Peptide

Peptides (from Gr. πεπτός, "digested", derived from πέσσειν, "to digest") are biologically occurring short chains of amino acid monomers linked by peptide (amide) bonds.

The covalent chemical bonds are formed when the carboxyl group of one amino acid reacts with the amine group of another. The shortest peptides are dipeptides, consisting of 2 amino acids joined by a single peptide bond, followed by tripeptides, tetrapeptides, etc. A polypeptide is a long, continuous, and unbranched peptide chain. Hence, peptides fall under the broad chemical classes of biological oligomers and polymers, alongside nucleic acids, oligosaccharides and polysaccharides, etc.

Peptides are distinguished from proteins on the basis of size, and as an arbitrary benchmark can be understood to contain approximately 50 or fewer amino acids.Proteins consist of one or more polypeptides arranged in a biologically functional way, often bound to ligands such as coenzymes and cofactors, or to another protein or other macromolecule (DNA, RNA, etc.), or to complex macromolecular assemblies. Finally, while aspects of the lab techniques applied to peptides versus polypeptides and proteins differ (e.g., the specifics of electrophoresis, chromatography, etc.), the size boundaries that distinguish peptides from polypeptides and proteins are not absolute: long peptides such as amyloid beta have been referred to as proteins, and smaller proteins like insulin have been considered peptides.

Amino acids that have been incorporated into peptides are termed "residues" due to the release of either a hydrogen ion from the amine end or a hydroxyl ion from the carboxyl end, or both, as a water molecule is released during formation of each amide bond. All peptides except cyclic peptides have an N-terminal and C-terminal residue at the end of the peptide (as shown for the tetrapeptide in the image).

Peptide classes
Peptides are divided into several classes, depending on how they are produced:

Milk peptides 
Two naturally occurring milk peptides are formed from the milk protein casein when digestive enzymes break this down; they can also arise from the proteinases formed by lactobacilli during the fermentation of milk.
Ribosomal peptides 
Ribosomal peptides are synthesized by translation of mRNA. They are often subjected to proteolysis to generate the mature form. These function, typically in higher organisms, as hormones and signaling molecules. Some organisms produce peptides as antibiotics, such as microcins. Since they are translated, the amino acid residues involved are restricted to those utilized by the ribosome.
However, these peptides frequently have posttranslational modifications... such as phosphorylation, hydroxylation, sulfonation, palmitoylation, glycosylation and disulfide formation. In general, they are linear, although lariat structures have been observed.More exotic manipulations do occur, such as racemization of L-amino acids to D-amino acids in platypus venom.

Nonribosomal peptides 
Nonribosomal peptides are assembled by enzymes that are specific to each peptide, rather than by the ribosome. The most common non-ribosomal peptide is glutathione, which is a component of the antioxidant defenses of most aerobic organisms.Other nonribosomal peptides are most common in unicellular organisms, plants, and fungi and are synthesized by modular enzyme complexes called nonribosomal peptide synthetases.
These complexes are often laid out in a similar fashion, and they can contain many different modules to perform a diverse set of chemical manipulations on the developing product.These peptides are often cyclic and can have highly complex cyclic structures, although linear nonribosomal peptides are also common. Since the system is closely related to the machinery for building fatty acids and polyketides, hybrid compounds are often found. The presence of oxazoles or thiazoles often indicates that the compound was synthesized in this fashion.

Peptones
See also Tryptone
Peptones are derived from animal milk or meat digested by proteolysis.In addition to containing small peptides, the resulting spray-dried material [clarification needed] includes fats, metals, salts, vitamins and many other biological compounds. Peptones are used in nutrient media for growing bacteria and fungi.
Peptide fragments 
Peptide fragments refer to fragments of proteins that are used to identify or quantify the source protein.Often these are the products of enzymatic degradation performed in the laboratory on a controlled sample, but can also be forensic or paleontological samples that have been degraded by natural effects.

Peptide synthesis

In organic chemistry, peptide synthesis is the production of peptides, which are organic compounds in which multiple amino acids are linked via amide bonds, also known as peptide bonds. The biological process of producing long peptides (proteins) is known as protein biosynthesis.

Chemistry
Peptides are synthesized by coupling the carboxyl group or C-terminus of one amino acid to the amino group or N-terminus of another. Due to the possibility of unintended reactions, protecting groups are usually necessary. Chemical peptide synthesis starts at the C-terminal end of the peptide and ends at the N-terminus. This is the opposite of protein biosynthesis, which starts at the N-terminal end.

Liquid-phase synthesis
Liquid-phase peptide synthesis is a classical approach to peptide synthesis. It has been replaced in most labs by solid-phase synthesis (see below). However, it retains usefulness in large-scale production of peptides for industrial purposes.

Solid-phase synthesis

Coupling step in solid-phase peptide synthesis
Solid-phase peptide synthesis (SPPS), pioneered by Robert Bruce Merrifield, caused a paradigm shift within the peptide synthesis community, and it is now the standard method for synthesizing peptides and proteins in the lab. SPPS allows for the synthesis of natural peptides which are difficult to express in bacteria, the incorporation of unnatural amino acids, peptide/protein backbone modification, and the synthesis of D-proteins, which consist of D-amino acids.

Small porous beads are treated with functional units ('linkers') on which peptide chains can be built. The peptide will remain covalently attached to the bead until cleaved from it by a reagent such as anhydrous hydrogen fluoride or trifluoroacetic acid. The peptide is thus 'immobilized' on the solid-phase and can be retained during a filtration process while liquid-phase reagents and by-products of synthesis are flushed away.

The general principle of SPPS is one of repeated cycles of deprotection-wash-coupling-wash. The free N-terminal amine of a solid-phase attached peptide is coupled (see below) to a single N-protected amino acid unit. This unit is then deprotected, revealing a new N-terminal amine to which a further amino acid may be attached. The superiority of this technique partially lies in the ability to perform wash cycles after each reaction, removing excess reagent with all of the growing peptide of interest remaining covalently attached to the insoluble resin.[citation needed]

The overwhelmingly important consideration is to generate extremely high yield in each step. For example, if each coupling step were to have 99% yield, a 26-amino acid peptide would be synthesized in 77% final yield (assuming 100% yield in each deprotection); if each step were 95%, it would be synthesized in 25% yield. Thus each amino acid is added in major excess (2~10x) and coupling amino acids together is highly optimized by a series of well-characterized agents.[citation needed]

There are two majorly used forms of SPPS – Fmoc and Boc. Unlike ribosome protein synthesis, solid-phase peptide synthesis proceeds in a C-terminal to N-terminal fashion. The N-termini of amino acid monomers is protected by either of these two groups and added onto a deprotected amino acid chain.

Automated synthesizers are available for both techniques, though many research groups continue to perform SPPS manually.

SPPS is limited by yields, and typically peptides and proteins in the range of 70 amino acids are pushing the limits of synthetic accessibility.[citation needed] Synthetic difficulty also is sequence dependent; typically amyloid peptides and proteins are difficult to make. Longer lengths can be accessed by using native chemical ligation to couple two peptides together with quantitative yields.

Since its introduction over 40 years ago, SPPS has been significantly optimized. First, the resins themselves have been optimized.Furthermore, the 'linkers' between the C-terminal amino acid and polystyrene resin have improved attachment and cleavage to the point of mostly quantitative yields.The evolution of side chain protecting groups has limited the frequency of unwanted side reactions. In addition, the evolution of new activating groups on the carboxyl group of the incoming amino acid have improved coupling and decreased epimerization. Finally, the process itself has been optimized. In Merrifield's initial report, the deprotection of the α-amino group resulted in the formation of a peptide-resin salt, which required neutralization with base prior to coupling. The time between neutralization of the amino group and coupling of the next amino acid allowed for aggregation of peptides, primarily through the formation of secondary structures, and adversely affected coupling. The Kent group showed that concomitant neutralization of the α-amino group and coupling of the next amino acid led to improved coupling. Each of these improvements has helped SPPS become the robust technique that it is today.

BOP SPPS
The use of BOP reagent was first described by Castro et al. in 1975.

