N-Methylated Analogs of hIAPP Fragments 18–22, 23–27, 33–37 Inhibit Aggregation of the Amyloidogenic Core of the Hormone

Amylin (hIAPP) aggregation leads to the formation of insoluble deposits and is one of the factors in the development of type II diabetes. The aim of this research was to find N-methylated analogs of the aggregating amylin fragments 18–22, 23–27, and 33–37, which would not themselves be susceptible to aggregation and would inhibit the aggregation of the amyloidogenic cores of the hormone. None of the analogs of fragment 18–22 containing one or two N-methylated amino acid residues showed any tendency to aggregate. Only the peptide H−F(N−Me)GA(N−Me) IL−OH ( 6 ) derived from the 23–27 hIAPP hot spot did not form fibrous structures. All analogs of the 33–37 amylin fragment were characterized by the ability to form aggregates, despite the presence of N-methylated amino acids in their structures. N-Methylated peptides 1 – 5 demonstrated inhibitory properties against the aggregation of fragment 18–22. Aggregation of the amyloidogenic core of 23–27 was significantly inhibited by N-methylated peptides 1 – 3 derived from the (18–22) H−HSSNN−OH fragment and by the H−F(N-Me)GA(N−Me)IL−OH ( 6 ) fragment derived from the 23–27 amylin hot spot. Fragment (33–37) H−GSNTY−NH₂ was found to be inhibited in the presence of N-methylated peptides 1 – 3 derived from the 18–22 fragment and by the double methylated peptide H−F(N−Me)GA(N−Me)IL−OH ( 6 ). Research on the possibility of using N-methylated analogs of amyloidogenic amylin cores as inhibitors of hormone aggregation is ongoing, with a focus on finding the minimum concentration of N-methylated peptides capable of inhibiting the aggregation of hIAPP hot spots.     

Non-Aggregating Amylin Fragments as an Inhibitors of the Aggregation Process of Susceptible to Aggregation Fragments 18–22, 23–27, and 33–37 of Hormone

Amylin aggregation is one of the factors in the development of diabetes mellitus, which is classified as a civilization disease. The aim of this research was to find whether non-aggregating fragments 1–7, 8–12, 13–17 and 28–32 of amylin would inhibit the aggregation of the amyloidogenic cores 18–22, 23–27, 33–37 of hormone. In the study of the inhibitory potential of non-aggregating amylin fragments, a set of independent methods were used to study aggregation properties (spectroscopic and fluorescence studies with the use of indicators, microscopic studies, circular dichroism studies) and the method of prediction of aggregation properties. The performed research allowed to select the cyclic fragment (1–7) H-KCNTATC-OH with disulfide bond as an inhibitor capable of inhibiting the aggregation of all amyloidogenic cores 18–22, 23–27, 33–37 of the hormone. Additionally, it was found that this peptide inhibits insulin hot spot aggregation, which may indicate its universal utility in inhibiting the process of aggregation of hormones regulating carbohydrate metabolism directly related to the development of diabetes. Research on the possibility of the extensive use of the cyclic fragment (1–7) of H-KCNTATC-OH as a peptide inhibitor of the polypeptide/protein aggregation process is ongoing.     

Amyloidogenicity of naturally occurring full‐length animal IAPP variants

The aggregation of the 37‐amino acid polypeptide human islet amyloid polypeptide (hIAPP), as either insoluble amyloid or as small oligomers, appears to play a direct role in the death of human pancreatic β‐islet cells in type 2 diabetes. hIAPP is considered to be one of the most amyloidogenic proteins known. The quick aggregation of hIAPP leads to the formation of toxic species, such as oligomers and fibers, that damage mammalian cells (both human and rat pancreatic cells). Whether this toxicity is necessary for the progression of type 2 diabetes or merely a side effect of the disease remains unclear. If hIAPP aggregation into toxic amyloid is on‐path for developing type 2 diabetes in humans, islet amyloid polypeptide (IAPP) aggregation would likely need to play a similar role within other organisms known to develop the disease. In this work, we compared the aggregation potential and cellular toxicity of full‐length IAPP from several diabetic and nondiabetic organisms whose aggregation propensities had not yet been determined for full‐length IAPP.     

