Saturday, March 28, 2009

Large Scale Custom Peptide Synthesis

Synthetic peptides have proven to be very valuable in most biomedical laboratories; since the mid-60’s , to the present biologically active peptides have been integrated into the growing number of API’s (active pharmaceutical ingredients) and as stand alone products also( Vasopressin, Gonadorelin, Leuprolide, Goserelin.among others); with the completion of the human genome, and the confirmation of abut 30,000 proteins produced in the human body, there are now many more targets for the biomedical researcher to explore.

Generally the biomedical researcher will explore the possibility to increase the native potency of a given peptide/protein, by rationale design; to this end. Small amounts of peptide are needed, more likely in the milligram scale. Once a desired activity/potency is found, then a larger scale of synthesis is need, usually in the gram/grams scale, in order to initiate small animal studies. Upon successful validation, then even larger scale of synthesis are desired, this can range from hundreds of grams to multikilo amounts.Bio-Synthesis has over 25 years of experience in the custom synthesis, from research grade scale, all the way to multikilo amounts.

Large scale peptide synthesis can be carried out either in solution or in solid phase; generally peptides shorter than 8 amino acids are prepared by solution chemistry, as it is usually more economical to do so. Peptides larger than 8 residues are generally assembled by solid phase chemistry; commercially peptides up to about 50 residues in length; once a pilot run is established and it is determined that a high load resin can work(about 1 mmmol/gram) good yields can be obtained for short peptides (8-20); above 20 residues a good balance of resin loading and possible steric hindrance problems needs to be analyzed. Solid phase synthesis can be carried out either in a fully automated fashion or manually; again manual synthesis for short peptides have the advantages of being able to scale up with more flexibility and be more able to troubleshoot unexpected problems; the operator can wash away piperidine during Fmoc deprotection, in the event of power failure or machine troubles (in the case of an automated instrument). Also the thermodymamic mixing can be better controlled in manual systems. We are not saying that manual systems are better, we are saying that for shorter and multikilo (10-20 kgs runs) solid manual methods are a good alternative. Large scale fully automated peptide instruments, have the obvious advantage of unattended operation and extensive documentation and for longer and mid-scale synthesis provide a generally reliable venue.

Large scale peptide synthesis manufactured under GMP conditions will also include the following documentation, as part of the quality control assays:

Bio-Synthesis has been producing synthetic peptides for over 25 years. Our expertise in custom synthetic polypeptide manufacturing allows us to produce the high-quality, large-scale, and GMP peptides with the highest success rate with long standing records. We have been delivered more than 100,000 peptides to customers worldwide, including very hydrophobic polypeptide, peptide with multiple disulfide bonds, multi-phosph0rylated peptides and extremely long peptides. Our large scale non-GMP ever delivered 5 kilograms of peptides on a single order and has the capacity of 10,000 peptides per month. Our capacity of GMP peptide is 10 kilograms.
Whenever your research work may require large scale synthesis of peptides, please keep us in mind, Bio-Synthesis Inc. staff will be glad to help you achieve your research/production goals.

Use of Bioconjugates in Apoptosis

The study of apotosis, the programmed death of cells, has direct applications to cancer a disease where tumor cells have developed mechanisms to avoid it and multiply without control. The fact that apoptosis is usually mediated by some cell receptors makes conjugates a valuable tool in elucidating apoptosis’ mechanisms as well drug development as the following examples show:

* For instance a conjugate of anti-CD33 antibody and the amphipathic peptide KLA target efficiently CD33-positive myeloid leukemia cell lines to cause their apoptotic death induced by the D-(KLAKLAK)2 proapoptotic peptide.
* Cells over-expressing erbB2 and resistant to apoptosis can be killed more efficiently by a conjugate composed of an erbB2-binding heptapeptide conjugated to the proapoptotic a-tocopheryl succinate (a-TOS) rather than the unconjugated a-TOS. Use of the conjugate resulted in breast carcinomas in a breast cancer prone transgenic mouse strain.
* Recently it has been shown that conjugates of cytochrome c and oligoarginine linked by a thioether resulted in an increased entry into the cell, compared to cyt. c alone, and increase in apoptotic activity. In contrast a conjugate linked by a disulfide bond although entered into the cell did not enhance the apoptotic activity.
* In some cases the carrier moiety can be a synthetic polymer such as poly-L-glutamic acid to which the drug paclitaxel has been conjugated. The actual mechanism of action is still being elucidated. Another carrier that has been used successfully are the cyclodextrins to which small pro-apoptotic agents can be linked.

Bio-Synthesis has acquired years of experience in chemical conjugation of peptides, porteins, oligonucleotides, lipid, bifunctional ligands, antibodies and other biological molecules onto solid surfaces. Our goal is to be your one expert source when seeking new solutions from early drug discovery to delivery. Contact us now!

Monday, March 23, 2009

Use of Conjugates in Signal Transduction

Many of the processes occurring in multi-cellular organisms are regulated via a complex network of extra-cellular signals and cell surface receptors. Attachment of the signal compound to its specific cell surface receptor results in conveying a signal through the receptor to the interior of the cell to start a reaction(s) until the signal is switched off. There is a large variety of signals, such as hormones, oligosaccharides, lipids, proteins and so on, while all of the receptors are of the protein transmembrane type, such as G protein-coupled receptors and receptor-tyrosine kinases. An effective way to elucidate the stimulatory mechanisms is by using compounds that have only the region(s) critical for linkage to the receptor and usually covalently bound to a reporter group such a fluorescent tag or an enzyme forming a conjugate. Identification in signal compounds of the regions critical for binding to their receptors, has allowed the development of agonist and antagonist compounds of those receptors that may have potential therapeutic applications Following is a description of some of these compounds, their structures and mode of action.

