Revolutionizing Peptide Synthesis: A Breakthrough Cocktail For Methionine-Containing Peptides

In the world of peptide synthesis, a game-changing innovation has emerged – a remarkable cocktail designed to enhance the cleavage and deprotection of methionine-containing peptides. This groundbreaking concoction, known as Reagent H, is set to transform the landscape of solid-phase peptide synthesis, particularly for those using the 9-fluorenylmethoxycarbonyl (Fmoc) methodology.

Unveiling Reagent H: Your Key to Methionine Side-Chain Protection

Reagent H, comprised of trifluoroacetic acid (81%), phenol (5%), thioanisole (5%), 1,2-ethanedithiol (2.5%), water (3%), dimethylsulphide (2%), and ammonium iodide (1.5% w/w), has been meticulously crafted to minimize the pesky oxidation of methionine side chains during synthesis. Its exceptional performance is exemplified in the synthesis of a model pentadecapeptide from the active site of DsbC, a pivotal player in protein disulfide bond formation.

The Triumph of Reagent H: Methionine Sulphoxide Conquered

When put to the test, Reagent H outshone its competitors, cocktails K, R, and B. The crude peptides obtained from these widely used mixtures contained a staggering 15% to 55% of methionine sulphoxide. However, Reagent H demonstrated its prowess by yielding pristine peptides devoid of methionine sulphoxide. Remarkably, even when 1.5% w/w NH4I was added to cocktails K, R, and B, they couldn’t match the perfection achieved by Reagent H, although their yield of the desired peptide fell short.

Unraveling the Mysteries: A Closer Look at Reagent H’s Mechanism

But how does Reagent H achieve this remarkable feat? We delve into the proposed mechanism behind its in situ oxidation of cysteine, shedding light on its impressive ability to safeguard methionine side chains while delivering high-quality peptides.

In the world of peptide synthesis, Reagent H stands as a beacon of hope for researchers seeking purity, precision, and protection in their work. Its ability to minimize methionine side-chain oxidation is nothing short of revolutionary, promising a brighter and more efficient future for peptide synthesis enthusiasts. Say goodbye to impurities and hello to perfection with Reagent H.

Peptides Help Determine the Effects of Aging on Vision

Many sensory declines come along with aging, one of much concern being sight. That being said, there is a need for much more research on the visual changes associated with such aging. Specifically, the changes of rod bipolar cells and their ribbon synapses due to aging are an area of interest, along with the complex calcium systems at work. Using a zebrafish model, peptides help determine the effects of aging on vision and the retina.

TAMRA-RBP peptides tag rod bipolar cells

Zebrafish were used for this experiment thanks to their unique roles as model organisms; they share 70% genomic similarity with humans, and their short lifespan offers the chance to study life cycles in a few short years while still comparable to human aging over decades. Researchers compared data between middle-aged (MA, 18-months-old) and older-aged (OA, 36-months-old) zebrafish, equating to human ages of approximately 38 and 75 years of age, respectively. Using TAMRA ribbon-binding peptides from LifeTein, the team was able to observe changes between the two ages of zebrafish.

What was discovered was a decreased number of synaptic ribbons and increased ribbon length in the OA models. Further, there were many alterations to the local calcium dynamics of the system, implying a more complex change to vision deterioration than initially expected. The model shows how subtle changes could have vast implications for disease models where these alterations may be amplified, and surely sheds more light on how human vision may decline with age.

Abhishek P Shrestha, Nirujan Rameshkumar, Johane Martins Boff, Rhea Rajmanna, Thadshayini Chandrasegaran, Frederick E Courtney, David Zenisek, Thirumalini Vaithianathan
bioRxiv 2023.09.01.555825; doi: https://doi.org/10.1101/2023.09.01.555825


Innovative Approach to Combat Breast Cancer: The Impact of F11R/JAM‑A Peptide Antagonist on Metastasis

LifeTein’s Innovative Peptide Antagonist: A New Ally in the Fight Against Breast Cancer Metastasis

Breast cancer stands as the most commonly diagnosed cancer in women worldwide and is a leading cause of cancer-related deaths. Among its subtypes, triple-negative breast cancer (TNBC) is particularly notorious for being aggressive and prone to metastasizing to vital organs like the brain, lungs, bones, and liver. Despite being more responsive to chemotherapy, TNBC’s propensity for metastasis poses a significant challenge in cancer treatment.

Recent studies, including notable research from the Medical University of Lodz in Poland, have identified a key factor in TNBC metastasis: increased F11R/JAM-A activity. This protein plays a crucial role in the early stages of cancer cell migration across blood vessels, a precursor to metastasis. Enter LifeTein, a pioneering force in peptide technology, which has made a groundbreaking contribution to this research area.