Human IAPP amyloidogenic properties and pancreatic β-cell death

A hallmark of type 2 diabetes mellitus (T2DM) is the presence of extracellular amyloid deposits in the islets of Langerhans. These deposits are formed by the human islet amyloid polypeptide, hIAPP (or amylin), which is a hormone costored and cosecreted with insulin. Under normal conditions, the hormone remains in solution but, in the pancreas of T2DM individuals, it undergoes misfolding giving rise to oligomers and cross-β amyloid fibrils. Accumulating evidence suggests that the amyloid deposits that accompany type 2 diabetes mellitus are not just a trivial epiphenomenon derived from the disease progression. Rather, hIAPP aggregation induces processes that impair the functionality and viability of β-cells and may lead to apoptosis. The present review article aims to summarize a few aspects of the current knowledge of this amyloidogenic polypeptide. In the first place, the physicochemical properties which condition its propensity to misfold and form aggregates. Secondly, how these properties confer hIAPP the capacity to interfere with some signaling of the pancreatic β-cell, interact with membranes, form channels or affect natural ion channels, including calcium channels. Finally, how misfolded hIAPP cytotoxicity results in apoptosis. A number of pathophysiological changes of the T2DM islet can be related to the amyloidogenic properties of hIAPP. However, in a certain way, the in vivo aggregation of the polypeptide also reflects a failure of chaperones and, in general, of cellular proteostasis, supporting the view that T2DM may also be considered as a conformational disorder.     

Micelle formation by a fragment of human islet amyloid polypeptide

Human islet amyloid polypeptide (hIAPP) is the major component of amyloid plaques found in the pancreatic islets of persons with type 2 diabetes mellitus. HIAPP belongs to the group of amyloidogenic proteins, characterized by their aggregation and deposition as fibrillar amyloid in various body tissues. The aggregation of amyloidogenic proteins is thought to occur via a common pathway, but currently no unifying kinetic model exists. In previous work, we presented a model of amyloid fibril formation formulated from our observations of the aggregation of an amyloidogenic fragment of hIAPP, amino acids 20-29. Our model is based on nucleation-dependent aggregation, modified by the formation of off-pathway hIAPP micelles. In the present study we confirm the presence of peptide micelles, and experimentally determine the critical micelle concentration in solutions of hIAPP fragments using three different techniques: conductivity, pH, and fluorescence. All three techniques yield a critical micelle concentration of 3-3.5 micro M peptide. Furthermore, based on changes in the fluorescence intensity of a labeled peptide fragment as well as a decrease in solution pH as a result of deprotonation of the amino terminus, we conclude that the amino terminus of the fragment undergoes a significant change of environment upon micellization.     

Lipid interaction and membrane perturbation of human islet amyloid polypeptide monomer and dimer by molecular dynamics simulations

The aggregation of human islet amyloid polypeptide (hIAPP or amylin) is associated with the pathogenesis of type 2 diabetes mellitus. Increasing evidence suggests that the interaction of hIAPP with β-cell membranes plays a crucial role in cytotoxicity. However, the hIAPP-lipid interaction and subsequent membrane perturbation is not well understood at atomic level. In this study, as a first step to gain insight into the mechanism of hIAPP-induced cytotoxicity, we have investigated the detailed interactions of hIAPP monomer and dimer with anionic palmitoyloleolyophosphatidylglycerol (POPG) bilayer using all-atom molecular dynamics (MD) simulations. Multiple MD simulations have been performed by employing the initial configurations where the N-terminal region of hIAPP is pre-inserted in POPG bilayer. Our simulations show that electrostatic interaction between hIAPP and POPG bilayer plays a major role in peptide-lipid interaction. In particular, the N-terminal positively-charged residues Lys1 and Arg11 make a dominant contribution to the interaction. During peptide-lipid interaction process, peptide dimerization occurs mostly through the C-terminal 20-37 region containing the amyloidogenic 20-29-residue segment. Membrane-bound hIAPP dimers display a pronounced ability of membrane perturbation than monomers. The higher bilayer perturbation propensity of hIAPP dimer likely results from the cooperativity of the peptide-peptide interaction (or peptide aggregation). This study provides insight into the hIAPP-membrane interaction and the molecular mechanism of membrane disruption by hIAPP oligomers.     