* Hyaluronic acid (HA) peptide conjugates for formyl peptide receptor like 1 (FPRL1) receptor. FPRL1 is a G protein-receptor that in phagocytes binds chemotactic peptides, some of them formylated, and mediate important biological functions, e.g. regulation of immune responses against pathogens by engulfing and destroying bacteria. These peptidic ligands can be either agonistic or antagonistic and therefore have a significant therapeutic potential. A potential application of antagonistic peptides would be the down-regulation of immunity to reduce inflammatory disease, e.g. arthritis and asthma. Recently it has been shown that conjugation of the peptide agonists or antagonists to HA results in good serum stability suitable for clinical use. The peptides were linked to the HA via a Michael addition reaction between a methacrylate group added to the HA and the thiol group from a cysteine residue present in the peptides. The efficacy of the conjugates increased if the HA size was reduced by enzymatic degradation, indicating some steric hindrance by large HA chains.
* The study of glycopeptide ligands to cell surface receptors has been advanced by the use of neoglycopolymers of which neoglycopeptides are an example. For instance, while some monomeric oligosaccharide act as inhibitors of L-selectin shedding, attachment of multiple copies of that oligosaccharide to a polymeric carrier, usually a polypeptide, resulted in the selectin’s shedding but independent of activation. An elegant example of how conjugates can help to dissociate the functions of the different moieties of a ligand.
* Lipid-modified peptides. Signal transducing proteins frequently have covalently attached lipids required for their biological functions. Lipidation of peptides derived from some G protein-coupled receptors, e.g. ras proteins, has provided information about the effectiveness of different lipid residues. For instance, it has been shown that geranylgeranylated or palmitoylated peptides have a higher membrane affinity than their analogous myristoylated or farnesylated peptides. Also increasing the degree of lipidation of a peptide showed that addition of a second lipid residue resulted in its stable insertion in the membrane. Thus, like in the glycosylation case the addition of lipids to model peptides allow the elucidation of the roles for the different protein’s moieties.
* Oligonucleotide-peptide conjugates. The objective of signal transduction is to stimulate a cell response that is usually the expression of a protein by transcription of DNA. In some viruses replication the formation of nucleopeptides is an obligatory step, also some processing of DNA by certain enzymes require the formation of intermediary nucleopeptides. Usually oligonucleotides are linked via the hydroxyl group of tyrosine, serine or threonine, either directly or via a spacer, depending on the requirements of the system. Synthesis of these conjugates is usually carried out by cross-linking an oligonucleotide to a peptide where both components had been prepared separately.

Use of conjugates in elucidating the roles on the different components that participate in signal transduction is a powerful tool in the development of new therapeutic agents to up-regulate or down-regulate certain biological responses. The design and production of these compounds frequently requires a deep knowledge of the chemistry used in their synthesis. BioSynthesis, with 25 years of experience in the areas of peptide and nucleic acid synthesis is well positioned to assist any researcher in the design and production of these agents.

Saturday, March 21, 2009

Large Scale Custom Peptide Synthesis

Peptide applications play an important role in biochemistry, molecular biology, immunology and medicine. In the human body, most if not all biological/physiological processes are regulated by various forms of molecular recognition. Most of these processes involve initiation or inhibition trough protein-protein interaction. As we know peptides and proteins due to the vast number of conformational possibilities are ideal to carry out such complex control functions.

Synthetic peptides have been widely used for the following purposes:

* Structure-function studies of polypeptides,
* As peptide hormones and hormone analogues,
* In the preparation of cross-reacting antibodies,
* In the design of novel enzyme
* And in drug discovery.

The last 40 years have seen an enormous growth in the methodologies available to obtain peptide and protein molecules. Through recombinant methods, most labs can now assemble genes, subcloned into expression vectors and obtain a wide range of endogenous proteins; likewise the pioneering work of Bruce Merrifield, makes it possible to obtain large scale with multikilo amounts of a number of biologically active peptides.
Bio-Synthesis has been producing synthetic peptides for over 25 years. Our expertise in custom synthetic polypeptide manufacturing allows us to produce the high-quality, large-scale, and GMP peptides with the highest success rate with long standing records. We have been delivered more than 100,000 peptides to customers worldwide, including very hydrophobic polypeptide, peptide with multiple disulfide bonds, multi-phosph0rylated peptides and extremely long peptides. Our large scale non-GMP ever delivered 5 kilograms of peptides on a single order and has the capacity of 10,000 peptides per month. Our capacity of GMP peptide 10 kilograms. Contact us more information on large scale custom peptide synthesis

Mass Spectrometry of Biomolecules

Mass spectrometers used for the analysis of macromolecules from biological sources, such as proteins, peptides, DNA or RNA oligomers, have experienced tremendious improvements in recent years. Electrospray ionization mass spectrometry (ESI-MS) and matrix assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) have become the methods of choice. Both techniques are powerful analytical tools by themself, but are most powerful if used in combination with techniques such as gel electrophoresis, electroblotting, LC, CE or protein sequencing. This is the major technology used for proteomic approaches.

Key Words



Abbreviations


Introduction

Since 1988, two new methods have emerged for getting proteins into the gas phase as intact molecular species bearing integral excess charges:

1. Matrix-assisted laser desorption MS and
2. Electrospray ionization MS

The laser desorption experiment is optimally combined with TOF mass measure¬ments (price range: $100,000.00 to 500,000.00 for a new instrument), whereas the electrospray method is optimally combined with a quadrupole mass filter (price range: $450,000.00 to 2,000,000.00). Both methods give mass accuracy of up to 1 part in 10,000 for proteins with MWs less than 30 to 40 kD and somewhat reduced mass accuracy for larger proteins.

Proteins with molecular masses of up to more than 100 kilodaltons can be analyzed at picomole sensitivities to give simple mass spectra corresponding to the intact molecule. Accurate measurements of the molecular weights (MWs) of biopolymers are necessary for this analytical technique. Most of the techniques developed to date for the measurements of the masses of proteins have accuracies limited to 5 to 10%. The most widely used of these techniques is sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE has assumed a pivotal role in biological research because of the power of simple visualization of the total protein content of a sample, together with crude information on the relative MWs and approximate amounts of the proteins present. A tech¬nique with high accuracy is matrix-assisted laser desorption/ionization time-of-flight (TOF) mass spectrometry (MS). This technique provides a measure of the mass of a pro¬tein with an accuracy of ~0.01%.