Solid supports
The name solid support implies that reactions are carried out on the surface of the support, but this is not the case. Reactions also occur within these particles, and thus the term "solid support" better describes the insolubility of the polymer. The physical properties of the solid support, and the applications to which it can be utilized, vary with the material from which the support is constructed, the amount of cross-linking, as well as the linker and handle being used. Most scientists in the field believe that supports should have the minimum amount of cross-linking to confer stability. This should result in a well-solvated system where solid-phase peptide synthesis can be carried out. Nonetheless, the characteristics of an efficient solid support include:

It must be physically stable and permit the rapid filtration of liquids, such as excess reagents
It must be inert to all reagents and solvents used during SPPS
It must swell extensively in the solvents used to allow for penetration of the reagents
It must allow for the attachment of the first amino acid
There are four primary types of solid supports:

Gel-type supports: These are highly solvated polymers with an equal distribution of functional groups. This type of support is the most common, and includes:
Polystyrene: Styrene cross-linked with 1–2% divinylbenzene
Polyacrylamide: A hydrophilic alternative to polystyrene
Polyethylene glycol (PEG): PEG-Polystyrene (PEG-PS) is more stable than polystyrene and spaces the site of synthesis from the polymer backbone
PEG-based supports: Composed of a PEG-polypropylene glycol network or PEG with polyamide or polystyrene
Surface-type supports: Many materials have been developed for surface functionalization, including controlled pore glass, cellulose fibers, and highly cross-linked polystyrene.
Composites: Gel-type polymers supported by rigid matrices.
Polystyrene resin

Polystyrene cross-linked with divinylbenzene. This is the most common solid support used in SPPS, and was the support pioneered by R. Bruce Merrifield.
Polystyrene resin is a versatile resin and it is quite useful in multi-well, automated peptide synthesis, due to its minimal swelling in dichloromethane. The initial support used by R. Bruce Merrifield was polysytrene cross-linked with 2% divinylbenzene. This support is sometimes referred to as the 'Merrifield resin.' This resin produces a hydrophobic bead that is solvated by a nonpolar solvent such as dichloromethane or dimethylformamide. Since then, new resins have been developed with the following advantages:

Enhanced swelling or rigidity (a property of mechanical strength)
Chemical inertness
Highly cross-linked (50%) polystyrene has been developed that possesses the features of increased mechanical stability, better filtration of reagents and solvents, and rapid reaction kinetics.

Polyamide resin
Polyamide resin is also a useful and versatile resin. It seems to swell much more than polystyrene, in which case it may not be suitable for some automated synthesizers, if the wells are too small.

PEG hybrid polystyrene resin
An example of this type of resin is the Tentagel resin. The base resin is polystyrene onto which is attached long chains (Mw ca. 3000 Da) of polyethylene glycol (PEG; also known as polyethylene oxide). Synthesis is carried out on the distal end of the PEG spacer making it suited for long and difficult peptides. In addition it is also attractive for the synthesis of combinatorial Peptide libraries and on resin screening experiments. It does not expand much during synthesis making it a preferred resin for robotic peptide synthesis.

PEG-based resin
ChemMatrix(R) is a new type of resin which is based on PEG that is crosslinked. ChemMatrix(R) has claimed a high chemical and thermal stability (is compatible with Microwave synthesis) and has shown higher degrees of swellings in acetonitrile, dichloromethane, DMF, N-methylpyrrolidone, TFA and water compared to the polystyrene-based resins. ChemMatrix has shown significant improvements to the synthesis of hydrophobic sequences. ChemMatrix may be useful for the synthesis of difficult and long peptides.

Improvements to solid supports used for peptide synthesis enhance their ability to withstand the repeated use of TFA during the deprotection step of SPPS.[8] Furthermore, different resins allow for different functional groups at the C-terminus. The oxymethylphenylacetamidomethyl (PAM) resin results in the conventional C-terminal carboxylic acid. On the other hand, the paramethylbenzhydrylamine (pMBHA) resin yields a C-terminal amide, which is useful in mimicking the interior of a protein.

Along with the development of Fmoc SPPS, different resins have also been created to be removed by TFA. Similar to the Boc strategy, two primary resins are used, based on whether a C-terminal carboxylic acid or amide is desired. The Wang resin is the most commonly used resin for peptides with C-terminal carboxylic acids.[9] If a C-terminal amide is desired, the Rink amide resin is used.

Protecting groups
Amino acids have reactive moieties at the N- and C-termini, which facilitates amino acid coupling during synthesis. Many amino acids also have reactive side chain functional groups, which can interact with free termini or other side chain groups during synthesis and peptide elongation and negatively influence yield and purity. To facilitate proper amino acid synthesis with minimal side chain reactivity, chemical groups have been developed to bind to specific amino acid functional groups and block, or protect, the functional group from nonspecific reactions. These protecting groups, while vast in nature, can be separated into three groups, as follows:

N-terminal protecting groups
C-terminal protecting groups (mostly used in liquid-phase synthesis)
Side chain protecting groups
Purified, individual amino acids are reacted with these protecting groups prior to synthesis and then selectively removed during specific steps of peptide synthesis.

N-terminal protecting groups
Amino acids are added in excess to ensure complete coupling during each synthesis step, and without N-terminal protection, polymerization of unprotected amino acids could occur, resulting in low peptide yield or synthesis failure. N-terminal protection requires an additional step of removing the protecting group, termed deprotection, prior to the coupling step, creating a repeating design flow as follows:

Protecting group is removed from the trailing amino acids in a deprotection reaction
Deprotection reagents are washed away to provide a clean coupling environment
Protected amino acids dissolved in a solvent such as dimethylformamide (DMF) combined with coupling reagents are pumped through the synthesis column
Coupling reagents are washed away to provide clean deprotection environment
Currently, two protecting groups (t-Boc, Fmoc) are commonly used in solid-phase peptide synthesis. Their lability is caused by the carbamate group which readily releases CO2 for an irreversible decoupling step.

t-Boc protecting group
Main article: Tert-butyloxycarbonyl protecting group

Boc cleavage
The original method for the synthesis of proteins relied on (tert-butyloxycarbonyl or more simply "Boc") to temporarily protect the α-amino group. In this method, the Boc group is covalently bound to the amino group to suppress its nucleophilicity. The C-terminal amino acid is covalently linked to the resin through a linker. Next, the Boc group is removed with acid, such as trifluoroacetic acid (TFA). This forms a positively charged amino group (in the presence of excess TFA; note image on the right illustrates neutral amino group), which is neutralized (via in-situ or non-in-situ methods) and coupled to the incoming activated amino acid.[6] Reactions are driven to completion by the use of excess (two- to four-fold) activated amino acid. After each deprotection and coupling step, a wash with dimethylformamide (DMF) is performed to remove excess reagents, allowing for high yields (~99%) during each cycle.

t-Boc protecting strategies retain usefulness in reducing peptide aggregation during synthesis. t-Boc groups can be added to amino acids with t-Boc anhydride and a suitable base. Some researchers prefer Boc SPPS for complex syntheses . In addition, when synthesizing nonnatural peptide analogs, which are base-sensitive (such as depsipeptides), the t-Boc protecting group is necessary, because Fmoc SPPS uses a base to deprotect the α-amino group.

Permanent side-chain protecting groups are typically benzyl or benzyl-based groups. Final removal of the peptide from the linkage occurs simultaneously with side-chain deprotection with anhydrous hydrogen fluoride via hydrolytic cleavage. The final product is a fluoride salt which is relatively easy to solubilize. Importantly, scavengers such as cresol are added to the HF in order to prevent reactive t-butyl cations from generating undesired products. In fact, the use of harsh hydrogen fluoride may degrade some peptides, which was the premise for the development of a milder, base-labile method of SPPS—namely, the Fmoc method.

Fmoc protecting group
See also: Fmoc chloride

Fmoc cleavage
The capacity for anhydrous hydrogen fluoride to degrade proteins during the final cleavage conditions led to a new α-amino protecting group based on 9-fluorenylmethyloxycarbonyl (Fmoc). The Fmoc method allows for a milder deprotection scheme. This method utilizes a base, usually piperidine (20–50%) in DMF in order to remove the Fmoc group to expose the α-amino group for reaction with an incoming activated amino acid.[7] Unlike the acid used to deprotect the α-amino group in Boc methods, Fmoc SPPS uses a base, and thus the exposed amine is neutral. Therefore, no neutralization of the peptide-resin is required, but the lack of electrostatic repulsions between the peptides can lead to increased aggregation. Because the liberated fluorenyl group is a chromophore, deprotection by Fmoc can be monitored by UV absorbance of the runoff, a strategy which is employed in automated synthesizers.

The advantage of Fmoc is that it is cleaved under very mild basic conditions (e.g. piperidine), but stable under acidic conditions, although this has not always held true in certain synthetic sequences. This allows mild acid-labile protecting groups that are stable under basic conditions, such as Boc and benzyl groups, to be used on the side-chains of amino acid residues of the target peptide. This orthogonal protecting group strategy is common in organic synthesis. Fmoc is preferred over BOC due to ease of cleavage; however it is less atom-economical, as the fluorenyl group is much larger than the tert-butyl group. Accordingly, prices for Fmoc amino acids were high until the large-scale piloting of one of the first synthesized peptide drugs, enfuvirtide, began in the 1990s, when market demand adjusted the relative prices of the two sets of amino acids.

Semipermanent side chain protecting groups are t-butyl-based, and final cleavage of the protein from the resin and removal of permanent protecting groups is performed with TFA in the presence of scavengers. Water and triisopropylsilane (TIPS) present in a 1:1 ratio are often used as scavengers. Thus, the Fmoc method is orthogonal in two directions: deprotection of any α-amino group, deprotection of side groups and final cleavage from the resin occur by independent mechanisms. The resulting final product is a TFA salt, which is more difficult to solubilize than the fluoride salts generated in Boc SPPS. This method is thus milder than the Boc method because the deprotection/cleavage-from-resin steps occur with different conditions rather than with different reaction rates.

Comparison of Boc and Fmoc solid-phase peptide synthesis
Both the Fmoc and Boc methods offer advantages and disadvantages. The selection of one technique over another is thus made on a case-by-case basis.