Neurotoxicity of Human Amylin in Rat Primary Hippocampal Cultures: Similarity to Alzheimer's Disease Amyloid-β Neurotoxicity

Amylin, a 37-amino-acid amyloidogenic peptide, bears biophysical similarities to the amyloid-β peptide (Aβ) deposited in Alzheimer's disease. Using embryonic rat hippocampal cultures we tested whether amylin induces neurotoxicity similar to that previously observed with Aβ(1–40). Treatment with human amylin (1–37) resulted in prominent toxicity as assessed by phasecontrast microscopy and quantification of lactate dehydrogenase in the medium. Amylin-induced neurotoxicity was morphologically similar to that induced by Aβ(1–40). In contrast, the nonamyloidogenic rat amylin showed negligible neurotoxicity despite having 95% sequence similarity to human amylin. Only full-length human amylin was toxic; various amylin peptide fragments including amino acid residues 20–29 were nontoxic at similar concentrations. These studies suggest that unrelated amyloidogenic peptides like human amylin and Aβ can adopt a similar neurotoxic conformation in vitro. Similar conformation-dependent neurotoxicity may drive the prominent neurite degeneration around compacted but not diffuse deposits of Aβ in Alzheimer's disease.     

Matrix Metalloproteinase-9 Protects Islets from Amyloid-induced Toxicity

Deposition of human islet amyloid polypeptide (hIAPP, also known as amylin) as islet amyloid is a characteristic feature of the pancreas in type 2 diabetes, contributing to increased β-cell apoptosis and reduced β-cell mass. Matrix metalloproteinase-9 (MMP-9) is active in islets and cleaves hIAPP. We investigated whether hIAPP fragments arising from MMP-9 cleavage retain the potential to aggregate and cause toxicity, and whether overexpressing MMP-9 in amyloid-prone islets reduces amyloid burden and the resulting β-cell toxicity. Synthetic hIAPP was incubated with MMP-9 and the major hIAPP fragments observed by MS comprised residues 1–15, 1–25, 16–37, 16–25, and 26–37. The fragments 1–15, 1–25, and 26–37 did not form amyloid fibrils in vitro and they were not cytotoxic when incubated with β cells. Mixtures of these fragments with full-length hIAPP did not modulate the kinetics of fibril formation by full-length hIAPP. In contrast, the 16–37 fragment formed fibrils more rapidly than full-length hIAPP but was less cytotoxic. Co-incubation of MMP-9 and fragment 16–37 ablated amyloidogenicity, suggesting that MMP-9 cleaves hIAPP 16–37 into non-amyloidogenic fragments. Consistent with MMP-9 cleavage resulting in largely non-amyloidogenic degradation products, adenoviral overexpression of MMP-9 in amyloid-prone islets reduced amyloid deposition and β-cell apoptosis. These findings suggest that increasing islet MMP-9 activity might be a strategy to limit β-cell loss in type 2 diabetes.     

Conformational preferences of the amylin nucleation site in SDS micelles: an NMR study

Human islet amyloid polypeptide (hIAPP), or amylin, is a 37 amino acid hormone secreted by pancreatic beta-cells. hIAPP constitutes approximately 90% of the amyloid deposits found in type II diabetic patients. It has been shown that the central region of the peptide (hIAPP(20-29)) constitutes the nucleation site for the amyloidogenic process with F23 playing a key role in the formation of the beta-pleated structures. In addition, it has been proposed that an important stage in the cytotoxicity of hIAPP is its interaction with the beta-cell membranes. As a first step toward the characterization of the interaction of hIAPP with cell membranes, we determined conformational preferences of hIAPP(20-29) in membrane-mimicking environments. We found that upon interacting with negatively charged micelles, the dominant conformation of hIAPP(20-29) is a distorted type I beta-turn centered on residues F23 and G24, with F23, A25, and I26 forming a small hydrophobic cluster that may facilitate the interaction of this peptide with the membrane bilayer. Moreover, we were able to elucidate the topological orientation of the peptide that is absorbed on the micelle surface, with the hydrophobic cluster oriented toward the hydrocarbon region of the micelles and both N- and C-termini exposed to the solvent.     

Cyclic N-Terminal Loop of Amylin Forms Non Amyloid Fibers

We report for the first time, to our knowledge, that the N-terminal loop (N_loop) of amylin (islet amyloid polypeptide (IAPP) residues 1–8) forms extremely long and stable non-β-sheet fibers in solution under the same conditions in which human amylin (hIAPP) forms amyloid fibers. This observation applies to the cyclic, oxidized form of the N_loop but not to the linear, reduced form, which does not form fibers. Our findings indicate a potential role of direct N_loop-N_loop interactions in hIAPP aggregation, which has not been previously explored, with important implications for the mechanism of hIAPP amyloid fiber formation, the inhibitory action of IAPP variants, and the competition between ordered and disordered aggregation in peptides of the calcitonin peptide family.     