LifeTein provided a specialized peptide antagonist, named P4D, designed to specifically target and inhibit F11R/JAM‑A. The effectiveness of P4D was rigorously tested in lab models. Remarkably, this antagonist not only curbed the proliferation of TNBC cells but also significantly reduced their survival by directly targeting F11R/JAM-A. The result was a notable hindrance in the metastasis process in the mouse models used for the study.

This breakthrough has significant implications. The success of P4D in these preliminary studies suggests potential for future clinical trials and paves the way for more targeted, effective treatments for TNBC, possibly extending to the development of tailored antibodies. LifeTein’s contribution to this field exemplifies its commitment to advancing cancer therapy, offering new hope to those battling with TNBC.

For more detailed insights, refer to the original study by Bednarek, R., Wojkowska, D.W., Braun, M. et al., titled “Triple negative breast cancer metastasis is hindered by a peptide antagonist of F11R/JAM‑A protein,” published in Cancer Cell International.

Bednarek, R., Wojkowska, D.W., Braun, M. et al. Triple negative breast cancer metastasis is hindered by a peptide antagonist of F11R/JAM‑A protein. Cancer Cell Int 23, 160 (2023).

Innovative Peptide Technology in Click Chemistry: Paving the Way for Smarter Drug Delivery

As methods of medicine advance, targeted drug delivery becomes a more appealing and achievable option over its non-selective counterpart. It can focus solely on increasing therapeutic concentration in the target area while greatly eliminating any exposure to healthy tissue, and thus drastically lowering side effects as well. The effective and simple mechanisms of click chemistry are a great way to design payloads for these targeted drug delivery methods. With the use of enzyme-degradable peptides in click chemistry drug delivery, lasting therapeutics can remain in the system for local sustained release over time as well.

Enzyme-degradable peptides for sustained drug delivery

The team at Rutgers focused on a two-phase method to set up the targeted drug delivery. First, ROS-sensitive PEGDA and acrylate-PEG-azide are aimed at the target area, driven by elevated free radical levels. Once the pretargeting is complete, a payload tethered to DBCO is delivered and captured via azide-DBCO reactions. Enzyme-degradable peptides were provided by LifeTein and incorporated into both steps for the ongoing release of the captured payloads.

The results showed success in the models tested, with the initial dosage still effective in capturing the payload several days later. This system demonstrated the versatility of a two-phase method, where long-term effects are even further avoided by incorporating enzyme-degradable peptides. The proof of concept displayed here has great promise for the future of drug delivery and just goes to show how applicable click chemistry is to even more fields.

Emily T. DiMartini, Kelly Kyker-Snowman & David I. Shreiber (2023) A click chemistry-based, free radical-initiated delivery system for the capture and release of payloads, Drug Delivery, 30:1, DOI: 10.1080/10717544.2023.2232952

Convergent Evolution of Holocentromeres Studied with Peptides

During cell division, microtubules in the chromosome attach to a region called the centromere. While most species have a single size-restricted centromere, or a monocentromere, some species exist with multiple centromeres distributed across the chromosome, called holocentromeres. What is even more interesting is how holocentric chromosomes are considered to have evolved from the monocentric organisms, and this transition occurred independently across distant lineages, such as green algae, protozoans, invertebrates, as well as flowering plant families. One group aimed to study these holocentromeres more via the lilioid Chionographis japonica. Their goal was to better understand the convergent evolution of holocentromeres studied with peptides.

Peptides help explore holocentromeres

The group determined that the chromosomal localization of the target centromere is usually marked with histone H3 (CENH3). With this knowledge, they utilized peptides and antibodies of CENH3 provided by LifeTein to create models of the transition of C. japonica from interphase to prophase and study the possible mechanisms as well. They found the holocentromere was made up only of a few, evenly spaced CENH3-positive megabase-sized satellite arrays. Overall, the reason for the convergent evolution of holocentromeres from a monocentromere may stem from multiple factors, but more experiments like the ones presented will surely provide further analysis into this complex and fascinating case of convergent evolution.
Kuo, YT., Câmara, A.S., Schubert, V. et al. Holocentromeres can consist of merely a few megabase-sized satellite arrays. Nat Commun 14, 3502 (2023). https://doi.org/10.1038/s41467-023-38922-7

Protease OMA1 Activity is Measured by MCA Fluorescent Peptide

– Fig. 1. Basis of the OMA1 activity using fluorescence-based peptide.
Fluorescence is released when OMA1 recognizes and cleaves the OPA1 8-mer
peptide (fluorescence reporter) presumably at the RA site, from the cited paper

The continual fission and fusion the Mitochondria undergoes to change its shape and function are a key trait of the organelle, one that is regulated by the enzyme OMA1. However, there is little known regarding OMA1 due to the lack of a consistent method to measure its activity. More information is needed to truly gauge the role of OMA1 as a therapeutic agent. This is where one group sought to measure this activity utilizing a fluorescence-based reporter cleavage assay, one where the protease OMA1 activity is measured by MCA fluorescent peptide.

