Lysine-containing Peptides Research Articles

An enrichment strategy based on hydrophobic tagging and reversed-phase chromatographic separation for the analysis of lysine-containing peptides

Lysine (K) is widely used in the design of lysine-targeted crosslinkers, structural elucidation of protein complexes, and analysis of protein-protein interactions. In "shotgun" proteomics, which is based on liquid chromatography-tandem mass spectrometry (LC-MS/MS), proteins from complex samples are enzymatically digested, generating thousands of peptides and presenting significant challenges for the direct analysis of K-containing peptides. In view of the lack of effective methods for the enrichment of K-containing peptides, this work developed a method which based on a hydrophobic-tag-labeling reagent C10-S-S-NHS and reversed-phase chromatography (termed as HYTARP) to achieve the efficient enrichment and identification of K-containing peptides from complex samples. The C10-S-S-NHS synthesized in this work successfully labeled standard peptides containing various numbers of K and the labeling efficiency achieved up to 96% for HeLa cell protein tryptic digests. By investigating the retention behavior of these labeled peptides in C18 RP column, we found that most K-labeled peptides were eluted once when acetonitrile percentage reached 57.6% (v/v). Further optimization of the elution gradient enabled the efficient separation and enrichment of the K-labeled peptides in HeLa digests via a stepwise elution gradient. The K-labeled peptides accounted for 90% in the enriched peptides, representing an improvement of 35% compared with the number of peptides without the enrichment. The dynamic range of proteins quantified from the enriched K-containing peptides spans 5-6 orders of magnitude, and realized the detection of low-abundance proteins in the complex sample. In summary, the HYTARP strategy offers a straightforward and effective approach for reducing sample complexity and improving the identification coverage of K-containing peptides and low-abundance proteins.

SpSrtA-Catalyzed Isopeptide Ligation on Lysine Residues.

Sortase-mediated ligation (SML) is widely used for protein bioconjugation. However, the sortase used in this strategy typically recognizes only the N-terminal oligoglycine, which is absent in most natural proteins. To broaden the spectrum of substrates compatible with SML, we focus on a novel sortase, sortase A from Streptococcus pneumoniae (SpSrtA), known for its expanded substrate specificity (N-terminal glycine, alanine, and serine). We present the first evidence showing that the reported SpSrtA mutant (SpSrtA*) can modify lysine residues in itself and other proteins. The modification sites of SpSrtA* were identified through LC-MS/MS analysis. Moreover, we discovered an optimal lysine-containing peptide tag by fusing it onto sfGFP, resulting in a labeling efficiency of 57%. Inspired by this, we applied the method to modify proteins on microorganism surfaces up to 13.5-fold. To enhance labeling efficiency, we fused the SpSrtA* onto a surface protein and achieved a 2.64-fold improvement. We further developed a high-throughput yeast display screening method for the directed evolution of SpSrtA*, achieving a 10-fold improvement in the labeling efficiency of this surface protein. Our study provides a novel strategy for modifying the lysine residues that will be a powerful addition to the protein bioconjugation toolbox.

Hybrid PNA-peptide hydrogels as injectable CEST-MRI agents.

The self-assembly of peptides and peptide analogues may be exploited to develop platforms for different biomedical applications, among which CEST-MRI (chemical exchange saturation transfer magnetic resonance imaging) represents one of the most attractive techniques to be explored as a novel metal-free contrast approach in imaging acquisitions. A lysine-containing peptide sequence (LIVAGK-NH2, named K2) was thus modified by insertion, at the N-terminus, of a peptide nucleic acid (PNA) base, leading to a primary amine suitable for the signal generation. a-K2, c-K2, g-K2 and t-K2 peptides were synthesized and characterized. The c-K2 sequence displayed gelling properties and the Watson and Crick pairing, arising from its combination with g-K2, allowed a significant increase in the mechanical responsivity of the hydrogel. These matrices were able to generate a CEST signal around 2.5 ppm from water and, after assessing their cytocompatibility on GL261 (murine glioma), TS/a (murine breast carcinoma), and 3T3-NIH (murine fibroblasts) cell lines, their capability to work as implants for in vivo detection, was proved by intratumor injection in Balb/c mice inoculated with TS/a murine breast cancer cells.

Analgesic Effect of the Lysine-Containing Short Peptide Is Due to Modulation of the NaV1.8 Channel Activation Gating System.