Cloning and expression of human islet amyloid polypeptide in cultured cells

Efforts to clone amyloidogenic proteins in the cells often have resulted in cell death. We report successful cloning and expression of recombinant human islet amyloid polypeptide (hIAPP) in cultured mammalian cells. Amylin gets secreted, forms fibrils that are toxic to target cells like beta cells of rat and human. The study involves cloning of full-length amylin in fluorescent protein vector followed by transfection into mammalian cells. The transfected cells with recombinant human amylin, secrete the translated protein corresponding to 37-amino acid native mature IAPP. The mature IAPP secreted out of the cell is purified and characterized by MALDI-TOF/TOF-MS and Western blotting. Purified IAPP forms fibrils as seen by Thioflavin-T fluorescence and AFM, and these fibrils were cytotoxic towards pancreatic cell line RIN5mf cells.     

Membrane fragmentation by an amyloidogenic fragment of human Islet Amyloid Polypeptide detected by solid-state NMR spectroscopy of membrane nanotubes

A key factor in the development of Type II diabetes is the loss of insulin producing pancreatic β-cells. The amyloidogenic human Islet Amyloid Polypeptide (hIAPP also known as human amylin) is believed to play a crucial role in this biological process. Previous studies have shown that hIAPP forms small aggregates that kill β-cells by disrupting the cellular membrane. In this study, we report membrane fragmentation by hIAPP using solid-state NMR experiments on nanotube arrays of anodic aluminum oxide containing aligned phospholipid membranes. In a narrow concentration range of hIAPP, an isotropic ³¹P chemical shift signal indicative of the peptide-induced membrane fragmentation was detected. Solid-state NMR results suggest that membrane fragmentation is related to peptide aggregation as the presence of Congo Red, an inhibitor of amyloid formation, prevented membrane fragmentation and the non-amyloidogenic rat-IAPP did not cause membrane fragmentation. The disappearance of membrane fragmentation at higher concentrations of hIAPP suggests an alternate kinetic pathway to fibril formation in which membrane fragmentation is inhibited.     

Cyclic N-Terminal Loop of Amylin Forms Non Amyloid Fibers

We report for the first time, to our knowledge, that the N-terminal loop (N_loop) of amylin (islet amyloid polypeptide (IAPP) residues 1–8) forms extremely long and stable non-β-sheet fibers in solution under the same conditions in which human amylin (hIAPP) forms amyloid fibers. This observation applies to the cyclic, oxidized form of the N_loop but not to the linear, reduced form, which does not form fibers. Our findings indicate a potential role of direct N_loop-N_loop interactions in hIAPP aggregation, which has not been previously explored, with important implications for the mechanism of hIAPP amyloid fiber formation, the inhibitory action of IAPP variants, and the competition between ordered and disordered aggregation in peptides of the calcitonin peptide family.     

Imaging Flow Cytometry Illuminates New Dimensions of Amyloid Peptide-Membrane Interactions

Membrane interactions of amyloidogenic proteins constitute central determinants both in protein aggregation as well as in amyloid cytotoxicity. Most reported studies of amyloid peptide-membrane interactions have employed model membrane systems combined with application of spectroscopy methods or microscopy analysis of individual binding events. Here, we applied for the first time, to our knowledge, imaging flow cytometry for investigating interactions of representative amyloidogenic peptides, namely, the 106–126 fragment of prion protein (PrP(106–126)) and the human islet amyloid polypeptide (hIAPP), with giant lipid vesicles. Imaging flow cytometry was also applied to examine the inhibition of PrP(106–126)-membrane interactions by epigallocatechin gallate, a known modulator of amyloid peptide aggregation. We show that imaging flow cytometry provided comprehensive population-based statistical information upon morphology changes of the vesicles induced by PrP(106–126) and hIAPP. Specifically, the experiments reveal that both PrP(106–126) and hIAPP induced dramatic transformations of the vesicles, specifically disruption of the spherical shapes, reduction of vesicle circularity, lobe formation, and modulation of vesicle compactness. Interesting differences, however, were apparent between the impact of the two peptides upon the model membranes. The morphology analysis also showed that epigallocatechin gallate ameliorated vesicle disruption by PrP(106–126). Overall, this study demonstrates that imaging flow cytometry provides powerful means for disclosing population-based morphological membrane transformations induced by amyloidogenic peptides and their inhibition by aggregation modulators.     