Boc SPPS uses special equipment to handle the final cleavage and deprotection step, which requires anhydrous hydrogen fluoride. Because the final cleavage of the peptide with Fmoc SPPS uses TFA, this special equipment is not necessary. The solubility of peptides generated by Boc SPPS is generally higher than those generated with the Fmoc method, because fluoride salts are higher in solubility than TFA salts. Next, problems with aggregation are generally more of an issue with Fmoc SPPS. This is primarily because the removal of a Boc group with TFA yields a positively charged α-amino group, whereas the removal of an Fmoc group yields a neutral α-amino group. The steric hindrance of the positively charged α-amino group limits the formation of secondary structure on the resin. Finally, the Fmoc method is considered orthogonal, since α-amino group deprotection is with base, while final cleavage from the resin is with acid. The Boc method utilizes acid for both deprotection and cleavage from the resin. Based on this comparison, one sees that both methods possess advantages and disadvantages. Thus, several factors help to decide which method may be preferable.

DMF must be 'peptide grade' i.e. little/no impurities and must also be 'fresh'. This is due to the fact that DMF undergoes photolysis to form carbon monoxide and dimethylamine. Dimethylamine may remove the Fmoc group and, therefore, lead to impurities.

Benzyloxy-carbonyl (Z) group
See also: Carboxybenzyl
The first use of (Z) group as protecting groups was done by Max Bergmann who synthesised oligopeptides.

Another carbamate-based group is the benzyloxy-carbonyl (Z) group. It is removed in harsher conditions: HBr/acetic acid or catalytic hydrogenation. Today it is almost exclusively used for side chain protection.

Alloc protecting group
The allyloxycarbonyl (alloc) protecting group is often used to protect a carboxylic acid, hydroxyl, or amino group when an orthogonal deprotection scheme is required. It is sometimes used when conducting on-resin cyclic peptide formation, where the peptide is linked to the resin by a side-chain functional group. The alloc group can be removed using tetrakis(triphenylphosphine)palladium(0) along with a 37:2:1 mixture of methylene chloride, acetic acid, and N-Methylmorpholine (NMM) for 2 hours. The resin must then be carefully washed 0.5% DIPEA in DMF, 3 × 10 ml of 0.5% sodium diethylthiocarbamate in DMF, and then 5 × 10 ml of 1:1 DCM:DMF.

Lithographic protecting groups
For special applications like protein microarrays lithographic protecting groups are used. Those groups can be removed through exposure to light.

Side chain protecting groups
Amino acid side chains represent a broad range of functional groups and are sites of nonspecific reactivity during peptide synthesis. Because of this, many different protecting groups are required that are usually based on the benzyl (Bzl) or tert-butyl (tBu) group. The specific protecting groups used during the synthesis of a given peptide vary depending on the peptide sequence and the type of N-terminal protection used (see next paragraph). Side chain protecting groups are known as permanent or semipermanent protecting groups, because they can withstand the multiple cycles of chemical treatment during synthesis and are only removed during treatment with strong acids after peptide synthesis is completed.

Protection schemes
Because multiple protecting groups are normally used during peptide synthesis, these groups must be compatible to allow deprotection of distinct protecting groups while not affecting other protecting groups. Protecting schemes are therefore established to match protecting groups so that deprotection of one protecting group does not affect the binding of the other groups. Because N-terminal deprotection occurs continuously during peptide synthesis, protecting schemes have been established in which the different types of side chain protecting groups (Bzl or tBu) are matched to either Boc or Fmoc, respectively, for optimized deprotection. These protecting schemes also incorporate each of the steps of synthesis and cleavage, as described in the table and in later sections of this page.

Activating groups
For coupling the peptides the carboxyl group is usually activated. This is important for speeding up the reaction. There are two main types of activating groups: carbodiimides and triazolols. However the use of pentafluorophenyl esters (FDPP,PFPOH) and BOP-Cl are useful for cyclising peptides.

Carbodiimides

Alanine attaching to DCC
These activating agents were first developed. Most common are dicyclohexylcarbodiimide (DCC) and diisopropylcarbodiimide (DIC). Reaction with a carboxylic acid yields a highly reactive O-acylisourea. During artificial protein synthesis (such as Fmoc solid-state synthesizers), the C-terminus is often used as the attachment site on which the amino acid monomers are added. To enhance the electrophilicity of carboxylate group, the negatively charged oxygen must first be "activated" into a better leaving group. DCC is used for this purpose. The negatively charged oxygen will act as a nucleophile, attacking the central carbon in DCC. DCC is temporarily attached to the former carboxylate group (which is now an ester group), making nucleophilic attack by an amino group (on the attaching amino acid) to the former C-terminus (carbonyl group) more efficient. The problem with carbodiimides is that they are too reactive and that they can therefore cause racemization of the amino acid.

Triazoles

To solve the problem of racemization, triazoles were introduced. The most important ones are 1-hydroxy-benzotriazole (HOBt) and 1-hydroxy-7-aza-benzotriazole (HOAt). Others have been developed. These substances can react with the O-acylurea to form an active ester which is less reactive and less in danger of racemization. HOAt is especially favourable because of a neighbouring group effect.[15] Recently, HOBt has been removed from many chemical vendor catalogues; although almost always found as a hydrate, HOBt may be explosive when allowed to fully dehydrate and shipment by air or sea is heavily restricted. Alternatives to HOBt and HOAt have been introduced. One of the most promising and inexpensive[citation needed] is ethyl 2-cyano-2-(hydroxyimino)acetate (trade name Oxyma Pure), which is not explosive and has a reactivity of that in between HOBt and HOAt.

Uronium-based peptide coupling reagents
Newer developments omit the carbodiimides totally. The active ester is introduced as a uronium or phosphonium salt of a non-nucleophilic anion (tetrafluoroborate or hexafluorophosphate): HBTU, HATU, HCTU, TBTU, PyBOP. Two uronium types of the coupling additive of Oxyma Pure is also available as COMU or TOTU reagent.

Regioselective disulfide formation

The formation of multiple native disulfides remains one of the primary challenges of native peptide synthesis by solid-phase methods. Random chain combination typically results in several products with nonnative disulfide bonds.[16] Stepwise formation of disulfide bonds is typically the preferred method, and performed with thiol protecting groups (PGs).[17] Different thiol PGs provide multiple dimensions of orthogonal protection. These orthogonally protected cysteines are incorporated during the solid-phase synthesis of the peptide. Successive removal of these PGs to allow for selective exposure of free thiol groups, leads to disulfide formation in a stepwise manner. The order of removal of these PGs must be considered so that only one group is removed at a time. Using this method, Kiso et al. reported the first total synthesis of insulin by this method in 1993.[18]

The thiol PGs must possess multiple characteristics. First, the PG must be reversible with conditions that do not affect the unprotected side chains. Second, the protecting group must be able to withstand the conditions of solid-phase synthesis. Third, the configuration of the removal of the thiol protecting group must be such that it leaves intact other thiol PGs, if orthogonal protection is desired. That is, the removal of PG A should not affect PG B. Some of the thiol PGs commonly used include the acetamidomethyl (Acm), tert-butyl (But), 3-nitro-2-pyridine sulfenyl (NPYS), 2-pyridine-sulfenyl (Pyr), and triphenylmethyl (Trt) groups. Importantly, the NPYS group can replace the Acm PG to yield an activated thiol.[19]

In the stepwise formation of disulfides to synthesize insulin by Kiso et al., the authors synthesize the A-chain with following protection: CysA6(But); CysA7(Acm); CysA11(But). Thus, CysA20 is unprotected. Synthesis of the B-chain is performed with the following protection: CysB7(Acm) CysB19(Pyr). The first disulfide bond, CysA20–CysB19, was formed by mixing the two chains in 8 M urea, pH 8 (RT) for 50 min. The second disulfide bond, CysA7–CysB7, was formed by treatment with iodine in aqueous acetic acid to remove the Acm groups. The third disulfide, the intramolecular CysA6–CysA11, was formed by the removal of the But groups by methyltrichlorosilane with diphenyl sulfoxide in TFA. Importantly, formation of the first disulfide in 8 M urea, pH 8 does not affect the other PGs, namely Acm and But groups. Likewise, formation of the second disulfide bond with iodine in aqueous acetic acid does not affect the But groups.

Important to the discussion of disulfide bond formation is the order in which disulfides are formed. From a logical standpoint, the order in which the thiol groups are exposed to form disulfides should be of little consequence, since the other cysteines are protected. Practically, however, the order in which disulfides are formed can have a significant effect on yields. This may be because the formation of the CysA20–CysB19 disulfide may place the thiol group of CysB7 in close proximity with both CysA6 and CysA7, leading to multiple disulfide products. This is one manifestation of the reality that solid-phase peptide synthesis is as much art as it is science.

Synthesizing long peptides

Stepwise elongation, in which the amino acids are connected step-by-step in turn, is ideal for small peptides containing between 2 and 100 amino acid residues. Another method is fragment condensation, in which peptide fragments are coupled. Although the former can elongate the peptide chain without racemization, the yield drops if only it is used in the creation of long or highly polar peptides. Fragment condensation is better than stepwise elongation for synthesizing sophisticated long peptides, but its use must be restricted in order to protect against racemization. Fragment condensation is also undesirable since the coupled fragment must be in gross excess, which may be a limitation depending on the length of the fragment.

