Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between samples using mass spectrometry. ReDi labeling is performed using either regular (light) or deuterated (heavy) forms of formaldehyde and sodium cyanoborohydride to add two methyl groups to each free amine. Here we demonstrate a robust protocol for ReDi labeling and quantitative comparison of complex protein mixtures. Protein samples for comparison are digested into peptides, labeled to carry either light or heavy methyl tags, mixed, and co-analyzed by LC-MS/MS. Relative protein abundances are quantified by comparing the ion chromatogram peak areas of heavy and light labeled versions of the constituent peptide extracted from the full MS spectra. The method described here includes sample preparation by reversed-phase solid phase extraction, on-column ReDi labeling of peptides, peptide fractionation by basic pH reversed-phase (BPRP) chromatography, and StageTip peptide purification. We discuss advantages and limitations of ReDi labeling with respect to other methods for stable isotope incorporation. We highlight novel applications using ReDi labeling as a fast, inexpensive, and accurate method to compare protein abundances in nearly any type of sample.     

Structural features of glutamine substrates for human plasma factor XIIIa (activated blood coagulation factor XIII)

The action of human plasma factor XIIIa (thrombin-activated blood coagulation factor XIII) and guinea pig liver transglutaminase on purified caseins, fibrin, the derivatized gamma chain of fibrin, and a number of synthetic glutamine peptides, and peptide derivatives is reported. There are wide variations in the properties of the individual proteins and peptides as substrates for amine incorporation by the two transglutaminases. beta-Casein and several of its derivatives are excellent substrates for factor XIIIa. However, beta-casein is a relatively poor substrate for the liver enzyme. The primary site of amine incorporation by factor XIIIa in beta-casein was identified as glutamine 167. This was accomplished by labeling with fluorescent amine followed by proteolytic digestion and identification of labeled peptides. An 11-residue peptide and a 15-residue peptide, each containing 1 glutamine residue and each modeled after the primary site of amine incorporation in beta-casein, were prepared. A 13-residue peptide modeled after the primary crosslinking site in fibrin gamma chain was also prepared. Each of these polypeptides proved to be an efficient substrate for factor XIIIa and displayed significantly better substrate properties than a number of small glutamine peptide derivatives that are good substrates for liver transglutaminase.     

Tools for investigating peptide-protein interactions: peptide incorporation of environment-sensitive fluorophores via on-resin derivatization

This protocol presents the peptide incorporation of environment-sensitive fluorophores derived from the dimethylaminophthalimide family. The procedure utilizes anhydride precursors of 4-dimethylaminophthalimide (4-DMAP) or 6-dimethylaminonaphthalimide (6-DMN), whose syntheses are described in a related protocol from these authors. In this protocol, the fluorophores are directly incorporated after solid-phase peptide synthesis (SPPS) via on-resin derivatization of peptides prepared using commercially available diamino acids, which are Alloc-protected on the side-chain amino group. The time required to complete the procedure depends on the size and number of peptides targeted. As an alternative to this approach, the corresponding fluorescent amino acids can be obtained in an Fmoc-protected form for convenient use as building blocks in SPPS. This option is described in a related protocol by these authors.     

Quantitative proteome analysis using differential stable isotopic labeling and microbore LC-MALDI MS and MS/MS

We demonstrate an approach for global quantitative analysis of protein mixtures using differential stable isotopic labeling of the enzyme-digested peptides combined with microbore liquid chromatography (LC) matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS). Microbore LC provides higher sample loading, compared to capillary LC, which facilitates the quantification of low abundance proteins in protein mixtures. In this work, microbore LC is combined with MALDI MS via a heated droplet interface. The compatibilities of two global peptide labeling methods (i.e., esterification to carboxylic groups and dimethylation to amine groups of peptides) with this LC-MALDI technique are evaluated. Using a quadrupole-time-of-flight mass spectrometer, MALDI spectra of the peptides in individual sample spots are obtained to determine the abundance ratio among pairs of differential isotopically labeled peptides. MS/MS spectra are subsequently obtained from the peptide pairs showing significant abundance differences to determine the sequences of selected peptides for protein identification. The peptide sequences determined from MS/MS database search are confirmed by using the overlaid fragment ion spectra generated from a pair of differentially labeled peptides. The effectiveness of this microbore LC-MALDI approach is demonstrated in the quantification and identification of peptides from a mixture of standard proteins as well as E. coli whole cell extract of known relative concentrations. It is shown that this approach provides a facile and economical means of comparing relative protein abundances from two proteome samples.     