Membrane fragmentation by an amyloidogenic fragment of human Islet Amyloid Polypeptide detected by solid-state NMR spectroscopy of membrane nanotubes

A key factor in the development of Type II diabetes is the loss of insulin producing pancreatic beta-cells. The amyloidogenic human Islet Amyloid Polypeptide (hIAPP also known as human amylin) is believed to play a crucial role in this biological process. Previous studies have shown that hIAPP forms small aggregates that kill beta-cells by disrupting the cellular membrane. In this study, we report membrane fragmentation by hIAPP using solid-state NMR experiments on nanotube arrays of anodic aluminum oxide containing aligned phospholipid membranes. In a narrow concentration range of hIAPP, an isotropic (31)P chemical shift signal indicative of the peptide-induced membrane fragmentation was detected. Solid-state NMR results suggest that membrane fragmentation is related to peptide aggregation as the presence of Congo Red, an inhibitor of amyloid formation, prevented membrane fragmentation and the non-amyloidogenic rat-IAPP did not cause membrane fragmentation. The disappearance of membrane fragmentation at higher concentrations of hIAPP suggests an alternate kinetic pathway to fibril formation in which membrane fragmentation is inhibited.     

A hIAPP-derived all-d-amino-acid inhibits hIAPP fibrillation efficiently at membrane surface by targeting α-helical oligomeric intermediates

A variety of peptides and peptide derivatives have been constructed using the “β-sheet core segment” of amyloid proteins as inhibitors of amyloidogenic fibrillation. A novel all-d-amino-acid from hIAPP β-sheet core segment (hIAPP 22–27) is demonstrated to inhibit hIAPP fibril formation efficiently both at the phospholipid membrane and in bulk solution. The inhibitor terminates hIAPP aggregation to the α-helical oligomeric intermediates at the membrane surface, whereas it stops the aggregation at the stage of β-sheet oligomeric intermediates in bulk solution. This is the first evidence that the inhibition mechanism of the inhibitor at membrane surface is significantly different from that in bulk solution.     

Complete characterization of the mutation landscape reveals the effect on amylin stability and amyloidogenicity

Type‐II diabetes is believed to be partially aggravated by the emergence of toxic amylin protein deposits in the extracellular space of the pancreas β‐cells. Amylin, the regulatory hormone that is co‐secreted with insulin, has been observed to misfold into toxic structures. Pramlintide, an FDA approved injectable amylin analog mutated at positions 25, 28, and 29 was therefore developed to create a more stable, soluble, less‐aggregating, and equipotent peptide that is used as an adjunctive therapy for diabetes. However, because Pramlintide is not ideal, researchers have been exploring other amylin analogs as therapeutic replacements. In this work, we assist the finding of optimal analogs by computationally revealing the mutational landscape of amylin. We computed the structure energies of all possible single‐point mutations and studied the effect they have on amylin stability and amyloidogenicity. Each of the 37 amylin residues was mutated in silico into the 19 canonical amino acids and an energy function computing the Lennard–Jones, Coulomb and solvation energy was used to analyze changes in stability. The mutation landscape identified amylin's conserved stable regions, residues that can be tweaked to further stabilize structure, regions that are susceptible to mutations, and mutations that are amyloidogenic. We used the single‐point mutational landscape data to generate estimations for higher‐order multiple‐point mutational landscapes and discovered millions of three‐point mutations that are more stable and less amyloidogenic than Pramlintide. The landscapes provided an explanation for the effect of the S20G and Q10R mutations on the onset of diabetes of the Chinese and Maori populations, respectively. Proteins 2015; 83:1014–1026. © 2015 Wiley Periodicals, Inc.     