A new development for producing longer peptide chains is chemical ligation: Unprotected peptide chains react chemoselectively in aqueous solution. A first kinetically controlled product rearranges to form the amide bond. The most common form of native chemical ligation uses a peptide thioester that reacts with a terminal cysteine residue.

In order to optimize synthesis of long peptides, Zealand Pharma (located in Denmark in Medicon Valley) invented a method for converting a difficult peptide sequence into an easy peptide sequence. The new technology, called SIP-technology, uses “structure-inducing probes” (SIP) to facilitate the synthesis of long peptides. The SIP-technology is a small pre-sequence peptide sequence (e.g. Lysine (Lysn); Glutamic Acid (Glun); (LysGlu)n) that is incorporated at the C-terminus of subsequent resin bound peptide to induce an alpha-helix-like structure in the peptide. The SIP technology constrains the parent peptide into a more ordered conformation using intramolecular hydrogen bonds. This allows the peptide structure to stabilize, and the utilized hydrogen bonds reduce the likelihood of aggregation and degradation by enzymes. In this way, the SIP technology is designed to optimize peptide synthesis, increase biological half-life, improve peptide stability and inhibit enzymatic degradation without altering pharmacological activity or profile of action.

Microwave assisted peptide synthesis

Although microwave irradiation has been around since the late 1940s, it was not until 1986 that microwave energy was used in organic chemistry. During the end of the 1980s and 1990s, microwave energy was an obvious source for completing chemical reactions in minutes that would otherwise take several hours to days. Through several technical improvements at the end of the 1990s and beginning of the 2000s, microwave synthesizers have been designed to provide both low and high energy pockets of microwave energy so that the temperature of the reaction mixture could be controlled. The microwave energy used in peptide synthesis is of a single frequency providing maximum penetration depth of the sample which is in contrast to conventional kitchen microwaves.

In peptide synthesis, microwave irradiation has been used to complete long peptide sequences with high degrees of yield and low degrees of racemization. Microwave irradiation during the coupling of amino acids to a growing polypeptide chain is not only catalyzed through the increase in temperature, but also due to the alternating electromagnetic radiation[citation needed] to which the polar backbone of the polypeptide continuously aligns. Due to this phenomenon, the microwave energy can prevent aggregation and thus increases yields of the final peptide product. There is however no clear evidence that microwave is better than simple heating and some peptide laboratories regard microwave just as a convenient method for rapid heating of the peptidyl resin. Heating to above 50–55 degrees Celsius also prevents aggregation and accelerates the coupling.

Despite the main advantages of microwave irradiation of peptide synthesis, the main disadvantage is the racemization which may occur with the coupling of cysteine and histidine. A typical coupling reaction with these amino acids are performed at lower temperatures than the other 18 natural amino acids. A number of peptides do not survive microwave synthesis or heating in general. One of the more serious side effects is dehydration (loss of water) which for certain peptides can be almost quantitative like pancreatic polypeptide (PP). This side effect is also seen by simple heating without the use of microwave.

Cyclic peptides

On resin cyclization
Peptide can be cyclized on solid support. A variety of cylization reagents can be used such as HBTU/HOBt/DIEA, PyBop/DIEA, PyClock/DIEA. Head-to-tail peptides can be made on the solid support. The deprotection of the C-terminus at some suitable point allows on-resin cyclization by amide bond formation with the deprotected N-terminus. Once cyclization has taken place, the peptide is cleaved from resin by acidolysis and purified. The strategy for the solid-phase synthesis of cyclic peptides in not limited to attachment through Asp, Glu or Lys side chains. Cysteine has a very reactive sulfhydryl group on its side chain. A disulfide bridge is created when a sulfur atom from one Cysteine forms a single covalent bond with another sulfur atom from a second cysteine in a different part of the protein. These bridges help to stabilize proteins, especially those secreted from cells. Some researchers use modified cysteines using S-acetomidomethyl (Acm) to block the formation of the disulfide bond but preserve the cysteine and the protein's original primary structure.

Peptides in molecular biology

Peptides received prominence in molecular biology for several reasons. The first is that peptides allow the creation of peptide antibodies in animals without the need of purifying the protein of interest.[17] This involves synthesizing antigenic peptides of sections of the protein of interest. These will then be used to make antibodies in a rabbit or mouse against the protein.

Another reason is that peptides have become instrumental in mass spectrometry, allowing the identification of proteins of interest based on peptide masses and sequence. In this case the peptides are most often generated by in-gel digestion after electrophoretic separation of the proteins.

Peptides have recently been used in the study of protein structure and function. For example, synthetic peptides can be used as probes to see where protein-peptide interactions occur- see the page on Protein tags.

Inhibitory peptides are also used in clinical research to examine the effects of peptides on the inhibition of cancer proteins and other diseases. For example, one of the most promising application is through peptides that target LHRH.These particular peptides act as an agonist, meaning that they bind to a cell in a way that regulates LHRH receptors. The process of inhibiting the cell receptors suggests that peptides could be beneficial in treating prostate cancer. However, additional investigations and experiments are required before the cancer-fighting attributes, exhibited by peptides, can be considered definitive.

Well-known peptide families

The peptide families in this section are ribosomal peptides, usually with hormonal activity. All of these peptides are synthesized by cells as longer "propeptides" or "proproteins" and truncated prior to exiting the cell. They are released into the bloodstream where they perform their signaling functions.

Tachykinin peptides
Main article: Tachykinin peptides
Substance P
Kassinin
Neurokinin A
Eledoisin
Neurokinin B
Vasoactive intestinal peptides
Main article: Secretin family
VIP (Vasoactive Intestinal Peptide; PHM27)
PACAP Pituitary Adenylate Cyclase Activating Peptide
Peptide PHI 27 (Peptide Histidine Isoleucine 27)
GHRH 1-24 (Growth Hormone Releasing Hormone 1-24)
Glucagon
Secretin

Pancreatic polypeptide-related peptides
NPY (NeuroPeptide Y)
PYY (Peptide YY)
APP (Avian Pancreatic Polypeptide)
PPY Pancreatic PolYpeptide
Opioid peptides[edit]
Main article: Opioid peptide
Proopiomelanocortin (POMC) peptides
Enkephalin pentapeptides
Prodynorphin peptides
Calcitonin peptides
Calcitonin
Amylin
AGG01
Other peptides
B-type Natriuretic Peptide (BNP) - produced in myocardium & useful in medical diagnosis
Lactotripeptides - Lactotripeptides might reduce blood pressure, although the evidence is mixed.[24]
Notes on terminology
Length:

A polypeptide is a single linear chain of many amino acids, held together by amide bonds.
A protein is one or more polypeptide (more than about 50 amino acids long).
An oligopeptide consists of only a few amino acids (between two and twenty).
Number of amino acids:

A monopeptide has one amino acid.
A dipeptide has two amino acids.
A tripeptide has three amino acids.
A tetrapeptide has four amino acids.
A pentapeptide has five amino acids.
A hexapeptide has six amino acids.
A heptapeptide has seven amino acids.
An octapeptide has eight amino acids (e.g., angiotensin II).
A nonapeptide has nine amino acids (e.g., oxytocin).
A decapeptide has ten amino acids (e.g., gonadotropin-releasing hormone & angiotensin I).
An undecapeptide (or monodecapeptide) has eleven amino acids, a dodecapeptide (or didecapeptide) has twelve amino acids, a tridecapeptide has thirteen amino acids, and so forth.
An icosapeptide has twenty amino acids, a tricontapeptide has thirty amino acids, a tetracontapeptide has forty amino acids, and so forth.
See also: IUPAC numerical multiplier
Function:

A neuropeptide is a peptide that is active in association with neural tissue.
A lipopeptide is a peptide that has a lipid connected to it, and pepducins are lipopeptides that interact with GPCRs.
A peptide hormone is a peptide that acts as a hormone.
A proteose is a mixture of peptides produced by the hydrolysis of proteins. The term is somewhat archaic.

Doping in sports
The term peptide has been incorrectly or unclearly used to mean illegal secretagogue and peptide hormones in sports doping matters: illegal secretagogue peptides are classified as Schedule 2 (S2) prohibited substances on the World Anti-Doping Agency (WADA) Prohibited List, and are therefore prohibited for use by professional athletes both in and out of competition. Such peptide secretagogues have been on the WADA prohibited substances list since at least 2008. The Australian Crime Commission (incorrectly using the term peptides) cited the alleged misuse of illegal peptide secretagogues used in Australian sport including growth hormone releasing peptides CJC-1295, GHRP-6, and GHSR (gene) hexarelin. There is ongoing controversy on the legality of using peptide secretagogues in sports.

Peptides (journal)

Peptides is a monthly peer-reviewed scientific journal covering the biochemistry, neurochemistry, pharmacology, and biological functions of peptides. It was established in 1980 and is published by Elsevier. The editor-in-chief is Karl-Heinz Herzig (University of Oulu).
Interferon activation of the transcription factor Stat91 involves dimerization through SH2-phosphotyrosyl peptide interactions.