Tools for investigating peptide-protein interactions: peptide incorporation of environment-sensitive fluorophores through SPPS-based 'building block' approach

This protocol presents the synthesis and peptide incorporation of environment-sensitive fluorescent amino acids derived from the dimethylamino-phthalimide family. The procedure uses anhydride precursors of 4-dimethylaminophthalimide (4-DMAP) or 6-dimethylaminonaphthalimide (6-DMN), whose syntheses are described in a related protocol by these authors. In this study, the corresponding fluorescent amino acids can be readily obtained in Fmoc-protected form for convenient use as building blocks in solid phase peptide synthesis (SPPS). The time required to complete the procedure depends on the size and the number of peptides targeted. Alternatively, the chromophores can be incorporated directly after SPPS via on-resin derivatization of peptides, which is an option described in a related protocol by these authors.     

Identifying and quantifying proteolytic events and the natural N terminome by terminal amine isotopic labeling of substrates

Analysis of the sequence and nature of protein N termini has many applications. Defining the termini of proteins for proteome annotation in the Human Proteome Project is of increasing importance. Terminomics analysis of protease cleavage sites in degradomics for substrate discovery is a key new application. Here we describe the step-by-step procedures for performing terminal amine isotopic labeling of substrates (TAILS), a 2- to 3-d (depending on method of labeling) high-throughput method to identify and distinguish protease-generated neo-N termini from mature protein N termini with all natural modifications with high confidence. TAILS uses negative selection to enrich for all N-terminal peptides and uses primary amine labeling-based quantification as the discriminating factor. Labeling is versatile and suited to many applications, including biochemical and cell culture analyses in vitro; in vivo analyses using tissue samples from animal and human sources can also be readily performed. At the protein level, N-terminal and lysine amines are blocked by dimethylation (formaldehyde/sodium cyanoborohydride) and isotopically labeled by incorporating heavy and light dimethylation reagents or stable isotope labeling with amino acids in cell culture labels. Alternatively, easy multiplex sample analysis can be achieved using amine blocking and labeling with isobaric tags for relative and absolute quantification, also known as iTRAQ. After tryptic digestion, N-terminal peptide separation is achieved using a high-molecular-weight dendritic polyglycerol aldehyde polymer that binds internal tryptic and C-terminal peptides that now have N-terminal alpha amines. The unbound naturally blocked (acetylation, cyclization, methylation and so on) or labeled mature N-terminal and neo-N-terminal peptides are recovered by ultrafiltration and analyzed by tandem mass spectrometry (MS/MS). Hierarchical substrate winnowing discriminates substrates from the background proteolysis products and non-cleaved proteins by peptide isotope quantification and bioinformatics search criteria.     

A convenient method for europium‐labeling of a recombinant chimeric relaxin family peptide R3/I5 for receptor‐binding assays

Relaxin family peptides have important biological functions, and so far, four G‐protein‐coupled receptors have been identified as their receptors (RXFP1–4). A chimeric relaxin family peptide R3/I5, containing the B‐chain of relaxin‐3 and the A‐chain of INSL5, is a selective agonist for both RXFP3 and RXFP4. In a previous study, europium‐labeled R3/I5, as a nonradioactive and low‐background receptor‐binding tracer, was prepared through a chemical synthesis approach. In the present study, we established a convenient alternative approach for preparing the europium‐labeled R3/I5 tracer based on a recombinant R3/I5 designed to carry a solubilizing tag at the A‐chain N‐terminus and a pyroglutamate residue at the B‐chain N‐terminus. Because of the presence of a single primary amine moiety, the recombinant R3/I5 peptide was site‐specifically mono‐labeled at the A‐chain N‐terminus by a diethylenetriaminepentaacetic acid/europium moiety through a convenient one‐step procedure. The diethylenetriaminepentaacetic acid/Eu³⁺‐labeled R3/I5 bound both receptors RXFP3 and RXFP4 with high binding affinities and low nonspecific binding. Thus, we have presented a valuable nonradioactive tracer for future interaction studies on RXFP3 and RXFP4 with various natural or designed ligands. The present approach could also be adapted for preparing and labeling of other chimeric relaxin family peptides. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Nanoparticle growth and surface chemistry changes in cell-conditioned culture medium