The present work continues our recent series of articles that aim to elucidate the ligand-receptor binding mechanism of short cationic peptides to the NaV1.8 channel in the nociceptive neuron. The applied methodological approach has involved several methods: the patch-clamp experimental evaluation of the effective charge of the NaV1.8 channel activation gating system, the organotypic tissue culture method, the formalin test, and theoretical conformational analysis. The lysine-containing short peptide Ac-KEKK-NH2 has been shown to effectively modulate the NaV1.8 channel activation gating system. As demonstrated by the organotypic tissue culture method, the studied short peptide does not trigger the downstream signaling cascades controlling neurite outgrowth and should not be expected to evoke adverse side effects. Conformational analysis of the Ac-KEKK-NH2 molecule has revealed that the distances between the positively charged amino groups of the lysine side chains are equal to 11-12 Å. According to the previously suggested mechanism of ligand-receptor binding of short peptides to the NaV1.8 channel molecule, Ac-KEKK-NH2 should exhibit an analgesic effect, which has been confirmed by the formalin test. The data obtained unequivocally indicate that the studied lysine-containing short peptide is a promising candidate for the role of a novel analgesic medicinal substance.

Switchable Enzyme-mimicking catalysts Self-Assembled from de novo designed peptides and DNA G-quadruplex/hemin complex.

Reconstruction of enzymatic active site in an artificial system is key to achieving high catalytic efficiency. Herein, we report the self-assembly of the lysine-containing peptides with guanine-rich DNA and hemin to form peroxidase-mimicking active sites and catalytic nanoparticles. The DNA strand self-folds into a G-quadruplex structure that provides a supramolecular scaffold and a potential axial ligand for hemin. The β-sheet forming capability of the lysine-containing peptides is found to affect the catalytic synergy between the G-quadruplex DNA and the peptide. It is hypothesized that the β-sheet formation of the peptides results in the enrichment of the lysine residues, which distribute on the distal side of hemin to promote the formation of Compound I, like distal arginine residue in natural heme pocket. Incorporation of the histidine residues into the lysine-containing peptides further enhanced the hemin activities, indicating the cooperation between the lysine and histidine. Furthermore, the peptide/DNA/hemin complexes can be switched between active and inactive state by reversible formation and deformation of the DNA G-quadruplex, which was attributed to the peptides-promoted conformational changes of the DNA components. This work opens an avenue to mimic the catalytic residues and their spatial distribution in the natural enzymes, and shed light on the design of the smart biocatalysts that can respond to the environmental stimuli.

Liposomal Amphotericin B Formulation Displaying Lipid-Modified Chitin-Binding Domains with Enhanced Antifungal Activity.

Fungal infections affect more than one billion people worldwide and cause more than one million deaths per year. Amphotericin B (AmB), a polyene antifungal drug, has been used as the gold standard for many years because of its broad antifungal spectrum, high activity, and low tendency of drug resistance. However, the side effects of AmB, such as nephrotoxicity and hepatotoxicity, have hampered its widespread use, leading to the development of a liposome-type AmB formulation, AmBisome. Herein, we report a simple but highly effective strategy to enhance the antifungal activity of AmBisome with a lipid-modified protein. The chitin-binding domain (LysM) of the antifungal chitinase, Pteris ryukyuensis chitinase A (PrChiA), a small 5.3 kDa protein that binds to fungal cell wall chitin, was engineered to have a glutamine-containing peptide tag at the C-terminus for the microbial transglutaminase (MTG)-catalyzed crosslinking reaction (LysM-Q). LysM-Q was site-specifically modified with a lysine-containing lipid peptide substrate of MTG with a palmitoyl moiety (Pal-K). The resulting palmitoylated LysM (LysM-Pal) exhibited negligible cytotoxicity to mammalian cells and can be easily anchored to yield LysM-presenting AmBisome (LysM-AmBisome). LysM-AmBisome exhibited a dramatic enhancement of antifungal activity toward Trichoderma viride and Cryptococcus neoformans, demonstrating the marked impact of displaying a cell-wall binder protein on the targeting ability of antifungal liposomal formulations. Our simple strategy with enzymatic protein lipidation provides a potent approach to upgrade other types of lipid-based drug formulations.

