The reaction of maleimides with thiols is widely used for bioconjugation and labeling biomolecules such as proteins and peptides. Maleimides are electrophilic compounds that show high selectivity towards thiols.
The Reaction of Maleimides With Thiols
1. Dissolve the peptide or other biomolecules containing thiol in a degassed buffer (PBS, Tris, or HEPES) at pH 7-7.5. 2. Add a 100x molar excess of TCEP (tris-carboxyethyl phosphine) reagent to reduce disulfide bonds. 3. Dissolve maleimide in DMSO or fresh DMF (1-10mg in 100uL). 4. Add dye solution such as cy5 maleimide to thiol solution (20x fold excess of dye), flush with an inert gas, and close tightly. 5. Mix thoroughly and keep at room temperature or 4C overnight. 6. Purify by gel filtration, HPLC, FPLC, or electrophoresis.
Interest in personalized treatment has been fuelled by the concept of tailoring therapy with the best response and highest safety margin to ensure better patient care. Personalized medicine holds promise for improving health care while also lowering costs.
Synthetic Peptides for Personalized Treatment
An immunogenic personal neoantigen vaccine for melanoma patients using synthetic peptides provides an opportunity to develop agents targeted to patient groups that do not respond to medications as intended and for whom the traditional health systems have otherwise failed.
The T cell epitopes with tumor-specific expression arising from non-silent somatic mutations are not expressed in normal tissues. These neoantigens are mutated peptides with the high-affinity binding of autologous HLA molecules.
The vaccination with neoantigens can induce new T cell specificities in cancer patients. Using the synthetic peptides as a personalized vaccine, researchers found that of 6 vaccinated patients, 4 had no recurrence at 25 months post-vaccination.
The T cells discriminated mutated from wild-type peptide antigens and directly recognized autologous tumors. Based on HLA binding predictions, immunizing peptides were selected from this study. Each patient received up to 20 long peptides in 4 pools.
Some fusion or chimeric proteins could never be produced from the E.coli expression system, especially when several hydrophobic sequences are involved in the functional domains. Obtaining peptides sized 100–200 amino acids using chemical synthesis is much faster and cheaper than cloning and overexpressing in Escherichia coli. In addition, the resulting peptide is always correct. Chemical synthesis can regularly incorporate non-genetically encoded structures, such as D-amino acids, into the protein. Synthetic peptides eliminate problems such as poor or no expression, cloning errors, tags like FLAG or 6-His, or the mistranslation of non-preferred codons in prokaryotic hosts. Artificial amino acids that have isosteric side chains can be used to investigate the functional importance of specific residues. The peptide design and synthesis using click chemistry can achieve all these chimeric proteins.
Histone methylation, a process that can signal either transcriptional repression or activation, is increasingly recognized for its interrelation with DNA methylation in mammals. For instance, the targeting of DNA methylation is intricately linked to H3K9 methylation, a key regulatory mechanism in gene expression. The p53 gene, known as the guardian of the genome and frequently mutated in human cancers, is regulated by various PTMs, including methylation.
Post-translational modifications (PTMs) of histone proteins, such as acetylation, methylation, and phosphorylation, are pivotal in regulating chromatin dynamics. Among these, the role of methylation, particularly at arginine or lysine residues, stands out for its complexity and significance. LifeTein, a leader in peptide synthesis, has contributed significantly to this field by synthesizing mono-, di-, or tri-methylated peptides. These peptides are instrumental in studying protein-protein interactions, especially in the context of histone methylation.
LifeTein’s contribution to this research is highlighted in a study focusing on the ASHH2 CW domain, which recognizes the methylation state at lysine 4 of histone 3 N-terminal tails. This domain is crucial in recruiting the ASHH2 methyltransferase enzyme to histones. The study utilized H3 histone tail mimicking peptides, specifically monomethylated (ARTK(me1)QTAR), dimethylated (ARTK(me2)QTAR), and trimethylated (ARTK(me3)QTAR) peptides, all synthesized by LifeTein with a remarkable 95% purity as confirmed by mass spectrometry.
The research documented the assignment of a shortened ASHH2 CW construct, CW42, which showed similar binding affinity and better expression yields than previous constructs. This advancement is significant in understanding how different methylation states affect protein-peptide interactions. The study also performed 1H–15N HSQC-monitored titrations to determine the saturation point of the protein-peptide complex. The findings revealed that the CW42 domain, when bound to the monomethylated histone tail mimic, showed similar perturbations in shifts as the di- and tri-methylated instances.