When biomolecules attach to engineered nanoparticle (ENP) surfaces, they confer the particles with a new biological identity. Physical format may also radically alter, changing ENP stability and agglomeration state within seconds. In order to measure which biomolecules are associated with early ENP growth, we studied ENPs in conditioned medium from A549 cell culture, using dynamic light scattering (DLS) and linear trap quadrupole electron transfer dissociation mass spectrometry. Two types of 100 nm polystyrene particles (one uncoated and one with an amine functionalized surface) were used to measure the influence of surface type. In identically prepared conditioned medium, agglomeration was visible in all samples after 1 h, but was variable, indicating inter-sample variability in secretion rates and extracellular medium conditions. In samples conditioned for 1 h or more, ENP agglomeration rates varied significantly. Agglomerate size measured by DLS was well correlated with surface sequestered peptide number for uncoated but not for amine coated polystyrene ENPs. Amine-coated ENPs grew much faster and into larger agglomerates associated with fewer sequestered peptides, but including significant sequestered lactose dehydrogenase. We conclude that interference with extracellular peptide balance and oxidoreductase activity via sequestration is worthy of further study, as increased oxidative stress via this new mechanism may be important for cell toxicity.     

On the chemical basis of the Lowry protein determination

The copper-catalyzed oxidation of peptides and proteins by phosphomolybdic/phosphotungstic acid (Folin phenol reagent) was studied with respect to redox stoichiometry of color formation and nature of the oxidation products. From peptides without reducing side chains two reducing equivalents were transferred under ideal conditions to Mo6+/W6+ for each unit of tetradentate copper complex with concomitant formation of an imino peptide. Tyrosine and tryptophan side chains contributed four additional reducing equivalents. Oxidation of proline-containing peptides was greatly impaired as judged from color formation due to the interference of the imino acid with complex formation. Reaction of the oxidized peptides with 2,4-dinitrophenyl (DNP)-hydrazine gave a peptide amine and the DNP-hydrazone of a 2-oxoacyl peptide. The oxidation products from tetraalanine were identified as dialanine amide and pyruvoylalanine DNP-hydrazone. From the time course of the development of the blue color on reduction of Folin reagent with tetraalanine it was inferred that the reaction consisted of an initial (less than 5 s) oxidation to a Cu3+ peptide complex followed by slow changes in absorbance, especially above 0.2 mM. Due to these complications the two-electron stoichiometry has to be considered only as a limiting case for peptide concentrations below 0.02 mM.     

Mass spectrometric analysis of single identified neurons of an insect

The combination of retrograde labelling with dextran-tetramethylrhodamine and MALDI-TOF mass spectrometry was used to analyse for the first time the peptidome of a series of morphologically identified single neurosecretory cells of an insect. Eight postero-lateral cells of the metathoracic ganglion of the American cockroach, Periplaneta americana, were used to demonstrate that: (1) the complete dissection procedure can be documented and (2) the mass spectrometric analysis of the dissected somata results in highly reproducible mass spectra. In total, 21 FMRFamide-related peptides were detected in each of the postero-lateral cells which release their neurosecretions via thoracic perisympathetic organs. Direct analysis of these neurohemal organs confirmed the co-storage of FMRFamide-related peptides. Two additional abundant peptides from thoracic perisympathetic organs which were not detectable in the postero-lateral cells were characterized using ESI-Q-TOF MS/MS. De novo sequencing yielded two related peptides (FERL/IEamides) without any similarity with known peptide families of insects.     

Site-specific DOTA/europium-labeling of recombinant human relaxin-3 for receptor-ligand interaction studies