Quantitative Assessment of Arsenite-Induced Perturbation of Ubiquitinated Proteome.

Arsenic contamination in food and groundwater constitutes a public health concern for more than 200 million people worldwide. Individuals chronically exposed to arsenic through drinking and ingestion exhibit a higher risk of developing cancers and cardiovascular diseases. Nevertheless, the underlying mechanisms of arsenic toxicity are not fully understood. Arsenite is known to bind to and deactivate RING finger E3 ubiquitin ligases; thus, we reason that a systematic interrogation about how arsenite exposure modulates global protein ubiquitination may reveal novel molecular targets for arsenic toxicity. By employing liquid chromatography-tandem mass spectrometry, in combination with stable isotope labeling by amino acids in cell culture (SILAC) and immunoprecipitation of di-glycine-conjugated lysine-containing tryptic peptides, we assessed the alterations in protein ubiquitination in GM00637 human skin fibroblast cells upon arsenite exposure at the entire proteome level. We observed that arsenite exposure led to altered ubiquitination of many proteins, where the alterations in a large majority of ubiquitination events are negatively correlated with changes in expression of the corresponding proteins, suggesting their modulation by the ubiquitin-proteasomal pathway. Moreover, we observed that arsenite exposure confers diminished ubiquitination of a rate-limiting enzyme in cholesterol biosynthesis, HMGCR, at Lys248. We also revealed that TRC8 is the major E3 ubiquitin ligase for HMGCR ubiquitination in HEK293T cells, and the arsenite-induced diminution of HMGCR ubiquitination is abrogated upon genetic depletion of TRC8. In summary, we systematically characterized arsenite-induced perturbations in a ubiquitinated proteome in human cells and found that the arsenite-elicited attenuation of HMGCR ubiquitination in HEK293T cells involves TRC8.

Arginine-containing dipeptides decrease affinity of gut trypsins and compromise soybean pest development

The design and production of molecules capable of mimicking the binding or/and functional sites of proteins inhibitors represent a promising strategy for the exploration and modulation of gut trypsin function in insect pests, specifically Lepidoptera. Here, for the first time, we characterized the trypsin activity present in the gut, performance and development of Anticarsia gemmatalis (Lepidoptera: Noctuidae) larvae when exposed to arginine-containing dipeptides. In silico assessment showed that arginine-containing dipeptides have a greater affinity for the active site of A. gemmatalis trypsins than lysine-containing peptides due to the presence of the double-charged guanidinium group that enhances the interaction at the S1 subsite of trypsins. Furthermore, the inhibitory and anti-insect potential of the peptides was demonstrated through kinetic and larval life cycle parameters, respectively. These dipeptides showed structural stability, binding to the active site, corroborated in vitro (competitive inhibition), and significant reduction of trypsin enzyme activity in the gut, survival, and weight of the A. gemmatalis larvae. Our findings reinforce the idea that small peptides are promising candidates for lepidopteran pest management. The optimization of DI2 and DI1 peptides, enhancing uptake and affinity to trypsins, may turn the use of these molecules feasible in agriculture.

Mechanism of Side Chain-Controlled Proton Conductivity in Bioinspired Peptidic Nanostructures.

Bioinspired peptide assemblies are promising candidates for use as proton-conducting materials in electrochemical devices and other advanced technologies. Progress toward applications requires establishing foundational structure-function relationships for transport in these materials. This experimental-theoretical study sheds light on how the molecular structure and proton conduction are linked in three synthetic cyclic peptide nanotube assemblies that comprise the three canonical basic amino acids (lysine, arginine, and histidine). Experiments find an order of magnitude higher proton conductivity for lysine-containing peptide assemblies compared to histidine and arginine containing assemblies. The simulations indicate that, upon peptide assembly, the basic amino acid side chains are close enough to enable direct proton transfer. The proton transfer kinetics is determined in the simulations to be governed by the structure and flexibility of the side chains. Together, experiments and theory indicate that the proton mobility is the main determinant of proton conductivity, critical for the performance of peptide-based devices.

Critical Evaluation of Low-Molecular Weight Respiratory Sensitizers and Their Protein Reactivity Potential Toward Lysine Residues.