Matrix-Assisted Laser Desorption MS

All mass spectrometers designed to analyze proteins consist of two essential compo¬nents, the ion source and the mass analyzer. In the ion source, a seemingly unlikely phase transition is effected: proteins introduced as solids or in solution are converted into intact, naked ionized molecules in the gas phase. Subsequently, in the mass analyzer, the mass-to-charge (m/z) ratios of the naked protein molecule ions are determined.

Methodologies have been developed whereby intact protein ions could be generated in large numbers by laser photon bombardment of protein-containing samples. An intense production of intact, naked ionized protein molecules can be achieved when dilute proteins imbedded in a solid matrix are bombarded with intense, short duration bursts or pulse of focused ultraviolet (UV) laser light, often 337 nm from a N2 laser. The solid matrix consists of low-MW organic molecules that strongly absorb the UV irradiation (See figure below for a schematic of the set up). Several compounds for use as matrices have been studied. The choice of the proper matrix is still semi-empirical and may depend on the nature of the sample studied.




Samples are prepared for mass analysis by adding a protein, a mix of proteins, a peptide or a mix of peptides (e.g. tryptic digest of a protein) to a concentrated aqueous solution containing a large molar excess of a matrix-forming material such as 3,5-dimethoxy-4-hydroxycinnamic acid (sinpinic acid) (see table 1 for matrices and their range of laser wavelengths). A small volume of this mixture containing ~1 pmole of the protein or proteins is dried on a sample probe and inserted into the mass spectrometer. Matrix materials to analyte ratio is 1000-10,000 : 1 M concentrations, respectively. In the mass spectrometer, the sample (consisting of a layer of matrix microcrystals containing isolated protein molecules) is bombarded with short duration (1 to 10 ns) pulses of UV laser light (typically, 337 nm from a nitrogen laser or 355 nm from a frequency-tripled Nd:YAG laser). The interaction of the laser pulse with the sample causes a fraction of the matrix and trapped proteins to go into the gas phase and causes the protein to be ionized. A static electric field is imposed upon ions generated from the sample by application of a high potential (typically +- 30 kV) to the sample probe with respect to a closely spaced accelerating electrode. Protein ions are thus accelerated through the orifice in the electrode and enter into a field-free flight-tube (typical length is 50 to 200 cm). The masses of the protein ions can be simply determined by TOF analysis. Because all ions are accelerated through a fixed potential difference, the velocity of the ions is proportional to (m/z)-1/2. As the ions pass through the field-free flight-tube, they separate into a series of spatially discrete individual ion packets, each traveling with a velocity characteristic of its m/z ratio. A detector positioned at the end of the field-free flight-tube produces a signal as each ion packet strikes it. A recording of the detector signal as a function of time yields a TOF spectrum. The difference between the start time, set by the occur¬rence of the laser pulse and common to all ions, and the arrival time of an individual ion at the detector is proportional to (m/z)+1/2 and can be used to calculate the ion's m/z ratio. Such a calculation can be used to convert the x-axis of the spectrum (TOFs) into a m/z ratio axis (a conventional mass spectrum). All ions of different m/z ratios arising from a single laser shot are measured; they simply arrive at the ion detector at different times. The MALDI systems are calibrated by measuring the ion arrival times of known reference standards. This calibration may be performed either externally for routine analysis or with an internal standard for higher accuracy analysis. The technique is primarily a qualitative technique, so the relative peak heights or areas may not accurately represent the ratios of components present in a sample. Additionally some components have higher desorption/ionization yields than others. This can be often observed in the MALDI analysis of protein digest mixtures where most, but not necessarily all, of the expected fragments are observed. Recent work of several investigators suggests that the technique can be used for semiquantitative analysis runs of mixtures of defined components.

Performance specifications of MALDI-TOF-MS for a high-quality commercial in¬strument include a resolution of 400 (mass range is 200 to 800 kilodaltons), a mass determination accuracy up to 1 part in 104, a sensitivity of better than 1 pmole, and a spectrum acquisition time of 1 min. The method appears almost universal for proteins that can be dissolved in appropriate solvents, such as a volume/volume ratio of 2:1 of 0.1% triflouroacetic acid-acetonitrile or 100% hexafluoro-isopropanol (for proteins with hydrophobic character). The technique has the ability to analyze complex mixtures of peptides and proteins in the presence of large molar ex¬cesses of salts, buffers, lipids, and other species. The limitations that need to be consid¬ered include the occurrence of adduct artifacts that limit the mass accuracy for masses greater than 30 to 40 kD, the requirement that both the protein and the matrix material be soluble in the solvent mixture used, and the poisoning effect on the mass spectra of traces of ionic detergent (such as SDS) or involatile additives (such as glycerol and dimethyl sulfoxide).

The physicochemical events leading to the transfer of proteins to the gas phase and their ionization in matrix-assisted laser desorption/ionization have not yet been fully explained. The matrix is believed to serve several functions, including absorption of energy from the laser light and the isolation of individual protein molecules within the large mo¬lar excess of the solid matrix. The protein-matrix mixture typically forms a microcrystalline layer spontaneously upon drying the sample on the insertion probe tip. Upon irradiation with a short duration pulse of laser light, one model for the mechanism assumes that the upper-most layer of matrix is induced to undergo a phase transition from the solid to the gas phase. The subsequent expansion of these matrix molecules into the vacuum drags the matrix-isolated protein molecules into the gas phase. During the transfer to the gas phase, the proteins undergo ionization through proton transfer reactions with the matrix by reaction processes that are not yet understood.

Table 1: Some commonly used matrices


Table 2: Structure of commonly used matrices


Correlation of Processed Proteins with their Genes

Once the cDNA sequence of a gene has been determined accurate, measurement of the MW of the corresponding protein can provide valuable information. If the measured mass of the protein agrees with that calculated from the gene sequence, it is likely that the deduced sequence is correct, the amino and carboxyl terminals of the mature protein have been correctly assigned, and the protein contains no post-translationally modified amino acid residues. A difference between the measured and predicted MWs implies either an error in the cDNA deduced sequence or a post-translational modification or processing of the protein. Sometimes, differences are observed between the measured and calculated MWs that are more difficult to interpret. In such cases, a useful strategy involves degradation of the protein by chemical or enzymatic means and measurement by MALDI-TOF-MS of the total mixture of peptide products so generated. Comparison of the accurately measured masses of the degradation products with those predicted from the cDNA sequence yields information on the sites and natures of modifications and errors.