Relaxin-3 (also known as INSL7) is a recently identified neuropeptide belonging to the insulin/relaxin superfamily. It has putative roles in the regulation of stress responses, food intake, and reproduction by activation of its cognate G-protein-coupled receptor RXFP3. It also binds and activates the relaxin family peptide receptors RXFP1 and RXFP4 in vitro. To obtain a europium-labeled relaxin-3 as tracer for studying the interaction of these receptors with various ligands, in the present work we propose a novel site-specific labeling strategy for the recombinant human relaxin-3 that has been previously prepared in our laboratory. First, the N-terminal 6 × His-tag of the single-chain relaxin-3 precursor was removed by Aeromonas aminopeptidase and all of the primary amines of the resultant peptide were reversibly blocked by citroconic anhydride. Second, the A-chain N-terminus of the blocked peptide was released by endoproteinase Asp-N cleavage that removed the linker peptide between the B- and A-chains. Third, an alkyne moiety was introduced to the newly released A-chain N-terminus by reaction with the highly active primary amine-specific N-hydroxysuccinimide ester. Fourth, after removal of the reversible blockage under mild acidic condition, europium-loaded DOTA with an azide moiety was introduced to the two-chain relaxin-3 carrying the alkyne moiety through click chemistry. Using this site-specific labeling strategy, homogeneous monoeuropium-labeled human relaxin-3 could be obtained with good overall yield. In contrast, conventional random labeling resulted in a complex mixture that was poorly resolved because human relaxin-3 has four primary amine moieties that all react with the modification reagent. Both saturation and competition binding assays demonstrated that the DOTA/Eu(3+)-labeled relaxin-3 retained high binding affinity for human RXFP3, RXFP4, and RXFP1 and was therefore a suitable non-radioactive and stable tracer to study the interaction of various natural or designed ligands with these receptors. Using this site-specific labeling strategy, other functional probes, such as fluorescent dyes, biotin, or nanoparticles could also be introduced to the A-chain N-terminal of the recombinant human relaxin-3. Additionally, we improved the time-resolved fluorescence assay for the DOTA-bound europium ion which paves the way for the use of DOTA as a lanthanide chelator for protein and peptide labeling in future studies.     

Solid-phase synthesis of amidine-substituted phenylbenzimidazoles and incorporation of this DNA binding and recognition motif into amino acid and peptide conjugates

Amidine-substituted phenylbenzimidazoles are well-established DNA-binding structural motifs that have contributed to the development of diverse classes of DNA-targeted agents; this ring system not only assists in increasing the overall DNA affinity of an agent, but can also influence its site selectivity. Seeking a means to conveniently exploit these attributes, a protocol for the on-resin synthesis of amino acid- and peptide-phenylbenzimidazole-amidine conjugates was developed to facilitate installation of phenylbenzimidazole-amidines into peptide chains during the course of standard solid-phase syntheses. Building from a resin-bound amino acid or peptide on Rink amide resin, 4-formyl benzoic acid was coupled to the resin-bound free amine followed by introduction of 3,4-diamino-N′-hydroxybenzimidamide (in the presence of 1,4-benzoquinone) to construct the benzimidazole heterocycle. Finally, the resin-bound N′-hydroxybenzimidamide functionality was reduced to an amidine via 1 M SnCl₂·2H₂O in DMF prior to resin cleavage to release final product. This procedure permits the straightforward synthesis of amino acids or peptides that are N-terminally capped by a phenylbenzimidazole-amidine ring system. Employing this protocol, a series of amino acid–phenylbenzimidazole-amidine (Xaa-R) conjugates was synthesized as well as dipeptide conjugates of the general form Xaa-Gly-R (where R is the phenylbenzimidazole-amidine and Xaa is any amino acid).     

Purification of nucleosidyl peptides by chromatography on dihydroxyboryl-substituted polyacrylamide and cellulose

A new column chromatographic method is presented for the purification of peptides which are covalently bound to nucleoside analogs (nucleosidyl peptides). The procedure involves complex formation between the cis-diol moiety of the nucleosidyl peptide and the dihydroxyborylphenyl group which is linked either to polyacrylamide or to cellulose as a support; thus, the nucleosidyl peptides can be reversibly bound to the column while all other peptides are eluted in the void volume. This approach is exemplified by the purification of two peptides of rabbit muscle pyruvate kinase labeled with 5′-p-fluorosulfonylbenzoyl adenosine and one peptide of bovine liver glutamate dehydrogenase modified with 5′-p-fluorosulfonylbenzoyl guanosine. The method may be generally applicable to the purification of peptides resulting from the affinity labeling of nucleotide sites in proteins.     

Automated identification of SUMOylation sites using mass spectrometry and SUMmOn pattern recognition software

Tandem mass spectrometry (MS/MS) allows for the rapid identification of many types of post-translational modifications (PTMs), especially those that can be detected by a diagnostic mass shift in one or more peptide fragment ions (for example, phosphorylation). But some PTMs (for example, SUMOs and other ubiquitin-like modifiers) themselves produce multiple fragment ions; combined with fragments from the modified target peptide, a complex overlapping fragmentation pattern is thus generated, which is uninterpretable by standard peptide sequencing software. Here we introduce SUMmOn, an automated pattern recognition tool that detects diagnostic PTM fragment ion series within complex MS/MS spectra, to identify modified peptides and modification sites within these peptides. Using SUMmOn, we demonstrate for the first time that human SUMO-1 multimerizes in vitro primarily via three N-terminal lysines, Lys7, Lys16 and Lys17. Notably, our method is theoretically applicable to any type of modification or chemical moiety generating a unique fragment ion pattern.     