Interest in the development of methods to evaluate the respiratory sensitization potential of low-molecular weight chemicals continues, but no method has yet been generally accepted or validated. A lack of chemical reference standards, together with uncertainty regarding relevant immunological mechanisms, has hampered method development. The first key event in the development of either skin or respiratory sensitization is the formation of stable adducts of the chemical with host proteins. This event is measured in the Direct Peptide Reactivity Assay using cysteine- and lysine-containing model peptides. It is hypothesized that protein reactivity and subsequent adduct formation may represent the earliest point of divergence in the pathways leading to either skin or respiratory sensitization. Direct Peptide Reactivity Assay data for 200 chemicals were compiled and grouped into respiratory, skin and nonsensitizers. Chemicals grouping was based on extensive literature research and expert judgment. To evaluate if chemical groups represent different peptide reactivity profiles, peptide reactivity data were clustered and compared with information on protein binding mechanisms and chemical categories available via the Organization for Economic Co-operation and Development. Toolbox. Respiratory sensitizers (n = 15) showed a significant (3-fold) higher lysine reactivity than skin sensitizers (n = 129). However, this difference was driven largely by the high representation of acid anhydrides among the respiratory sensitizers that showed clear lysine selectivity. Collectively, these data suggest that preferential reactivity for either cysteine or lysine is associated primarily with chemical structure, and that lysine preference is not a unifying characteristic of chemical respiratory allergens.

Участие лизинсодержащих олигопептидов в регуляции свертывания крови

Установлено регуляторное влияние лизинсодержащих пептидов Arg-Lys-Lys-Arg-Pro-Gly-Pro (RKKRPGP) и Lys-Arg-Lys-Pro-Gly-Pro (KRKPGP) на процессы свертывания крови животных. Многократное в течение 7 сут через каждые 24 ч пероральное или интраназальное введение пептидов в ежедневной дозе 100 мкг/кг массы тела крыс приводило к снижению антитромбоцитарной активности на 29 – 49 %, активности фактора XIIIa — на 15 – 49 %. При этом повышалась антикоагулянтная активность плазмы крови крыс по тесту активированного частичного тромбопластинового времени на 16 – 33 %, по тесту тромбинового времени — на 23 – 57 %. Кроме того, под влиянием пептида KRKPGP снижалась концентрация свертываемого тромбином фибриногена на 47 – 49 %. Поскольку оба пептида при пероральном и интраназальном применении в разной степени проявляли в здоровом организме антитромбоцитарную, антикоагулянтную активность и снижали активность фактора свертывания ХШа, сделан вывод, что лизинсодержащие пептиды участвуют в регуляции свертывания крови и могут считаться перспективными антитромботическими веществами.

Specific Binding of Primary Ammonium Ions and Lysine-Containing Peptides in Protic Solvents by Hexahomotrioxacalix[3]arenes.

The binding of ammonium ions by two homooxacalix[3]arene-based receptors was studied using NMR spectroscopy and in silico methods. Both receptors are shown to endocomplex, even in a protic environment, a large variety of primary ammonium ions, including biomolecules. The binding mode is similar for all guests with the ammonium ion deeply inserted into the polyaromatic cavity and its NH3+ head nearly in the plane defined by the three oxygen atoms of the 18-crown-3 moiety, thus enabling it to establish three H-bonds with the ethereal macrocycle. The remarkable electronic, size, and shape complementarity between primary ammonium ions and the two cavity-based receptors leads to an unprecedented specificity for primary ammonium ions over secondary, tertiary, and quaternary ones. These binding properties were exploited for the selective liquid-liquid extraction of primary ammonium salts from water and for the selective recognition of lysine-containing peptides, opening new perspectives in the field of peptide sensing.

High-Resolution Lysine Acetylome Profiling by Offline Fractionation and Immunoprecipitation.

Acetylation of lysine side chains at their ε-amino group is a reversible posttranslational modification (PTM), which can affect diverse protein functions. Lysine acetylation was first described on histones, and nowadays gains more and more attention due to its more general occurrence in proteomes, and its possible crosstalk with other protein modifications. Here we describe a workflow to investigate the acetylation of lysine-containing peptides on a large scale. For this high-resolution lysine acetylome analysis, dimethyl-labeled peptide samples are pooled and offline-fractionated using hydrophilic interaction liquid chromatography (HILIC). The offline fractionation is followed by an immunoprecipitation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for data acquisition and subsequent data analysis.