Stat91 (a 91 kd protein that acts as a signal transducer and activator of transcription) is inactive in the cytoplasm of untreated cells but is activated by phosphorylation on tyrosine in response to a number of polypeptide ligands, including interferon alpha (IFN-alpha) and IFN-gamma. We report here that the inactive Stat91 in the cytoplasm of untreated cells is a monomer and that upon IFN-gamma-induced phosphorylation it forms a stable homodimer. Only the dimer is capable of binding to a specific DNA sequence directing transcription.
Through dissociation and reassociation assays, we show that dimerization of Stat91 is mediated through SH2-phosphotyrosyl peptide interactions. Dimerization involving SH2 recognition of specific phosphotyrosyl peptides may well provide a prototype for interactions among family members of STAT proteins to form different transcription complexes.

Binding of a high affinity phosphotyrosyl peptide to the Src SH2 domain: Crystal structures of the complexed and peptide-free forms

The crystal structure of the Src SH2 domain complexed with a high affinity 11-residue phosphopeptide has been determined at 2.7 A resolution by X-ray diffraction.
The peptide binds in an extended conformation and makes primary interactions with the SH2 domain at six central residues: PQ(pY)EEI. The phosphotyrosine and the isoleucine are tightly bound by two well-defined pockets on the protein surface, resulting in a complex that resembles a two-pronged plug engaging a two-holed socket.
The glutamate residues are in solvent-exposed environments in the vicinity of basic side chains of the SH2 domain, and the two N-terminal residues cap the phosphotyrosine-binding site. The crystal structure of Src SH2 in the absence of peptide has been determined at 2.5 A resolution, and comparison with the structure of the high affinity complex reveals only localized and relatively small changes.


The antigenic identity of peptide-MHC complexes: a comparison of the conformations of five viral peptides presented by HLA-A2.

Complexes of five peptides (from HIV-1, influenza A virus, HTLV-1, and hepatitis B virus proteins) bound to the human class I MHC molecule HLA-A2 have been studied by X-ray crystallography.
While the peptide termini and their second and C-terminal anchor side chains are bound similarly in all five cases, the main chain and side chain conformations of each peptide are strikingly different in the center of the binding site, and these differences are accessible to direct TCR recognition. Each of the central peptide residues is seen to point up for some bound peptides, but down or sideways for others.
Thus, although fixed at its ends, the structure of an MHC-bound peptide appears to be a highly complex function of its entire sequence, potentially sensitive to even small sequence differences. In contrast, MHC structural variation is relatively limited. These results offer a structural framework for understanding the role of nonanchor peptide side chains in both peptide-MHC binding affinity and TCR recognition.
Clearance of Alzheimer's Abeta peptide: the many roads to perdition.
The amyloid hypothesis of Alzheimer's disease (AD) maintains that the accumulation of the amyloid β protein (Aβ) is a critical event in disease pathogenesis.
A great deal of both academic and commercial research has focused on the mechanisms by which Aβ is generated. However, investigations into the mechanisms underlying Aβ clearance have blossomed over the last several years. This minireview will summarize pathways involved in the removal of cerebral Aβ, including enzymatic degradation and receptor-mediated efflux out of the brain.
The amyloid hypothesis of Alzheimer's disease (AD) maintains that the accumulation of the amyloid beta protein (Abeta) is a critical event in disease pathogenesis. A great deal of both academic and commercial research has focused on the mechanisms by which Abeta is generated. However, investigations into the mechanisms underlying Abeta clearance have blossomed over the last several years. This minireview will summarize pathways involved in the removal of cerebral Abeta, including enzymatic degradation and receptor-mediated efflux out of the brain.
Selective coupling of methotrexate to peptide hormone carriers through a gamma-carboxamide linkage of its glutamic acid moiety: benzotriazol-1-yloxytris
A convenient synthetic method is described for the preparation of peptide-methotrexate (MTX) conjugates in which MTX is coupled selectively through the gamma-carboxyl group of its glutamic acid moiety to a free amino group in peptide analogs. The syntheses of a somatostatin analog-MTX conjugate (MTX-D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2) (AN-51) and two conjugates of analogs of luteinizing hormone-releasing hormone (LH-RH) with MTX [Glp-His-Trp-Ser-Tyr-D-Lys(MTX)-Leu-Arg-Pro-Gly-NH2] (AJ-04) and [Ac-Ser-Tyr-D-Lys(MTX)-Leu-Arg-Pro-NH-Et] AJ-51 are presented as examples.
Benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent) was used in the synthesis for activation of 4-amino-4-deoxy-N10-methylpteroic acid, which reacted with the potassium salt of glutamic acid alpha-tert-butyl ester in dimethyl sulfoxide to form the suitably protected MTX derivative. This synthesis provides an example of the high suitability of BOP reagent for the salt-coupling method.
The selectively protected MTX derivative was then coupled to the different peptide carriers and deprotected under relatively mild conditions by trifluoroacetic acid. The conjugates of MTX with hormonal analogs are suitable for targeting to various tumors that possess receptors for the peptide moieties.
Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma.

A novel hypotensive peptide was discovered in human pheochromocytoma by monitoring the elevating activity of platelet cAMP. 
Since this peptide is abundant in normal adrenal medulla as well as in pheochromocytoma tissue arising from adrenal medulla, it was designated "adrenomedullin". 
The peptide, consisting of 52 amino acids, has one intramolecular disulfide bond and shows slight homology with calcitonin gene related peptide. It was found to elicit a potent and long lasting hypotensive effect. 
The peptide circulates in blood in a considerable concentration, but it was not found in brain. These data suggest that adrenomedullin is a new hormone participating in blood pressure control. Occurrence of adrenomedullin indicates the possible existence of a novel system for circulation control.

Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector.

Attempts to generate reliable and versatile vectors for gene therapy and biomedical research that express multiple genes have met with limited success. Here we used Picornavirus 'self-cleaving' 2A peptides, or 2A-like sequences from other viruses, to generate multicistronic retroviral vectors with efficient translation of four cistrons. 
Using the T-cell receptor:CD3 complex as a test system, we show that a single 2A peptide-linked retroviral vector can be used to generate all four CD3 proteins (CD3epsilon, gamma, delta, zeta), and restore T-cell development and function in CD3-deficient mice. 
We also show complete 2A peptide-mediated 'cleavage' and stoichiometric production of two fluorescent proteins using a fluorescence resonance energy transfer-based system in multiple cell types including blood, thymus, spleen, bone marrow and early stem cell progenitors.


Identification of an immunodominant peptide of HER-2/neu protooncogene recognized by ovarian tumor-specific cytotoxic T lymphocyte lines.

Synthetic peptide analogues of sequences in the HER-2 protooncogene (HER-2) were selected based on the presence of HLA-A2.
1 anchor motifs to identify the epitopes on HER-2 recognized by ovarian tumor-reactive CTL. 19 synthetic peptides were evaluated for recognition by four HLA-A2 ovarian-specific cytotoxic T lymphocyte (CTL) lines obtained from leukocytes associated with ovarian tumors. The nonapeptide E75 (HER-2, 369-377:KIFGSLAFL) was efficient in sensitizing T2 cells for lysis by all four CTL lines. This peptide was specifically recognized by cloned CD8+ CTL isolated from one of the ovarian-specific CTL lines. 
E75-pulsed T2 cells inhibited lysis by the same CTL clone of both an HLA-A2+ HER-2high ovarian tumor and a HER-2high cloned ovarian tumor line transfected with HLA-A2, suggesting that this or a structurally similar epitope may be specifically recognized by these CTL on ovarian tumors. Several other HER-2 peptides were recognized preferentially by one or two CTL lines, suggesting that both common and private HER-2 epitopes may be immunogenic in patients with ovarian tumors. 
Since HER-2 is a self-antigen, these peptides may be useful for understanding mechanisms of tumor recognition by T cells, immunological tolerance to tumor, and structural characterization of tumor antigens.


Extensive neurite outgrowth and active synapse formation on self-assembling peptide scaffolds.

A new type of self-assembling peptide (sapeptide) scaffolds that serve as substrates for neurite outgrowth and synapse formation is described. These peptide-based scaffolds are amenable to molecular design by using chemical or biotechnological syntheses. They can be tailored to a variety of applications. The sapeptide scaffolds are formed through the spontaneous assembly of ionic self-complementary beta-sheet oligopeptides under physiological conditions, producing a hydrogel material. The scaffolds can support neuronal cell attachment and differentiation as well as extensive neurite outgrowth. Furthermore, they are permissive substrates for functional synapse formation between the attached neurons. That primary rat neurons form active synapses on such scaffold surfaces in situ suggests these scaffolds could be useful for tissue engineering applications. The buoyant sapeptide scaffolds with attached cells in culture can be transported readily from one environment to another. Furthermore, these peptides did not elicit a measurable immune response or tissue inflammation when introduced into animals. These biological materials created through molecular design and self assembly may be developed as a biologically compatible scaffold for tissue repair and tissue engineering.


Comment on "Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin's effects on food intake".