Glycoprotein Analysis

The determination of the carbohydrate portion of glycoproteins provides an analytical challenge to researchers, especially when only small amounts of sample are available. The reason for this is the very large number of isomers that are possible when these structures are built up from their constituent monosaccharides. Unlike peptides and oligonucleotides, which are composed of linear head-to-tail combinations of different amino acids and nucleotides, respectively, oligosaccharides contain many isobaric monosaccharides that not only can be linked through different hydroxy groups, but also can form complex branching patterns. See Figure 2 below for an example.

Monosaccharide composition and sequence analysis alone is not sufficient to determine the detailed primary structure. Only nuclear magnetic resonance (NMR) has the capability of identifying an oligosaccharide structure, but it lacks the sensitivity to address many biological problems. Mass spectrometry is much more sensitive but requires that the molecules be made volatile before analysis. MALDI-TOF-MS has been introduced for the ionization of large peptides and proteins. However, it has been shown that other compounds, including oligosaccharides, could be ionized (Mock et al., 1991).

All methods and strategies developed so far may be applied without or, if necessary, with the needed modifications to enable the use of the techniques mentioned above for mass and structure elucidation when glycoproteins are studied. Combination of HPLC or CE with MS peptide mapping of a protein is ideal for evaluating the presence of modifications, including those labile to the conditions of the Edman degradation. To obtain the best signals for oligosaccharides, the dried mixture of sample and matrix can be redissolved on the target with ethanol and allowed to recrystallize.



Figure 2: Top: Structures of ß-D-Galactopyranose (GAL), ß-D-Mannopyranose (Man), N-Acetyl-ß-D-glucopyranose (GlcNAc), and 6-Deoxy-b-L-galactopyranose (L-Fucose, Fuc), the four constituent monosaccharides of a common N-linked biantennary oligosaccharide shown below. Common abbreviations are shown in parentheses. Linkage in N-linked oligosaccharides is always through the 1-position of the monosaccharides to the left of the structure to any other position on the adjacent monosaccharides as shown at the bottom of the figure. Structures may be abbreviated with symbols replacing the monosaccharides as shown at the bottom of the figure. o = Gal, n = Man, f = GlcNAc, Ñ = Fuc.


Phosphoprotein Analysis

Phosphoproteins play a central role in many intracellular processes, including signal transduction and regulation of cell division. The site and extent of phosphorylation of key proteins are believed to play an important regulatory role in many intracellular signaling pathways.

The following is an example to demonstrate the analytical capabilities of the technique: The enzyme "cAMP-dependent protein kinase" is a complex with a mass of 178 kD and is made up of two regulatory and two catalytic subunits (Knighton et al., 1991, Chrivia et al., 1988). If cAMP is present, which binds to the regulatory subunit, the complex dissociates and releases the enzymatically active catalytic subunits. The 3-D molecular structure of the catalytic subunit of cAMP-dependent protein kinase has been determined by x-ray crystallography. The protein consists of 350 amino acids with a molecular mass of 40,440 daltons.

The alpha catalytic subunit of cAMP-dependent protein kinase from the mouse was cloned and expressed in Escherichia coli. The recombinant protein was isolated as a mixture of molecular species, all containing the same peptide chain but differing from each other in the degree of phosphorylation at specific residues. Three isoforms of the recombinant enzyme were prepared in highly purified form for structural analysis. Each was believed to be a homogeneous molecular species. The final stage of purification resulted in the protein samples being in a high salt buffer. The use of on-line reverse-phase HPLC-electrospray MS allowed the determination of the MWs of the three isoforms. The experiment simultaneously evaluated the purity of the proteins and desalted them in a form suitable to electrospray MS analysis.

The isoforms differed from one another by the mass of a single-PO3H group (80 daltons). The predicted sequence of the mature form of the recombinant polypeptide chain asks for a calculated mass of 40,440 daltons. Isoform I had a molecular mass of 40,759 daltons, 319 daltons above the calculated mass and therefore containing four phosphate groups (319/80). Isoform II had a mass of 40,678 daltons (that is, -81 daltons with respect to I and +238 daltons with respect to the parent polypeptide chain) and thus contained three phosphate groups. Isoform III had a mass of 40,600 daltons (that is, -78 daltons with respect to II, -159 daltons with respect to I, and +160 daltons with respect to the parent) and had two phosphate groups. The data confirmed that the isoforms were of high purity and were homogeneous molecular species. The differences in phosphate content of the three isoforms were consistent with prior data.

Although the above work was done using electrospray MS similar results may be obtained using MALDI-TOF-MS. The results showed that phosphoproteins can be analyzed by MS. The accuracy of MW measurement is well within the limits for useful determination of the degree of phosphorylation of intact proteins of typical size. The combination with enzymatic digestion and HPLC- or CE-MS peptide mapping is feasible to allow for a powerful technique for the structural characterization of phosphoproteins.


PROTEIN SEQUENCE DETERMINATION

Determination of the amino acid sequence of a protein molecule plays a central role in much biological research. Typically, the biological researcher's first direct observation of a protein would be by 1-D or 2-D PAGE. A critical step in the study of many biologically im¬portant proteins is the determination of limited stretches of amino acid sequence data from 5 to 100 picomole amounts of the natural protein isolated from a biological source. These limited sequence data are frequently the key information used to identify and clone the gene corresponding to the protein of interest. The nucleic acid sequence of the gene is then determined and translated to afford the complete amino acid sequence of the translation product. After cloning and expression of the identified gene, amino acid sequence data are used to confirm the structure of the protein produced. At the present time, amino acid sequence data are almost invariably generated by automated Edman degradation of a pro¬tein from the amino terminal, either of the intact protein or of peptides separated after proteolytic digestion of the protein.