β-Mercaptan-Assisted Chemical Ligation of Lysine-Containing Peptides

β-Mercaptan-Assisted Chemical Ligation of Lysine-Containing Peptides

Gold(I) Cationization Promotes Ring Opening in Lysine-Containing Cyclic Peptides.

A strategy to sequence lysine-containing cyclic peptides by MSn is presented. Doubly protonated cyclic peptides ions are transformed into gold (I) cationized peptide ions via cation switching ion/ion reaction. Gold(I) cationization facilitates the oxidation of neutral lysine residues in the gas phase, weakening the adjacent amide bond. Upon activation, facile cleavage N-terminal to the oxidized lysine residue provides a site-specific ring opening pathway that converts cyclic peptides into acyclic analogs. The ensuing ion contains a cyclic imine as the new N-terminus and an oxazolone, or structural equivalent, as the new C-terminus. Product ions are formed from subsequent fragmentation events of the linearized peptide ion. Such an approach simplifies MS/MS data interpretation as a series of fragment ions with common N- and C-termini are generated. Results are presented for two cyclic peptides, sunflower trypsin inhibitor and the model cyclic peptide, β-Loop. The power of this strategy lies in the ability to generate the oxidized peptide, which is easily identified via the loss of HAuNH3 from [M + Au]+. While some competitive processes are observed, the site of ring opening can be pinpointed to the lysine residue upon MS4 enabling the unambiguous sequencing of cyclic peptides.

α-Selective Lysine Ligation and Application in Chemical Synthesis of Interferon Gamma.

A traceless β-mercaptan-assisted α-selective ligation of N-terminal lysine-containing peptides has been developed. In this ligation-desulfurization-based protocol, the ε-amine of lysine is free of protection, thus improving the overall synthetic efficiency and avoiding harsh reactions in preparing large peptides and proteins. The applicability of this methodology has been demonstrated in the synthesis of an acid-labile therapeutic protein, interferon gamma, and the anticancer activity of synthetic protein has also been evaluated.

Comparative Characterization of the Atlantic salmon, Salmo salar L., Di/Tripeptide Transporters PepT1a and PepT1b

Amino acids and small peptides in the form of di/tripeptides are key nutrients in animal diets and essential contributors to growth. In mammals, di/tripeptides represent a highly significant fraction of dietary protein after digestion and are transported across the brush‐border membrane of the intestinal epithelium via a single transport protein, the Peptide Transporter 1 (PepT1), a member of the Solute Carrier 15 (Slc15) family. PepT1 is a low‐affinity/high‐capacity transporter that uses an inwardly‐directed proton electrochemical gradient to drive the uptake of di/tripeptides, and peptidomimetics, across the cell membrane. However, in teleost fishes two pept1 genes, namely pept1b and pept1a, have been identified. PepT1b has been studied in many teleost species, including the Atlantic (A.) salmon (Salmo salar L.), whereas PepT1a remain unexplored. The aim of our study was to characterize, the A. salmon PepT1a vs. PepT1b in terms of tissue/organ distribution and function, and possibly extrapolate their physiological role(s).Despite the extra (4th) whole genome duplication (WGD) in the Actinopterygian lineage, A. salmon has only two pept1 genes (namely pept1a and pept1b), like in other telosts that didn't undergo the extra WGD, e.g., the zebrafish (Danio rerio). At the amino acid level, the two paralogues share 77% similarity, 64% identity. Tissue distribution and relative abundance of A. salmon pept1a and pept1b revealed that both transcripts have a similar expression profile. A. salmon pept1a is highly expressed in the proximal intestine (pyloric caeca ≈ anterior midgut > midgut >> posterior midgut), but ~5‐fold less than pept1b, which is expressed in the same gut regions. No expression signals were detected in the anterior and posterior stomach or the anterior and posterior hindgut.As assessed by heterologous expression in Xenopus laevis oocytes and Two‐Electrode Voltage Clamp measurements, A. salmon PepT1a is, like PepT1b, a low‐affinity/high‐capacity system, able to sustain an electrogenic, sodium‐independent and pH‐dependent transport of di/tripeptides. Transport associated currents show that the protein exhibits a K0.5 value for the reference dipeptide Gly‐Gln of 0.5 mM at pH 7.6 and of 0.07 mM at pH 6.5.The pre‐steady state and steady‐state transport currents analysis indicate important functional differences between A. salmon PepT1a and PepT1b transporters. The difference in expression levels and a significantly dissimilar substrate specificity for certain di/tripeptides, such as lysine‐containing peptides, supports the idea of distinct roles in peptide recognition and transport for PepT1a and PepT1b.Support or Funding InformationFunded by RFFV; 247978 SalmoFeedPlus and CargillThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