Making Ends Meet: Microwave-Accelerated Synthesis of Cyclic and Disulfide Rich Proteins Via In Situ Thioesterification and Native Chemical Ligation

The development of synthetic methodologies for cyclic peptides is driven by the discovery of cyclic peptide drug scaffolds such as the plant-derived cyclotides, sunflower trypsin inhibitor 1 (SFTI-1) and the development of cyclized conotoxins. Currently, the native chemical ligation reaction between an N-terminal cysteine and C-terminal thioester group remains the most robust method to obtain a head-to-tail cyclized peptide. Peptidyl thioesters are effectively generated by Boc SPPS. However, their generation is challenging using Fmoc SPPS because thioester linkers are not stable to repeated piperidine exposure during deprotection. Herein we describe a Fmoc-based protocol for synthesizing cyclic peptides adapted for microwave assisted solid phase peptide synthesis. The protocol relies on the linker Di-Fmoc-3,4-diaminobenzoic acid, and we demonstrate the use of Gly, Ser, Arg and Ile as C-terminal amino acids (using HBTU and HATU as coupling reagents). Following synthesis, an N-acylurea moiety is generated at the C-terminal of the peptide; the resin bound acylurea peptide is then deprotected and cleaved from the resin. The fully deprotected peptide undergoes thiolysis in aqueous buffer, generating the thioester in situ. Ultimately, the head-to-tail cyclized peptide is obtained via native chemical ligation. Two naturally occurring cyclic peptides, the prototypical Möbius cyclotide kalata B1 and SFTI-1 were synthesized efficiently, avoiding potential branching at the diamino linker, using the optimized protocol. In addition, we demonstrate the possibility to use the approach for the synthesis of long and synthetically challenging linear sequences, by the ligation of two truncated fragments of a 50-residue long plant defensin.     

Optimized selective N-methylation of peptides on solid support.

Peptides containing N(alpha)-methylamino acids exhibit interesting therapeutic profiles and are increasingly recognized as potentially useful therapeutics. Unfortunately, their synthesis is hampered by the high price and nonavailability of many N(alpha)-methylamino acids. An efficient and practical three-step procedure for selective N-methylation of peptides on solid support is described. The procedure was based on the well known solid-phase N-methylation of N(alpha)-arylsulfonyl peptides, which was improved by using dimethylsulfate and the less expensive DBU as base. Every step of the procedure, amine activation by an o-nitrobenzenesulfonyl group, selective N-methylation and removal of the sulfonamide group, was optimized in respect of time and economy. The described optimized three-step procedure is performed in 35 min without solvent changes, instead of 3 h. Tripeptides (Fmoc-Phe-MeXaa-Leu-OH) containing N-methylated common amino acids were also prepared using the optimized procedure to demonstrate its compatibility with these amino acids. The described procedure allows an efficient synthesis of N(alpha)-methylamino acid containing peptides in a very short time using Fmoc solid-phase peptide synthesis.     

Surface immobilization chemistry influences peptide‐based detection of lipopolysaccharide and lipoteichoic acid

Antimicrobial peptides (AMPs) have recently gained attention as potentially valuable diagnostic and therapeutic agents. The utilization of these peptides for diagnostic purposes relies on the ability to immobilize them on the surface of a detection platform in a predictable and reliable manner that facilitates target binding. The method for attachment of peptides to a solid support is guided by peptide length, amino acid composition, secondary structure, and the nature of the underlying substrate. While immobilization methods that target amine groups of amino acid sequences are widely used, they can result in heterogeneous conjugation at multiple sites on a peptide and have direct implications for peptide presentation and function. Using two types of commercial amine‐reactive microtiter plates, we described the effects of analogous immobilization chemistries on the surface attachment of AMPs and their differential binding interaction with Gram‐specific bacterial biomarkers, lipopolysaccharide and lipoteichoic acid. As might be expected, differences in overall binding affinities were noted when comparing AMPs immobilized on the two types of plates. However, the two‐amine‐targeted linking chemistries also affected the specificity of the attached peptides; lipopolysaccharide generally demonstrated a preference for peptides immobilized on one type of plate, while (when observed at all) lipoteichoic acid bound preferentially to AMPs immobilized on the other type of plate. These results demonstrate the potential for tuning not only the binding affinities but also the specificities of immobilized AMPs by simple alterations in linking strategy. Published 2012. This article is a U.S. Government work and is in the public domain in the USA.     