Identification of a novel human islet amyloid polypeptide beta-sheet domain and factors influencing fibrillogenesis

Human islet amyloid polypeptide (hIAPP) accumulates as pancreatic amyloid in type 2 diabetes and readily forms fibrils in vitro. Investigations into the mechanism of hIAPP fibril formation have focused largely on residues 20 to 29, which are considered to comprise a primary amyloidogenic domain. In rodents, proline substitutions within this region and the subsequent beta-sheet disruption, prevents fibril formation. An additional amyloidogenic fragment within the C-terminal sequence, residues 30 to 37, has been identified recently. We have extended these observations by examining a series of overlapping peptide fragments from the human and rodent sequences. Using protein spectroscopy (CD/FTIR), electron microscopy and X-ray diffraction, a previously unrecognised amyloidogenic domain was localised within residues 8 to 20. Synthetic peptides corresponding to this region exhibited a transition from random coil to beta-sheet conformation and assembled into fibrils having a typical amyloid-like morphology. The comparable rat 8-20 sequence, which contains a single His18Arg substitution, was also capable of assembling into amyloid-like fibrils. Examination of peptide fragments corresponding to residues 1 to 13 revealed that the immediate N-terminal region is likely to have only a modulating influence on fibril formation or conformational conversion. The contributions of charged residues as they relate to the amyloid-forming 8-20 sequence were also investigated using IAPP fragments and by assessing the effects of pH and counterions. The identification of these principal amyloidogenic sequences and the effects of associated factors provide details on the IAPP aggregation pathway and structure of the peptide in its fibrillar state.     

Destabilization of human IAPP amyloid fibrils by proline mutations outside of the putative amyloidogenic domain: is there a critical amyloidogenic domain in human IAPP?

Islet amyloid polypeptide (IAPP; amylin) is responsible for amyloid formation in type-2 diabetes. Not all organisms form islet amyloid, and amyloid formation correlates strongly with variations in primary sequence. Studies of human and rodent IAPP have pointed to the amino acid residues 20-29 region as the important amyloid-modulating sequence. The rat 20-29 sequence contains three proline residues and does not form amyloid, while the human sequence contains no proline and readily forms amyloid. This has led to the view that the 20-29 region constitutes a critical amyloidogenic domain that dictates the properties of the entire sequence. The different behavior of human and rat IAPP could be due to differences in the 20-29 region or due simply to the fact that multiple proline residues destabilize amyloid fibrils. We tested how critical the 20-29 region is by studying a variant identical with the human peptide in this segment but with three proline residues outside this region. We designed a variant of the amyloidogenic 8-37 region of human IAPP (hIAPP(8-37) 3xP) with proline substitutions at positions 17, 19 and 30. Compared to the wild-type, the 3xP variant was much easier to synthesize and had dramatically greater solubility. Fourier transform infra red spectroscopy, transmission electron microscopy, Congo red staining and thioflavin-T binding indicate that this variant has a reduced tendency to form beta-sheet structure and forms deposits with much less structural order than the wild-type. Far-UV CD studies show that the small amount of beta-sheet structure developed by hIAPP(8-37) 3xP after long periods of incubation dissociates readily into random-coil structure upon dilution into Tris buffer. The observation that proline substitutions outside the putative core domain effectively abolish amyloid formation indicates that models of IAPP aggregation must consider contributions from other regions.     

Synthesis and purification of amyloidogenic peptides

The polypeptide hormone amylin forms amyloid deposits in patients with type 2 diabetes mellitus. Amyloid-forming peptides are often very difficult to synthesize and purify. Amylin and fragments of amylin are no exception. In this paper we describe the efficient synthesis and purification of two amyloidogenic fragments of human amylin. One fragment corresponds to residues 17 to 37 of the full-length hormone and the other corresponds to residues 24 to 37. These fragments have previously been identified in vivo and have been shown to form amyloid in vitro. The strategy used to elucidate appropriate conditions for the synthesis and purification of these peptides is generally applicable to other peptides that are difficult to synthesize. These peptides were prepared using solid-phase peptide synthesis with Fmoc alpha-amino protection. The effects of varying the solvent, side-chain-protecting group and choice of cleavage conditions were examined. The use of NMP as the main solvent and cleavage with trifluoroacetic acid, phenol, ethanedithiol, thioanisole, and water proved to be optimal. 1,1,1,3, 3,3-Hexafluoro-2-propanol (HFIP) was found to be the best solvent for solubilizing the crude peptides. A wide range of HPLC conditions for the purification of the peptides were examined and an acetonitrile-based solvent system with HCl as the ion pairing agent provided efficient purification.