Protein ladder sequencing with one-step MS readout

Chait and Kent (1992) and others have described a new approach to determine the amino acid sequence of picomole amounts of a protein which takes advantage of the ability of MALDI-TOF-MS to accurately and rapidly measure protein mixtures. Manual Edman chemistry is used prior to mass analysis to generate, in a controlled fashion, a family of sequence-defined fragments from a polypeptide chain. The sequence-defined fragments are analyzed and read out using laser desorption MS to simultaneously generate the complete data set in a single operation as a protein ladder. Mass differences between consecutive peaks define the identity of a particular amino acid, based on the distinctive mass of each genetically coded amino acid. Problems arise in the differentiation between leucine and isoleucine (same mass) and glutamine and lysine (Gln and Lys; 0.04 D mass difference). The family of fragments found defines the sequence of amino acids in the original peptide chain. One way of generating the sequence-defined set of fragments from a peptide or protein is to carry out the Edman degradation in the presence of a terminating agent (This is not automated yet. The reactions are done in a volume containing > 5 picomoles. Even so, only picomole amounts are needed for the experiment. A terminating reagent may not be needed if reaction times and temperature are optimized.). The protein ladder sequencing method relies on the capabilities of matrix-assisted laser desorption MS to measure the MWs of proteins and peptide with high accuracy. This method is as yet unproved for very small amounts of proteins of unknown sequence isolated from biological sources, but the potential exists for speeding up and simplifying protein sequencing.

Future Developments

There is a need for further improvement of methods for sample preparation for Edman chemistry sequencing as well as for ion production for matrix-assisted laser desorption MS. It is desirable to be able to use water as a matrix, which would allow the direct examination of biological specimens.

The resolution for TOF instrumentation for proteins is constantly increasing towards the resolution imposed by the envelope of the isotope distribution over the full mass range of interest. Furthermore, the speed for data collection and analysis is also in a state of constant improvement. Many researchers are working towards this goal at present. This will lead to further improvement of the described techniques in regard to resolutiona and through-put in the near future.

For rest of information, please click on PDF links below.

Peptidomimetics

There are many instances where the native information within a natural peptide ligand can be conferred/duplicated or mimetized into a non-peptide molecule, preferably of low molecular weight, hence the basis for the field of peptidomimetics(PM’s). The desire to convey the three dimensional information present in a peptide into small nonpeptide molecules is what encompasses the field of peptidomimetics.

Many research groups, both in academia and in pharmaceutical companies search constantly for non-peptide compounds that have better bioavailability and stability, perhaps even with greater receptor selectivity. The known structure-activity interactions and conformational foldings of peptide structures aid a great deal in the design of novel peptidomimetics. There are a number of factors that help in the rational design of PM’s such us: binding site optimal fit, conformational stabilization, (given by rigid elements and the positioning of specific functional groups), polar or hydrophobic regions (inside strategic reactive pockets) that favor the basic atomic interactions provided by hydrogen , electrostatic and hydrophobic bonding.

The goal in PM’s is to obtain molecules that mimic the specific molecular interactions of natural proteins and their ligands. The protein to protein interaction of biologivally active peptides at the receptor level can be obtained by small molecules, in an agonistic fashion or can be blocked, in an antagonistic fashion.

To obtain PM’s generally the biological researcher will have to screen compound libraries(either natural products or synthetic products). Combinatorial chemistry, amethod that was heavily used in the mid to late 90’s can be a tool to generate vast numbers of peptidic and non-peptidic molecules As an example of a PM’s , an inhibitor of angiotensin-converting enzyme (ACE), was developed, this PM’s is called Captopril**. Also, morphine , an opiod alkaloid, represents a classic example of a nonpeptidic compound found that mimics an endogenous peptide. Morphine replicates the biological effect of beta endorphin, on the respective receptor. A number of important aspects regarding conformational resctriction, peptide bond replacement, addition of turn mimetics and combinatorial library screening ,are investigated in order to search and find novel ligands, within the field of peptidomimetics

Ref: M.A. Ondetti,B.Rubin, D.W. Cushman,Science 1977,196, 441

Applications of Peptides and Proteins

In the human body, most if not all biological/ physiological processes are regulated by various forms of molecular recognition. Most of these processes involve initiation or inhibition trough protein-protein interaction. As we know peptides and proteins due to the vast number of conformational possibilities are ideal to carry out such complex control functions. The last 40 years have seen an enormous growth in the methodologies available to obtain peptide and protein molecules. Through recombinant methods, most labs can now assemble genes, subcloned them into expression vectors and obtain a wide range of endogenous proteins; likewise the pioneering work of Bruce Merrifield, makes it possible to obtain multikilo amounts of a number of biologically active peptides.

However, the number of peptides that have entered the pharmaceutical market is relatively low; perhaps due to a number of reasons, among them: antigenicity, immunogenicity , bioavailability and stability of the product upon administration into the patient. To some degree the relative scarcity of large scale peptide production plants that can make the peptide products in large amounts (perhaps at the ton levels) at more affordable costs, have also been a factor.

The good news is that in the last few years, after the completion of the human genome project, there have been more targets that are being worked on and the numbers of GMP facilities and the cost for GMP peptides have been continuously improving. Our forecast is that synthetic peptide chemistry will be an important source of many medically/clinically relevant peptides and proteins in the years to come.

Bio-Synthesis has been producing synthetic peptides for over 25 years. Our expertise in custom synthetic polypeptide manufacturing allows us to produce the high-quality, large-scale, and GMP peptides with the highest success rate with long standing records. We have been delivered more than 100,000 peptides to customers worldwide, including very hydrophobic polypeptide, peptide with multiple disulfide bonds, multi-phosph0rylated peptides and extremely long peptides. Our large scale non-GMP ever delivered 5 kilograms of peptides on a single order and has the capacity of 10,000 peptides per month. Our capacity of GMP peptide is 10 kilograms.