5-Formylcytosine mediated DNA–protein cross-links block DNA replication and induce mutations in human cells

5-Formylcytosine (5fC) is an epigenetic DNA modification introduced via TET protein-mediated oxidation of 5-methyl-dC. We recently reported that 5fC form reversible DNA–protein conjugates (DPCs) with histone proteins in living cells (Ji et al. (2017) Angew. Chem. Int. Ed., 56:14130–14134). We now examined the effects of 5fC mediated DPCs on DNA replication. Synthetic DNA duplexes containing site-specific DPCs between 5fC and lysine-containing proteins and peptides were subjected to primer extension experiments in the presence of human translesion synthesis DNA polymerases η and κ. We found that DPCs containing histones H2A or H4 completely inhibited DNA replication, but the replication block was removed when the proteins were subjected to proteolytic digestion. Cross-links to 11-mer or 31-mer peptides were bypassed by both polymerases in an error-prone manner, inducing targeted C→T transitions and –1 deletions. Similar types of mutations were observed when plasmids containing 5fC-peptide cross-links were replicated in human embryonic kidney (HEK) 293T cells. Molecular simulations of the 11-mer peptide-dC cross-links bound to human polymerases η and κ revealed that the peptide fits well on the DNA major groove side, and the modified dC forms a stable mismatch with incoming dATP via wobble base pairing in the polymerase active site.

Computation-Guided Rational Design of a Peptide Motif That Reacts with Cyanobenzothiazoles via Internal Cysteine-Lysine Relay.

Site-selective protein modification based on covalent reactions of peptide tags and small molecules is a key capability for basic research as well as for the development of new therapeutic bioconjugates. Here, we describe the computation-guided rational design of a cysteine- and lysine-containing 11-residue peptide sequence that reacts with 2-cyanobenzothiazole (CBT) derivatives. Our data show that the cysteine residue reversibly reacts with the nitrile group on the CBT moiety to form an intermediate thioimidate, which undergoes irreversible SN transfer to the lysine residue, yielding an amidine-linked product. The concepts outlined herein lay a foundation for future development of peptide tags in the context of site-selective modification of lysine residues within engineered microenvironments.

Evaluation of Fluorine-18-Labeled α1(I)-N-Telopeptide Analogs as Substrate-Based Radiotracers for PET Imaging of Melanoma-Associated Lysyl Oxidase

Accumulating evidence suggests an unequivocal role of lysyl oxidases as key players of tumor progression and metastasis, which renders this enzyme family highly attractive for targeted non-invasive functional imaging of tumors. Considering their function in matrix remodeling, malignant melanoma appears as particularly interesting neoplasia in this respect. For the development of radiotracers that enable PET imaging of the melanoma-associated lysyl oxidase activity, substrates derived from the type I collagen α1 N-telopeptide were labeled with fluorine-18 using N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) as prosthetic reagent. With regards to potential crosslinking to tumor-associated collagen in vivo, their interaction with triple-helical type I collagen was studied by SPR. A mouse model of human melanoma was established on the basis of the A375 cell line, for which the expression of the oncologically relevant lysyl oxidase isoforms LOX and LOXL2 was demonstrated in Western blot and immunohistochemical experiments. The radiopharmacological profiles of the peptidic radiotracers were evaluated in normal rats and A375 melanoma-bearing mice by ex vivo metabolite analysis, whole-body biodistribution studies and dynamic PET imaging. Out of three 18F-labeled telopeptide analogs, the one with the most favorable substrate properties has shown favorable tumor uptake and tumor-to-muscle ratio. Lysyl oxidase-mediated tumor uptake was proven by pharmacological inhibition using β-aminopropionitrile and by employing negative-control analogs of impeded or abolished targeting capability. The latter were obtained by substituting the lysine residue by ornithine and norleucine, respectively. Comparing the tumor uptake of the lysine-containing peptide with that of the non-functional analogs indicate the feasibility of lysyl oxidase imaging in melanoma using substrate-based radiotracers.