Zhang et al. (Research Articles, 11 November 2005, p. 996) reported that obestatin, a peptide derived from the ghrelin precursor, activated the orphan G protein-coupled receptor GPR39. 
 We sought to evaluate B-type natriuretic peptide (BNP), alone and in comparison to cardiac troponin I (cTnI) and high-sensitivity C-reactive protein (hs-CRP), for risk assessment at initial presentation with ST-segment elevation myocardial infarction (STEMI). 
BACKGROUND: Elevated levels of BNP drawn two to four days after acute myocardial infarction are associated with higher mortality. Sparse data are available on its use at first presentation with STEMI. METHODS: We obtained samples from 438 patients presenting within 6 h of STEMI enrolled in the Enoxaparin Tenecteplase-Tissue-Type Plasminogen Activator With or Without Glycoprotein IIb/IIIa Inhibitor as Reperfusion Strategy in ST-Segment Elevation Myocardial Infarction (ENTIRE)-Thrombolysis In Myocardial Infarction (TIMI)-23 trial. 
However, we found that I125-obestatin does not bind GPR39 and observed no effects of obestatin on GPR39-transfected cells in various functional assays (cyclic adenosine monophosphate production, calcium mobilization, and GPR39 internalization). Our results indicate that obestatin is not the cognate ligand for GPR39.
Glucagon-like peptide I stimulates insulin gene expression and increases cyclic AMP levels in a rat islet cell line.

Insulin secretion is controlled by a complex set of factors. Although blood glucose levels serve as the major stimulus of insulin secretion in mammals, insulin release is also modulated by amino acids, catecholamines, glucagon, and other, intestinal hormones. 
The identification of factors that modulate insulin production has engendered much interest because of their potential importance in the altered dynamics of insulin secretion in response to glucose characteristic of maturity-onset diabetes mellitus. Decoding of the glucagon gene has uncovered two additional glucagon-like peptides encoded in proglucagon, the polypeptide precursor of glucagon. One of these peptides, glucagon-like peptide I, is processed from proglucagon in two forms, of 31 and 37 amino acids. 
We report that the smaller of the two glucagon-like peptides potently increases cAMP levels, insulin mRNA transcripts, and insulin release in cultured rat insulinoma cells. These results indicate that glucagon-like peptide I may be a physiologic modulator of insulin gene expression.

Use of biosynthetic human C-peptide in the measurement of insulin secretion rates in normal volunteers and type I diabetic patients.

We undertook this study to examine the accuracy of plasma C-peptide as a marker of insulin secretion. The peripheral kinetics of biosynthetic human C-peptide (BHCP) were studied in 10 normal volunteers and 7 insulin-dependent diabetic patients. 
Each subject received intravenous bolus injections of BHCP as well as constant and variable rate infusions. After intravenous bolus injections the metabolic clearance rate of BHCP (3.8 +/- 0.1 ml/kg per min, mean +/- SEM) was not significantly different from the value obtained during its constant intravenous infusion (3.9 +/- 0.1 ml/kg per min). The metabolic clearance rate of C-peptide measured during steady state intravenous infusions was constant over a wide concentration range. 
During experiments in which BHCP was infused at a variable rate, the peripheral concentration of C-peptide did not change in proportion to the infusion rate. Thus, the infusion rate of BHCP could not be calculated accurately as the product of the C-peptide concentration and metabolic clearance rate. However, the non-steady infusion rate of BHCP could be accurately calculated from peripheral C-peptide concentrations using a two-compartment mathematical model when model parameters were derived from the C-peptide decay curve in each subject. 
Application of this model to predict constant infusions of C-peptide from peripheral C-peptide concentrations resulted in model generated estimates of the C-peptide infusion rate that were 101.5 +/- 3.4% and 100.4 +/- 2.8% of low and high dose rates, respectively. 
Estimates of the total quantity of C-peptide infused at a variable rate over 240 min based on the two-compartment model represented 104.6 +/- 2.4% of the amount actually infused. Application of this approach to clinical studies will allow the secretion rate of insulin to be estimated with considerable accuracy. The insulin secretion rate in normal subjects after an overnight fast was 89.1 pmol/min, which corresponds with a basal 24-h secretion of 18.6 U.


Measurement of fibrinopeptide A in human blood.

Since thrombin cleaves fibrinopeptides A (FPA) and B from the NH(2)-terminal end of the fibrinogen molecule, measurement of fibrinopeptide levels in plasma may provide a direct index of thrombin action. Recently a radioimmunoassay for FPA has been developed, and in the present paper, we describe the application of this assay to the measurement of FPA levels in clinical blood samples. Since fibrinogen cross-reacts with antibodies to FPA, dialysis was used to extract the peptide from plasma. 
In vitro generation of FPA was prevented by removing the fibrinogen from the plasma by precipitation with ethanol before dialysis. The processing technique permitted recovery of 75% of FPA added to blood in vitro. Evidence that the immunoreactivity measured in plasma is due to FPA was provided by the results of experiments in which two antisera to FPA with different specificities showed comparable results and addition of thrombin caused no change in immunoreactivity. 
In contrast, extracts of streptokinasetreated plasma showed a five-fold increase in activity when treated with thrombin and markedly different immunoreactivity with the two antisera. Plasma FPA levels in 30 normal men were below 2 ng/ml, with a mean of 0.5 ng/ml. FPA levels in 12 patients with reduced fibrinogen levels or reduced platelet counts or both ranged between 4 and 289 ng/ml. FPA levels in 13 patients with normal or elevated fibrinogen levels, including 6 patients with clinical evidence of venous thrombosis or pulmonary embolism or both, ranged between 5 and 23 ng/ml. FPA and fibrinogen degradation product levels did not correlate, and in several patients, elevated FPA levels were found in the presence of normal fibrinogen degradation product levels. 
After infusion of FPA-containing solutions in four normal individuals, FPA showed a disappearance rate from the plasma consistent with a t((1/2)) of 3-5 min. Heparin infusions in six patients with venous thrombosis or pulmonary embolism or both and elevated FPA levels were followed by a prompt decline in FPA level at a mean rate equivalent to a 3-5 min t((1/2)).


Calcitonin gene-related peptide coexists with substance P in capsaicin sensitive neurons and sensory ganglia of the rat.

Immunohistochemical and radioimmunoassay studies revealed that both CGRP- and SP-like immunoreactivity in the caudal spinal trigeminal nucleus and tract, the substantia gelatinosa and the dorsal cervical spinal cord as well as in cell bodies of the trigeminal ganglion and the spinal dorsal root ganglion is markedly depleted by capsaicin which is known to cause degeneration of a certain number of primary sensory neurons. 
Higher brain areas and the ventral spinal cord were not affected by capsaicin treatment. Furthermore CGRP and substance P-like immunoreactivity were shown to be colocalized in the above areas and to coexist in cell bodies of the trigeminal ganglion and the spinal dorsal root ganglia. It is suggested that CGRP, like substance P, may have a neuromodulatory role on nociception and peripheral cardiovascular reflexes.


Pancreatic beta-cells are rendered glucose-competent by the insulinotropic hormone glucagon-like peptide-1(7-37).

Non--dependent (, ) is a disorder of characterized by , peripheral resistance, impaired hepatic , and diminished -dependent of from pancreatic beta-cells. () is an intestinally derived hormone that may be useful for the treatment of because it acts in vivo to increase the level of circulating , and thus lower the concentration of blood . 
This therapeutic effect may result from the ability of to compensate for a defect in the signalling pathway that regulates from beta-cells. In support of this concept we report here that confers sensitivity to -resistant beta-cells, a phenomenon we term competence. 
Induction of competence by results from its synergistic interaction with to inhibit metabolically regulated channels that are also targeted for inhibition by sulphonylurea drugs commonly used in the treatment of . competence allows depolarization, the , and + influx, events that are known to trigger.

Glucagon-like peptide I stimulates insulin gene expression and increases cyclic AMP levels in a rat islet cell line.

Insulin secretion is controlled by a complex set of factors. Although blood glucose levels serve as the major stimulus of insulin secretion in mammals, insulin release is also modulated by amino acids, catecholamines, glucagon, and other, intestinal hormones. 
The identification of factors that modulate insulin production has engendered much interest because of their potential importance in the altered dynamics of insulin secretion in response to glucose characteristic of maturity-onset diabetes mellitus. Decoding of the glucagon gene has uncovered two additional glucagon-like peptides encoded in proglucagon, the polypeptide precursor of glucagon. One of these peptides, glucagon-like peptide I, is processed from proglucagon in two forms, of 31 and 37 amino acids. 
We report that the smaller of the two glucagon-like peptides potently increases cAMP levels, insulin mRNA transcripts, and insulin release in cultured rat insulinoma cells. These results indicate that glucagon-like peptide I may be a physiologic modulator of insulin gene expression.


Use of biosynthetic human C-peptide in the measurement of insulin secretion rates in normal volunteers and type I diabetic patients.