Cyclic Peptides

Synthetic or naturally occurring peptides can be classified as linear or cyclic peptides. Cyclic peptides (CP’s) comprise those peptides where there is a bond formed, between two residues, whereby a closed loop is formed. They can be classified as homodetic ( where all bonds are true peptide bonds) and heterodetic (where there are both peptide bonds and other types of bonding,such as ester or cystine linkages) based upon the type of bonding found in them. It has been found that CP’s often have increased metabolic stability, better receptor selectivity, controlled bioavailability and increased activity profiles. Also CP’sare metabolized more slowly due to their higher resistance to chemical degradation; on the other hand, due to their hydrophobicity they are excreted more readily than their corresponding linear counterparts. Many hormones, antibiotics,antimycotics and toxins in nature present themselves as CP’s. Many of these have been isolated, their structures resolved and produced by direct synthesis. More recently there have been more CP’s found in plants, fungi, bacteria snails and a number of other marine organisms. They are useful in the study of receptor-ligand interaction due to their restricted conformation and decreased flexibility; therefore there is continous interest in finding better synthetic routes and experimenting with modifications including but not limited to ring size, side chains and amino acid substitutions. Since the amounts found in their natural sources is often very minute, total synthetic routes remain the main/only option to obtain sufficient quantities for more in depth studies and analyses. Cyclic peptides can be of the following topologies: head to tail, side chain to side chain, side chain to tail ( or head) and branched. Ring closure in side chain to side chain CP’s can be attained by disulfide or amide bond formation between sulfhydryl groups or the appropriate functionalities. Of particular interest are connotoxins, small CP’s generally less than 12 amino acids, which have the unique characteristics of possessing 6 cysteines, which form 3 specific cystine bonds, conferring them their unique activities; some of these connotoxins have been found to have affinities 100X greater than morphine (phenathrene opiod receptor agonist) making them good candidates in the pain reducing research field, as they do not appear to induce addicition

In summary CP’s remain a very active area of synthetic interest, as more and more natural occurring CP”s are found in many flora and fauna, both terrestrial and aquatic.

Saturday, March 14, 2009

Synthetic Peptides in Cosmetics

In the last few years the cosmetic industry have added value to their top cosmetic lines by the addition of synthetic peptides. The inclusion of synthetic peptides has added glamour to the upper line of cosmetics and certainly has added a steeper price. But, do peptides really enhance skin care; to better answer that question, we need to understand a bit of biology; it is known that there is a number of growth factors, that is proteins that are normally produced in the human body that are responsible for the proliferation of certain types of human cells; for example epidermal growth factor (EGF) is a 53 amino acid long protein that regulates cell growth, proliferation and differentiation, when it binds to the corresponding EGF receptor. This protein was discovered by Stanley Cohen, who won the Nobel Prize for this work, in 1986. Since 1989 it has been used in the cosmetic industry. The protein contains 6 cysteines, which form 3 disulfide bridges, which confer the specific 3 D-structure responsible for its bioactivity.

Furthermore, there are other peptides such us the RGD peptide, that was derived from fibronectin; this short trimer functions as a cell adhesion factor, so when cells divide, the presence of RGD peptide, allows them to form multilayers; therefore it is important in tissue formation.

Unbeknown to most of us, the skin is the biggest organ of the human body and covers its entirety; as such it has not only protective functions, but also immunological, metabolic and thermoregulatory functions. The skin is made up of three distinct layers: the epidermis, dermis and hypodermis. The epidermis is the external structure of the skin and has a protective function. The cosmetic industry values any and all compounds that have a positive impact on the skin, therefore active ingredients that protect, and maintain skin health are highly desirable.

Consequently synthetic peptides do have a biological basis for perhaps having a positive impact on skin well being; however most creams that incorporate peptides in their formulas do not necessarily conduct FDA controlled studies, that include statistically significant number of individuals, in order to appreciate the effects claimed for a given cream. So, whereas certain synthetic peptides do have positive biological impact, when produced by our own body and administered in clinical relevant dosages, the positive effects of peptides incorporated in facial creams and the like, is difficult to assess as, most do not have controlled, placebo double blinded studies to back them up.

Difficult to Reduce Proteins

Difficult to Reduce Proteins

Most proteins attain their 3D conformation, driven by the presence of cysteine amino acids, which form specific disulfide Bonds, which twist and turn the proteins to spatial conformations, many of which ultimately will react with their corresponding receptors on the cell’s surface of their receptors, thus initiating a number of biochemical cascades such us phosphorylation events , and direct trigger signals that make the cell produce specific molecules with specific functions . At times the biochemical researcher needs to reduce the protein, for a variety of reasons, weather to carry on protein sequence analysis, folding-refolding studies, protein activation, denaturation, solubilization and for other reasons.

Commonly used reducing reagents are, dithiothreitol (DTT), 2-mercaptoethanol (MCE)and 2-mercaptoethylamine, and Tris(carboxyethyl) phosphine (TCEP).

The researcher can select which reducing agent to use based upon his/her specific needs, for example, low concentrations of 2-Mercaptoethylamine•HCl are used to reduce the disulfide bridges of antibodies; on the other hand 0.5% solutions of BME are used when preparing samples for isolectric focusing applications. A good alternative to BME is DTT that can be used at concentrations ranging from 10-100mM (pH 7-9).

TCEP has the advantage of being across a larger pH range (2-11) and posses a greater redox potential as compared to DTT. In real case situations, one can run into difficult to reduce proteins, whereby one hast to use even harsher reducing conditions like 10 mM DTT and 8M urea, 4hrs ambient or 6M guanidine, 80 mM DTT, 60min at 40C, followed by alkylation.Hence you have to be careful not to carbamylate(do not heat urea)and deamidation can occur at higher temperature and pH

The following is a good reference:

Wu, J., and Watson, J. T. (1997) A novel methodology for assignment of disulfide bond pairings in proteins, Protein Sci. 6, 391-398.