Pharmacodynamic synergy strictly dependent on the co-operative aggregation of enantiomers of cyclic peptides in the bacterial cell membrane

The antimicrobial activity of the peptide enantiomers cyclo[D-Tle-D-Lys-D-Tle-L-Ala-D-Tle-L-Ala-D-Tle-L-Ala] and cyclo[L-Tle-L-Lys-L-Tle-D-Ala-L-Tle-D-Ala-L-Tle-D-Ala] against Bacillus megaterium was investigated. Both these peptides showed very low activity in both an agar diffusion assay and a broth microdilution assay. However, when both peptides were present during the experiments a potent inhibition with an IC(50) value of 2 μM was observed. Furthermore, the peptides also showed low hemolytic activity. Neither peptide had any hemolytic activity in concentrations up to 1mM but when erythrocytes were exposed to both peptides a weak hemolytic activity could be observed with a HC(50) value of 316 μM.     

Developmental expression of neuromodulators in the central complex of the grasshopper Schistocerca gregaria

The central complex is a major integrative region within the insect brain with demonstrated roles in spatial orientation, the regulation of locomotor behavior, and sound production. In the hemimetabolous grasshopper, the central complex comprises the protocerebral bridge, central body (CB), ellipsoid body, noduli, and accessory lobes, and this modular organization develops entirely during embryogenesis. From a biochemical perspective, a range of neuroactive substances has been demonstrated in these modules of the adult central complex, but little is known about their developmental expression. In this study, we use matrix‐assisted laser desorption/ionization‐imaging mass spectrometry on single brain slices to confirm the presence of several peptide families (tachykinin, allatostatin, periviscerokinin/pyrokinin, FLRFamide, and neuropeptide F) in the adult central complex and then use immunohistochemistry and histology to examine their developmental expression, together with that of the indolamin serotonin, and the endogenous messenger nitric oxide (NO; via its synthesizing enzyme). We find that each neuromodulator is expressed according to a unique, stereotypic, pattern within the various modules making up the central complex. Neuropeptides such as tachykinin (55%) and allatostatin (65%), and the NO‐synthesizing enzyme diaphorase (70%), are expressed earlier during embryonic development than the biogenic amine serotonin (80%), whereas periviscerokinin‐like peptides and FLRFamide‐like peptides begin to be expressed only postembryonically. Within the CB, these neuroactive substances are present in tangential projection neurons before they appear in columnar neurons. There is also no colocalization of serotonin‐positive and peptide‐positive projections up to the third larval instar during development, consistent with the clear dorsoventral layering of the neuropil we observe. Our results provide the first neurochemical fingerprint of the developing central complex in an hemimetabolous insect. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

Altering Peptide fibrillization by polymer conjugation

A strategy is presented that exploits the ability of synthetic polymers of different nature to disturb the strong self-assembly capabilities of amyloid based β-sheet forming peptides. Following a convergent approach, the peptides of interest were synthesized via solid-phase peptide synthesis (SPPS) and the polymers via reversible addition-fragmentation chain transfer (RAFT) polymerization, followed by a copper(I) catalyzed azide-alkyne cycloaddition (CuAAC) to generate the desired peptide-polymer conjugates. This study focuses on a modified version of the core sequence of the β-amyloid peptide (Aβ), Aβ(16-20) (KLVFF). The influence of attaching short poly(N-isopropylacrylamide) and poly(hydroxyethylacrylate) to the peptide sequences on the self-assembly properties of the hybrid materials were studied via infrared spectroscopy, TEM, circular dichroism and SAXS. The findings indicate that attaching these polymers disturbs the strong self-assembly properties of the biomolecules to a certain degree and permits to influence the aggregation of the peptides based on their β-sheets forming abilities. This study presents an innovative route toward targeted and controlled assembly of amyloid-like fibers to drive the formation of polymeric nanomaterials.