We undertook this study to examine the accuracy of plasma C-peptide as a marker of insulin secretion. The peripheral kinetics of biosynthetic human C-peptide (BHCP) were studied in 10 normal volunteers and 7 insulin-dependent diabetic patients. 
Each subject received intravenous bolus injections of BHCP as well as constant and variable rate infusions. After intravenous bolus injections the metabolic clearance rate of BHCP (3.8 +/- 0.1 ml/kg per min, mean +/- SEM) was not significantly different from the value obtained during its constant intravenous infusion (3.9 +/- 0.1 ml/kg per min). The metabolic clearance rate of C-peptide measured during steady state intravenous infusions was constant over a wide concentration range. 
During experiments in which BHCP was infused at a variable rate, the peripheral concentration of C-peptide did not change in proportion to the infusion rate. Thus, the infusion rate of BHCP could not be calculated accurately as the product of the C-peptide concentration and metabolic clearance rate. However, the non-steady infusion rate of BHCP could be accurately calculated from peripheral C-peptide concentrations using a two-compartment mathematical model when model parameters were derived from the C-peptide decay curve in each subject. 
Application of this model to predict constant infusions of C-peptide from peripheral C-peptide concentrations resulted in model generated estimates of the C-peptide infusion rate that were 101.5 +/- 3.4% and 100.4 +/- 2.8% of low and high dose rates, respectively. 
Estimates of the total quantity of C-peptide infused at a variable rate over 240 min based on the two-compartment model represented 104.6 +/- 2.4% of the amount actually infused. Application of this approach to clinical studies will allow the secretion rate of insulin to be estimated with considerable accuracy. The insulin secretion rate in normal subjects after an overnight fast was 89.1 pmol/min, which corresponds with a basal 24-h secretion of 18.6 U.


Measurement of fibrinopeptide A in human blood.

Since thrombin cleaves fibrinopeptides A (FPA) and B from the NH(2)-terminal end of the fibrinogen molecule, measurement of fibrinopeptide levels in plasma may provide a direct index of thrombin action. Recently a radioimmunoassay for FPA has been developed, and in the present paper, we describe the application of this assay to the measurement of FPA levels in clinical blood samples. Since fibrinogen cross-reacts with antibodies to FPA, dialysis was used to extract the peptide from plasma. 
In vitro generation of FPA was prevented by removing the fibrinogen from the plasma by precipitation with ethanol before dialysis. The processing technique permitted recovery of 75% of FPA added to blood in vitro. Evidence that the immunoreactivity measured in plasma is due to FPA was provided by the results of experiments in which two antisera to FPA with different specificities showed comparable results and addition of thrombin caused no change in immunoreactivity. 
In contrast, extracts of streptokinasetreated plasma showed a five-fold increase in activity when treated with thrombin and markedly different immunoreactivity with the two antisera. Plasma FPA levels in 30 normal men were below 2 ng/ml, with a mean of 0.5 ng/ml. FPA levels in 12 patients with reduced fibrinogen levels or reduced platelet counts or both ranged between 4 and 289 ng/ml. FPA levels in 13 patients with normal or elevated fibrinogen levels, including 6 patients with clinical evidence of venous thrombosis or pulmonary embolism or both, ranged between 5 and 23 ng/ml. FPA and fibrinogen degradation product levels did not correlate, and in several patients, elevated FPA levels were found in the presence of normal fibrinogen degradation product levels. 
After infusion of FPA-containing solutions in four normal individuals, FPA showed a disappearance rate from the plasma consistent with a t((1/2)) of 3-5 min. Heparin infusions in six patients with venous thrombosis or pulmonary embolism or both and elevated FPA levels were followed by a prompt decline in FPA level at a mean rate equivalent to a 3-5 min t((1/2)).


Calcitonin gene-related peptide coexists with substance P in capsaicin sensitive neurons and sensory ganglia of the rat.

Immunohistochemical and radioimmunoassay studies revealed that both CGRP- and SP-like immunoreactivity in the caudal spinal trigeminal nucleus and tract, the substantia gelatinosa and the dorsal cervical spinal cord as well as in cell bodies of the trigeminal ganglion and the spinal dorsal root ganglion is markedly depleted by capsaicin which is known to cause degeneration of a certain number of primary sensory neurons. 
Higher brain areas and the ventral spinal cord were not affected by capsaicin treatment. Furthermore CGRP and substance P-like immunoreactivity were shown to be colocalized in the above areas and to coexist in cell bodies of the trigeminal ganglion and the spinal dorsal root ganglia. It is suggested that CGRP, like substance P, may have a neuromodulatory role on nociception and peripheral cardiovascular reflexes.


Pancreatic beta-cells are rendered glucose-competent by the insulinotropic hormone glucagon-like peptide-1(7-37).

Non--dependent (, ) is a disorder of characterized by , peripheral resistance, impaired hepatic , and diminished -dependent of from pancreatic beta-cells. () is an intestinally derived hormone that may be useful for the treatment of because it acts in vivo to increase the level of circulating , and thus lower the concentration of blood . 
This therapeutic effect may result from the ability of to compensate for a defect in the signalling pathway that regulates from beta-cells. In support of this concept we report here that confers sensitivity to -resistant beta-cells, a phenomenon we term competence. 
Induction of competence by results from its synergistic interaction with to inhibit metabolically regulated channels that are also targeted for inhibition by sulphonylurea drugs commonly used in the treatment of . competence allows depolarization, the , and + influx, events that are known to trigger.
RAGE and amyloid-beta peptide neurotoxicity in Alzheimer's disease.

Amyloid-beta peptide is central to the pathology of Alzheimer's disease, because it is neurotoxic--directly by inducing oxidant stress, and indirectly by activating microglia. 
A specific cell-surface acceptor site that could focus its effects on target cells has been postulated but not identified. Here we present evidence that the 'receptor for advanced glycation end products' (RAGE) is such a receptor, and that it mediates effects of the peptide on neurons and microglia. Increased expressing of RAGE in Alzheimer's disease brain indicates that it is relevant to the pathogenesis of neuronal dysfunction and death.
A beta peptide immunization reduces behavioural impairment and plaques in a model of Alzheimer's disease.

Much evidence indicates that abnormal processing and extracellular deposition of amyloid-β peptide (Aβ), a proteolytic derivative of the β-amyloid precursor protein (βAPP), is central to the pathogenesis of Alzheimer's disease (reviewed in ref. 1). 
In the PDAPP transgenic mouse model of Alzheimer's disease, immunization with Aβ causes a marked reduction in burden of the brain amyloid. 
Evidence that Aβ immunization also reduces cognitive dysfunction in murine models of Alzheimer's disease would support the hypothesis that abnormal Aβ processing is essential to the pathogenesis of Alzheimer's disease, and would encourage the development of other strategies directed at the `amyloid cascade'. 
Here we show that Aβ immunization reduces both deposition of cerebral fibrillar Aβ and cognitive dysfunction in the TgCRND8 murine model of Alzheimer's disease without, however, altering total levels of Aβ in the brain. This implies that either a ~50% reduction in dense-cored Aβ plaques is sufficient to affect cognition, or that vaccination may modulate the activity/abundance of a small subpopulation of especially toxic Aβ species.


Adrenomedullin, a multifunctional regulatory peptide.

Abstract Since the discovery of adrenomedullin in 1993 several hundred papers have been published regarding the regulation of its secretion and the multiplicity of its actions. It has been shown to be an almost ubiquitous peptide, with the number of tissues and cell types synthesizing adrenomedullin far exceeding those that do not. 
In Section II of this paper we give a comprehensive review both of tissues and cell lines secreting adrenomedullin and of the mechanisms regulating gene expression. The data on circulating adrenomedullin, obtained with the various assays available, are also reviewed, and the disease states in which plasma adrenomedullin is elevated are listed. 
In Section III the pharmacology and biochemistry of adrenomedullin binding sites, both specific sites and calcitonin gene-related peptide (CGRP) receptors, are discussed. In particular, the putative adrenomedullin receptor clones and signal transduction pathways are described. In Section IV the various actions of adrenomedullin are discussed: its actions on cellular growth, the cardiovascular system, the central nervous system, and the endocrine system are all considered. Finally, in Section V, we consider some unresolved issues and propose future areas for research.


Glucagon-like peptide 1 promotes satiety and suppresses energy intake in humans.

We examined the effect of intravenously infused glucagon-like peptide 1 (GLP-1) on subjective appetite sensations after an energy-fixed breakfast, and on spontaneous energy intake at an ad libitum lunch. 20 young, healthy, normal-weight men participated in a placebo-controlled, randomized, blinded, crossover study. Infusion (GLP-1, 50 pmol/ kg.h or saline) was started simultaneously with initiation of the test meals. Visual analogue scales were used to assess appetite sensations throughout the experiment and the palatability of the test meals. 
Blood was sampled throughout the day for analysis of plasma hormone and substrate levels. After the energy-fixed breakfast, GLP-1 infusion enhanced satiety and fullness compared with placebo (treatment effect: P < 0.03). Furthermore, spontaneous energy intake at the ad libitum lunch was reduced by 12% by GLP-1 infusion compared with saline (P = 0.002). Plasma GLP-1, insulin, glucagon, and blood glucose profiles were affected significantly by the treatment (P < 0.002). In conclusion, the results show that GLP-1 enhanced satiety and reduced energy intake and thus may play a physiological regulatory role in controlling appetite and energy intake in human


Peptide-pulsed dendritic cells induce antigen-specific CTL-mediated protective tumor immunity.