Tuesday, March 10, 2009

Cobalt Inhibits the Interaction between Hypoxia Inducible Factor-α and von Hippel-Lindau Protein by Direct Binding to Hypoxia Inducible Factor-α

This article reveal to us the fine points of the interaction between two unique proteins hypoxia-inducible factor (HIF) (which activates the expression of genes that contain a hypoxia response element ) and hypoxia response element (HRE) We are shown the importance of von Hippel-Lindau protein (pVHL) which mediates the ubiquitination and fast degradation of HIF (both HIF1 and 2). Proline hydroxylation (post translationally) in the oxygen dependent degradation (ODD) domain of HIF is necessary for the correct interaction between HIF and VHL. The mechanism by which cobalt mimics hypoxia, causing accumulation of HIF-1 and HIF-2 is still not well understood. The authors then show initial evidence that cobalt inhibits pVHL binding to HIF, when HIF, is hydroxylated. Removing 17 amino acids within the ODD domain of HIF-2 ( normally required for pVHL binding) inhibited cobalt binding and stabilized HIF-2, during normoxia. The authors conclude that cobalt mimics hypoxia by accessing the VHL-binding domain of HIF,and in so doing prevents HIF degradation.

Dephosphorylation and deactivation of Ca2+/calmodulin-dependent protein kinase II in bTC3-cells is mediated

This article is illustrative of how proteins phosphorylate/dephosphorylate in vivo. The authors utilized a-toxin-permeabilized bTC3 cell for the identification of protein phosphatases responsible for the dephosphorylation and deactivation of calmodulin-dependent protein kinase II (CaM kinase II) in situ.They show that an increase in calmodulin from .05 to 10 mM induced the almost-total conversion of CaM kinase II into a calmodulin-independent form typical of autophosphorylated, activated enzyme. Removing calmodulin caused CaM Kinase II to quickly go back to prestimulated levels. This observed reversal was diminished, but not prevented, by the action of inhibitors of protein phosphatase-1 (PP-1) and PP-2A, okadaic acid and calyculin A, and also by the selective chelation of Mg++ upon addition of EDTA. When both okadaic acid and EDTA were present one can observe arrest of enzyme deactivation. Then we learn that CaM kinase II phosphatase is more sensitive to calyculin A relative to okadaic acid, typical of PP-1 activity. This work hints to the fact that CaM kinase II dephosphorylation /deactivation in pancreatic b-cells is controlled by the joint action of phosphatase(okadaic acid sensitive) and PP-2C. (a Mg++- dependent phosphatase)

Conformational and Linear B-Cell Epitopes of Asp f 2, a Major Allergen of Aspergillus fumigatus

Antibodies can be generated against most commonly linear epitopes and less frequently to discontinous/conformational epitopes. In this work the authors describe Asp f 2 a major Aspergillus fumigatus (AF) allergen involved in allergic bronchopulmonary aspergillosis (ABPA). It is important to know the corresponding B-cell epitopes in order to the understandi its immunoregulation and immunodiagnosis. In order to identify the IgE binding epitopes from the linear sequence of Asp f 2, a series of decamer peptides comprising the whole molecule of Asp f 2 were synthesized on a solid support (ie; derivatized cellulose membranes) and tested IgE binding in ABPA patient and control sera. Also short synthetic peptides (three to five amino acids long) were assembled based on amino acid residues within the IgE binding regions and tested for epitope-antibody interactions. A total of nine IgE binding regions were identifed within the 268 amino acid domain and the following epitopes ,ATQRRQI, RKYFG, HWR, YTTRR, DHFAD, ALEAYA, and THEGGQ were found in the hydrophilic domain of Asp f 2. Three recombinant fragments, Asp f 2A that includes the N-terminal epitope region, Asp f 2B with no N- and C-terminal regions of the protein, and Asp f 2C comprising C-terminal epitopes, showed that either the N- or C-terminal region of the protein is necessary for the proper folding and conformation for IgE to effect antibody binding.

Base pairing involving deoxyinosine: implications for probe design

The thermal stability of oligodeoxyribonucleotide duplexes containing deoxyinoslne (1) residues matched with each of the four normal DNA bases were determined by optical melting techniques. The duplexes containing at least one I were obtained by nixing equimolar amounts of an oligonucleotide of sequence dCA3XA3G with one of sequence dCT3YT3G where X and Y were A, C, G, T, or I. Comparison of optical melting curves yielded relative stabilities for the I-containlng standard base pairs in an otherwise identical base-pair sequence. I:C pairs were found to be less stable than A:T pairs in these duplexes. Large neighboring-base effects upon stability were observed. For example, when (X,Y)=(I,A), the duplex is eight-fold more stable than when (X,Y)=(A,I). Independent of sequence effects the order of stabilities is: I:C > I:A > I:T = I:G. This order differs from that of deoxyguanosine which pairs less strongly with dA; otherwise each deoxy-lnosine base pair is less stable than its deoxyguanosine counterpart in the same sequence environment. Implications of these results for design of DNA oligonucleotide probes are discussed.

CD36 Peptides That Block Cytoadherence Define the CD36 Binding Region for Plasmodium falciparum-Infected Erythrocytes

Cell adhesion interactions are important in the understanding of various cellular processes where specific recoggition of proteín surfaces is required. Erythrocytes parasitized (PE)by P. Falciparum segregate/sequester themselves, from the circulation by adhering to microvascular endothelial cells (MEC). This segregation contributes directly to the virulence and severe pathology of malaria derived from P. falciparum; it is known that cell surface protein CD36 is an important host receptor for PE adherence, and this adhesion is mediated by the malarial variant antigen, P. falciparum erythrocyte membrane protein 1 (PfEMP1) and also by its cysteine-rich interdomain region 1 (CIDR-1). A group of peptides from the immunodominant domain of CD36 (residues 139-184) ranging in sizes from 17 to 29 amino acids, interfered with the CD36-PfEMP1 protein-protein interaction. All these peptides diminished binding at low micromolar range. Two such peptides, CD36 145-171 and CD36 156-184, selectively blocked PE adhesion to CD36 with no effect on its binding to the host receptor intercellular adhesion molecule-1 (ICAM-1).

It is also described that an adhesion blocking peptide from the ICAM-1 sequence interferes with PfEMP1– ICAM-1 interaction with no effect on its adhesion to CD36. These experiments validate earlier evidence that PfEMP1 is also a receptor for ICAM.