In summary, LifeTein’s synthetic methylated peptides have been instrumental in advancing our understanding of histone methylation. Their high-purity peptides have enabled researchers to delve deeper into chromatin dynamics and gene regulation complexities, paving the way for future discoveries in epigenetic therapies and cancer treatment.
Noble metal gold (Au) and silver (Ag) nanoparticles (NPs) are used to conjugate with M3 peptides. The AuNPs-sGFP and AuNPs-M3 peptide forms an active SERS hot spot through self-assembly and GFP complementation. The nanoparticles self-assemble into surface-enhanced Raman-scattering (SERS) nanoclusters. The nanocluster can be a contrast agent for multimodal SERS and photoacoustic microscopy with single-cell sensitivity.
AuNPs coated with M3 peptides-GFP
Reference: M3 peptide was purchased from LifeTein.
Cellular imaging by targeted assembly of hot-spot SERS and photoacoustic nanoprobes using split-fluorescent protein scaffolds
Using D-amino acids as the building blocks for bioactive peptides can dramatically increase their potency. In this study, the authors generated a database of ∼2.8 million D-peptides using a mirror image of every structure in the Protein Data Bank (PDB). The critical or hotspot residues were studied. Residues critical to target binding and activity can be ideally done experimentally, such as by alanine scanning mutagenesis. It can also be done computationally through thermodynamic integration or free energy perturbation.
D-Amino Acids for Bioactive Peptides
Two peptides were tested to prove the concept: GLP-1 and Parathyroid Hormone. Both (L)- and (D)-peptides were synthesized by Lifetein LLC.
GLP-1 is a helical GPCR agonist as a diabetes mellitus and obesity treatment. Hotspot and junction residues are annotated in green and blue, respectively. The authors investigated the ability of (D)-GLP1 peptide to induce activation of GLP1R and compared the response with native (L)-GLP1 peptide. It was found that the D-GLP-1 performed well and was resistant to protease degradation. The retro-inversion (RI) reversing the (D)-peptide sequence was used in the experiment.
2. Parathyroid Hormone (PTH) is an FDA-approved treatment for osteoporosis. The (D)-PTH activates PTH1R with potency and efficacy comparable to (L)-PTH. And more than 85% of the (D)-PTH analog is still detectable at six hours.
PTH 1-34: SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF
Conclusion: The D-Protein Data Bank (PDB) can be used to search and find therapeutically active topologies. The D-PDB could be a vital tool for finding stable lead molecules in early-stage drug discovery.
Zonula occludens toxin (Zot) and its biologically active fragment, delta G, have been shown to reversibly open tight junctions (TJ) in endothelial and epithelial cells. AT1002, a six-mer synthetic peptide H-FCIGRL-OH of ZO toxin, was identified and synthesized to retain the Zot permeating effect on intercellular TJ. It was found that AT1002 disrupts the epithelial barrier while larazotide acetate restores barrier function by rearrangement of actin. In addition, AT1002 enhances the transport of molecular weight markers or agents with low bioavailability with no cytotoxicity. So, this synthetic peptide AT1002 is a tight junction modulator with promising permeation-enhancing activity.
A Synthetic Peptide Showed Enhanced Nasal Drug Delivery
The C-terminal amidated AT1002 FCIGRL-NH2 showed enhanced nasal drug delivery and may lead to the development of a practical drug delivery technology for drugs with low bioavailability.
LifeTein synthesized the synthetic peptide AT1002.
It was found that the HIV-1 Integrase binds the Viral RNA genome and is essential during Virion Morphogenesis. The L50 Peptide: Cyclo-(-Arg-Val-Arg-Thr-Arg-Gly-LysArg-Arg-Ile-Arg-Arg-DPro-Pro-) was synthesized by LifeTein and used for the inhibition assays. The L50 is a Tat-derived peptide sequence, which selectively engages both the loop and 3-nt bulge but not the double-stranded stem of the transactivation response. The TAT-derived peptide is a well-defined cell penetration peptide.
The SrtA substrates Biotin–aminohexanoic acid–LPETGS and SELPETGG were used to interact with immune cells ‘Labelling Immune Partnerships by SorTagging Intercellular Contacts’ (LIPSTIC). The peptide-receptor interactions enable the direct measurement of dynamic cell-cell interactions. The peptides are flexible tools with different receptor-ligand pairs and a range of detectable labels.
Peptide Biotin–aminohexanoic acid–LPETGS, SELPETGG was purchased from LifeTein.
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