Cytotoxic T lymphocytes (CTLs) are a critical component of the immune response to tumors. Tumor-derived peptide antigens targeted by CTLs are being defined for several human tumors and are potential immunogens for the induction of specific antitumor immunity. Dendritic cells (DC) are potent antigen-presenting cells (APCs) capable of priming CTL responses in vivo. Here we show that major histocompatibility complex class I-presented peptide antigen pulsed onto dendritic APCs induces protective immunity to lethal challenge by a tumor transfected with the antigen gene. The immunity is antigen specific, requiring expression of the antigen gene by the tumor target, and is eliminated by in vivo depletion of CD8+ T cells. Furthermore, mice that have rejected the transfected tumor are protected from subsequent challenge with the untransfected parent tumor. These results suggest that immunization strategies using antigen-pulsed DC may be useful for inducing tumor-specific immune responses.


Human beta-defensin 2 is a salt-sensitive peptide antibiotic expressed in human lung.

Previous studies have implicated the novel peptide antibiotic human beta-defensin 1 (hBD-1) in the pathogenesis of cystic fibrosis. We describe in this report the isolation and characterization of the second member of this defensin family, human beta-defensin 2 (hBD-2). A cDNA for hBD-2 was identified by homology to hBD-1. 
hBD-2 is expressed diffusely throughout epithelia of many organs, including the lung, where it is found in the surface epithelia and serous cells of the submucosal glands. A specific antibody made of recombinant peptide detected hBD-2 in airway surface fluid.
The paper discusses general problems in using PEG for conjugation to high or low molecular weight molecules. Methods of binding PEG to different functional groups in macromolecules is reported together with their eventual limitations. Problems encountered in conjugation, such as the evaluation of the number of PEG chains bound, the localisation of the site of conjugation in polypeptides and the procedure to direct PEGylation to the desired site in the molecule are discussed. 
Finally, the paper reports on more specific methods regarding reversible PEGylation, cross-linking reagents with PEG arms, PEG for enzyme solubilization in organic solvent and new polymers as alternative to PEG.

Early prediction of severity in acute pancreatitis by urinary trypsinogen activation peptide: a multicentre study.

At 24 h after symptom onset, the median urinary TAP concentration was 37 nmol/L (IQR 17-110) for severe and 15 nmol/L (5-35) for mild disease (p<0.001).There is a pressing clinical requirement for an early simple test of severity in acute pancreatitis. 
We investigated the use of an assay of trypsinogen activation peptide (TAP).We undertook a multicentre study in 246 patients (172 with acute pancreatitis [35 with severe disease], 74 controls). We assessed the predictive value of urinary TAP concentrations measured by a validated competitive immunoassay. We compared the results with those for plasma C-reactive protein and three clinicobiochemical scoring systems. 
TAP and C-reactive protein concentrations were analysed at set times after symptom onset and compared with the clinicobiochemical systems scores at key times during hospital stay. The respective values for plasma C-reactive protein were 24 mg/L (3-34) and 25 mg/L (6-75; p=0.208). The sensitivity, specificity, positive predictive, and negative predictive values of the test to show severe acute pancreatitis compared with mild acute pancreatitis at 24 h were: for TAP (>35 nmol/L), 58%, 73%, 39%, and 86%, respectively, and for C-reactive protein (>150 mg/L), 0%, 90%, 0%, and 75%. 48 h after admission the values for the clinicobiochemical scoring systems were: APACHE II (> or =8), 56%, 64%, 30%, and 85%; Ranson score (> or =3), 89%, 64%, 38%, and 96%; and Glasgow score (> or =3), 77%, 75%, 44%, and 93%. 
At 48 h, the values for C-reactive protein were 86%, 61%, 37%, and 94% and for TAP were 83%, 72%, 44%, and 94%. Combined testing of C-reactive protein and TAP was not superior to TAP alone for accuracy.Urinary TAP provided accurate severity prediction 24 h after onset of symptoms. This single marker of severity in acute pancreatitis deserves routine clinical application.

Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector.

Attempts to generate reliable and versatile vectors for gene therapy and biomedical research that express multiple genes have met with limited success. Here we used Picornavirus 'self-cleaving' 2A peptides, or 2A-like sequences from other viruses, to generate multicistronic retroviral vectors with efficient translation of four cistrons. 
Using the T-cell receptor:CD3 complex as a test system, we show that a single 2A peptide-linked retroviral vector can be used to generate all four CD3 proteins (CD3epsilon, gamma, delta, zeta), and restore T-cell development and function in CD3-deficient mice. We also show complete 2A peptide-mediated 'cleavage' and stoichiometric production of two fluorescent proteins using a fluorescence resonance energy transfer-based system in multiple cell types including blood, thymus, spleen, bone marrow and early stem cell progenitors.

Plasma brain natriuretic peptide as a biochemical marker of high left ventricular end-diastolic pressure in patients with symptomatic left ventricular dysf...
Background: Plasma atrial natriuretic peptide (ANP), mainly from the atrium, brain natriuretic peptide (BNP), mainly from the ventricle, norepinephrine (NE), and endothelin-1 (ET-1) levels are increased with the severity of congestive heart failure (CHF). 
Although a close correlation between the left ventricular end-diastolic pressure (LVEDP) and plasma ANP in patients with left ventricular dysfunction has been reported, it is not yet known which cardiac natriuretic peptide is a better predictor of high LVEDP in patients with CHF. Methods: To investigate the biochemical predictors of the high LVEDP in patients with left ventricular dysfunction, we measured plasma ANP, BNP, NE, and ET-1 levels and the hemodynamic parameters in 72 patients with symptomatic left ventricular dysfunction. 
Stepwise multivariate regression analyses were also used to determine whether the plasma levels of ANP, BNP, NE, and ET-1 could predict high LVEDP. Results: Although significant positive correlations were found among the plasma levels of ANP, BNP, ET-1, and NE and the LVEDP, only BNP ( p = 0.0001) was an independent and significant predictor of high LVEDP in patients with CHF. In all eight patients with severe CHF measured for hemodynamics before and after the treatments, the plasma BNP levels decreased in association with the decrease of LVEDP, whereas other factors increased in some patients despite the decrease of LVEDP. 
Conclusions: These findings suggest that plasma BNP is superior to ANP as a predictor of high LVEDP in patients with symptomatic left ventricular dysfunction. (Am Heart J 1998;135:825-32.)


Cutting edge: 1,25-dihydroxyvitamin D3 is a direct inducer of antimicrobial peptide gene expression.

The hormonal form of vitamin D(3), 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), is an immune system modulator and induces expression of the TLR coreceptor CD14. 1,25(OH)(2)D(3) signals through the vitamin D receptor, a ligand-stimulated transcription factor that recognizes specific DNA sequences called vitamin D response elements. In this study, we show that 1,25(OH)(2)D(3) is a direct regulator of antimicrobial innate immune responses. The promoters of the human cathelicidin antimicrobial peptide (camp) and defensin beta2 (defB2) genes contain consensus vitamin D response elements that mediate 1,25(OH)(2)D(3)-dependent gene expression. 1,25(OH)(2)D(3) induces antimicrobial peptide gene expression in isolated human keratinocytes, monocytes and neutrophils, and human cell lines, and 1,25(OH)(2)D(3) along with LPS synergistically induce camp expression in neutrophils. Moreover, 1,25(OH)(2)D(3) induces corresponding increases in antimicrobial proteins and secretion of antimicrobial activity against pathogens including Pseudomonas aeruginosa. 1,25(OH)(2)D(3) thus directly regulates antimicrobial peptide gene expression, revealing the potential of its analogues in treatment of opportunistic infections.
Magainin Spacer Peptide Angiotensin II Antipeptide Angiotensin III Antipeptide CD36 Peptide P (139-155) CD36 Peptide P (93-110) Anti-Inflammatory Peptide 1 Anti-Inflammatory Peptide 3 Cecropin A-melittin hybrid peptide [CA(1-7)M(2-9)NH2] Anti-Inflammatory Peptide 2 Cecropin A-melittin hybrid peptide [CA(1-7)M(2-9)NH2] [Tyr0]-C-Peptide, human C-Peptide, dog Tyr-C-Peptide, dog Proinsulin C-Peptide (55-89), ?human Gastric Inhibitory Peptide (1-30), amide, porcine Gastric Inhibitory Peptide (GIP), human Gastric Inhibitory Peptide, porcine Glucagon-Like Peptide II, rat Glucagon-Like Peptide II, rat GLP-1/Glucagon-Like Peptide, amide, human Laminin Penta Peptide, amide Mast Cell Degranulating Peptide HR-2 Mast Cell Degranulating Peptide Mast Cell Degranulating Peptide HR-1 OVA peptide (257-264) Vasonatrin Peptide (1-27) Atrial Natriuretic Peptide (3-28), human Atrial Natriuretic Peptide (1-28), rat Neuropeptide S (1-10) (human) Neuropeptide W-23 (human) opeptide W-30 (rat) Neuropeptide S (human) Neuropeptide W-30 (human) Neuropeptide S (rat) Neuropeptide W-23 (human) Neuropeptide FF Neuropeptide W-30 (human) Brain Injury Derived Neurotrophic Peptide opeptide W-30 (rat) Neuropeptide FF Neuropeptide S (1-10) (human) Neuropeptide S (rat) Brain Injury Derived Neurotrophic Peptide Neuropeptide K, porcine Head Activator Neuropeptide Neuron Specific Peptide Neuropeptide K, porcine
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