In summary it is shown that region 139-184 ,comprise the binding/adhesion for P. falciparum. These peptides can be be very useful for understanding the PE/PfEMP1 interaction with CD36 and for possible development of anti-adhesion molecular therapies.

Anti-angiogenin Activity of the Peptides Complementary to the Receptor-binding Site of Angiogenin

The authors describe the discovery of synthetic human angiogenic antagonists; they did so, by inferring the antisense RNA sequence codes matching to the to the receptor-binding site (RBS) of angiogenin in either 5’-3 (chANG) or 3’-5’ (chGNA) direction. These two peptides bind to angiogenin with good specificity and with a Kd of 44 nM., and in the process disrupt the interaction of angiogenin with actin, which is considered the angiogenin-binding protein on the surface of endothelial cells.These peptides block the neovascularization promoted by angiogenin in the chick chorioallantoic membrane assay (CCMA). The antagonistic activity of these peptides is angiogenin-specific, and the peptides do not seem to have an effect on embryonic angiogenesis or blood vessels already present. These peptides (chANG and chGNA) also block the angiogenesis created by the angiogenin-secreting PC 3 human prostate adenocarcinoma cells.
In conclusion, neutralization of the extracellular angiogenins secreted by PC 3 cells is achieved by the inhibition of the tumor-induced angiogenesis (peptide mediated) Therefore, chANG and chGNA synthetic peptides show potential to be effective in the treatment of angiogenin secreting human tumors.

Anthrax Toxin-Mediated Delivery In Vivo and In Vitro of a Cytotoxic T-Lymphocyte Epitope from Ovalbumin

The field of immunotherapy is a growing area where the use of synthetic peptides can be very helpful. As we all know Anthrax has received a lot of attention for its potential to be a low cost potential biochemical warfare agent. In this article the researchers describe how a non-toxic strain of anthrax toxin is able to deliver a cytotoxic T-lymphocyte (CTL) epitope in vivo, so that a unique CTL response is primed against the epitope.
This bacterial origin epitope, was then fused to the amino-terminal fragment (LFn) from the lethal-factor component of the toxin, and this chimeric protein was then injected, along with the protective antigen (PA) portion , into BALB/c mice. The authors then describe that indeed this fusion protein ( PA plus LFn) is able to deliver a different epitope, for example OVA257–264 from ovalbumin. Delivery was carried out in a different mouse haplotype, H-2Kb and took place in vitro as well as in vivo. An specific CTL clone, OVA257-264GA-4, was shown to recognize EL-4 cells treated in vitro with PA containing as little as 30 fmol of the LFn-OVA257–264 fusion protein. Also, OVA257–264-specific CTL can proliferate by incubation with splenocytes reacted with PA plus LFn-OVA257–264. In summary this work shows the potential for PA-LFn to serve as a universal delivery vehicle for CTL epitopes in vivo and also as a safe tool for the in-vitro expansion of patient-derived CTL for applications in adoptive immunotherapy.

Absolute Quantification of Specific Proteins in Complex Mixtures Using Visible Isotope-Coded

This article illustrate to us the use of a novel type of protein tagging reagent, that the authors call , “the visible isotope-coded affinity tag” (VICAT) which permits the quantification of the absolute amount of a target protein/s within a highly complex biological sample such as a eukaryotic cell lysate. This method can tag the thiol groups of cysteines or thioacetylated amino groups and also can introduce a biotin affinity handle (a visible moiety ) which allows tracking of the chromatographic position of the target peptide, without the need of a mass spectrometrer. A photocleavable linker (for tag removal) and an isotopic tag are also introduced, which enables distinguishing between the sample and reference peptides. The authors show the application of VICAT reagents to determine the absolute abundance of human group V phospholipase A2, in eukaryotic cell lysates;combining isolectric focusing of peptides on immobilized gel strip followed by microliquid chromatography/electrospray ionization mass spectrometry, they show that 66 fmol of phospholipase A2 per 100 ugs of cell protein are found in human lung macrophages. On the other hand Western blotting did not provide conclusive results. It is envisioned that VICAT reagents may be helpful for various applications including but not limited to the analysis of lead disease markers that could be detected in serum samples.

How short is short in RNAi Research

The original work of Mello and Fire from the Univ. of Massachusetts demonstrated in C. elegans, that gene expression is controlled by RNA interference (RNAi) . The initial patent applications filed by the Univ. of Massachussetts, with both Mello and Fire as the inventors, and covering this use of RNAi, clained only dsRNA longer than 25 bp’s. Later on, work done in mamalians showed that the long ds RNA was capable of inducing the release of IFN and other pro-inflammatory cytokines, causing dangerous reactions in the animals we tested. We now know that long dsRNA is recognized in the endosomes by TLRs, inducing an undesirable immune response in the animals; a situation that created a new challenge in the study of RNAi. Subsequently other researchers, such as Tuschl et.al. now at Rockefeller Univ, realized that shorter dsRNAi fragments of 25bp of smaller, while still capable of inhibiting the expression of a targeted gene, failed to induce an innate immunity response. In other words, the mammalian system is still capable of utilizing the diced dsRNA produced by the enzyme Dicer, which normally chops down the long dsRNA to sizes of 21-23 nts with 2 bases overhanging at the 3’ends of each strand. These 2 bases overhanging in dsRNA suggests that perhaps Dicer cleaves the long dsRNA in a fashion analogous to restriction enzymes. This short dsRNA can then interact with the RISC complex, where the guide strand is prepared and readied up to base pair with the target mRNA for its cleavage.

The RNAi situation is a good example of the unexpected in science. Although at the time of the initial discovery it was hard to predict that very small fragments of RNA could be pivotal in such important newly found mechanism, currently, even shorter dsRNA fragments, e.g. 15-18 bps’ are being tested. These new third generation modifiers such as LNA’s, UNA’s and others, because of their size have significant therapeutic potential.

There is a significant amount of ongoing research to elucidate the fine details of this novel gene control mechanism, including but not limited to studies of how miRNA precursors are transported to specific compartments of the cell, as these events may play important roles in the processing of the precursor by Dicer to render the active mature form of dsRNA.