthocyanins. This technology was used to optimize the optimal extraction process of anthocyanins in purple cabbage. The best parameters were: sample 21 5 g, temperature 99 ℃, extraction time 7 min, solvent was V (water) BV (ethanol) BV (methanol) = 94: 5: 1.

Aqueous solution extraction

Anthocyanins extracted by organic solvents often have toxic residues and environmental pollution during the production process. In view of this, aqueous solution extraction came into being. This method generally soaks plant material with hot water under normal pressure or high pressure, and then adsorbs it with non-polar macroporous resin; or directly extracts with deoxygenated hot water, and then uses ultrafiltration or reverse osmosis to concentrate to obtain the crude extract.

Microbial fermentation extraction

This method applies the biological fermentation technology to the extraction of anthocyanins, which is a super strong penetration and effective combination between biological science and chemical production. The microbial fermentation method uses microorganisms or enzymes to degrade and separate the cell wall of anthocyanin-containing cells, so that the anthocyanins in the cell body are fully dissolved in the extract, thereby increasing the yield and rate of extraction.

Other extraction methods

Including high-voltage pulsed electric field-assisted extraction, two-phase aqueous extraction, and ultra-high pressure auxiliary extraction. The first two can be applied to the extraction of proteins, nucleic acids, and polysaccharides.Ultra-high pressure-assisted extraction has been successfully used in the extraction of anthocyanins from grapes. Increased by nearly 50%. Anthocyanins has a sort of functions, including, anti-mutation function, anti-oxidation, scavenging free radical function, application in food, etc.