OMA1 activity measured by (MCA-AFRATDHG-(lys)DNP) peptide

The group arrived at this specific sequence as it includes the specific point on protein OPA1 (between the arginine and alanine) that OMA1 cleaves. They would then be able to spectrofluorometrically measure the fluorescent MCA moiety after the cleavage takes place. The assay proved successful in measuring the activity of OMA1, and in an inexpensive manner. The work clearly lays out the foundation for future studies of OMA1, in both its normal and abnormal pathology.

Julia Tobacyk, Nirmala Parajuli, Stephen Shrum, John P. Crow, Lee Ann MacMillan-Crow, The first direct activity assay for the mitochondrial protease OMA1, Mitochondrion, Volume 46, 2019, Pages 1-5, ISSN 1567-7249, https://doi.org/10.1016/j.mito.2019.03.001.

Multi-Epitopic Peptide Vaccines that Combat Crimean–Congo Hemorrhagic Fever Virus

Tick infestations are a recurring roadblock of human development around the world, with estimated damages in the global economic landscape being as high as 30 billion USD. Specifically, India has long been susceptible to tick-borne diseases, due to multiple species invading the livestock. These regional parasites are major vectors for Crimean–Congo hemorrhagic fever virus (CCHFV), a disease with a devastating case fatality rate of 10–40%. While the main way of combatting the infestation of ticks and their carried disease has always been pesticides, often to an invasive degree of their own, scientists are working diligently for ways to produce a vaccine for this deadly and prevalent outbreak. One such method that has been explored is multi-epitopic peptide vaccines that combat Crimean–Congo hemorrhagic fever virus, specifically through the potential immune stimulatory responses they cause.

Multi-epitopic peptides help boost immune system

LifeTein provided the group with the two designed multi-epitopic peptides, VT1 and VT2. Using the two peptides, the group put them into two working vaccines in an effort to explore how effective they were at fighting back the ticks. With rabbits, they found strong immunity conferred by the vaccine, displayed by quick larval detachment, delayed tick feeding, low engorgement weights, and overall efficacy against both tick larvae and adults. The results show just how effective treatment with the vaccines are against ticks carrying CCHFV, and the compatibility with rabbits is a great starting point.

In an ideal experiment, the group would have tested on cattle, since that is a much more affected group by these ticks. Regardless, the suitability and stability displayed warrants more attention be put into these multi-epitopic peptide vaccines. Their efficacy displayed against infestations as such is sure to save the global economy billions, as well as countless lives. Immunization in this route is surely more appealing than that of constant and overwhelming pesticides being put in place at every conceivable turn. LifeTein is excited to see where else peptide-based vaccines can be implemented and what other unique properties they can bring to the table.

Nandi A, Manisha, Solanki V, Tiwari V, Sajjanar B, Sankar M, Saini M, Shrivastava S, Bhure SK, Ghosh S. Protective Efficacy of Multiple Epitope-Based Vaccine against Hyalomma anatolicum, Vector of Theileria annulata and Crimean–Congo Hemorrhagic Fever Virus. Vaccines. 2023; 11(4):881. https://doi.org/10.3390/vaccines11040881


LifeTein Cell Penetrating Peptides and Machine Learning

Cell-penetrating peptides (CPPs) are a longstanding part of the biochemical world, with their potential for delivering bioactive agents into cells, their importance will likely never diminish. However, not many advancements are being made to CPPs, rather scientists and teams alike are always looking to develop more efficient CPPs when possible. This is where a hot topic of recent comes into play, researchers utilized a deep-learning-based CPP prediction method, one they have named AiCPP, to effectively develop novel CPPs while reducing false-positive predictions. Using LifeTein cell-penetrating peptides and machine learning, AiCPP was able to combine data with other lists of CPPs to generate successful new sequences that the team at hand was able to test effectively.

LifeTein CPPs help train AI to generate new sequences

The group utilized a sliding window approach on their wealth of data and included a list of peptides with low similarity to CPPs as a negative to reduce false positives obtained. These techniques helped AiCPP stand out against other machine learning methods before it, and they found AiCPP can further optimize CPP sequences with higher efficacy as well. Valuable information can be gathered by studying the patterns by which this machine learning determines are effective for CPPs.

Though novel, the limitations of utilizing this and other machine-learning methods should be understood as well. Foremost, this and other CPP prediction studies do not answer any important questions about the mechanisms of cell permeation, or how each CPP specifically achieves this. It is also worth noting that the effectiveness of a given CPP is limited to the type of cell is it trying to penetrate, for example, MCF-7 used in the referenced study. Though, these shortcomings are possible to overcome, and in the future, AiCPP may become more advanced and be able to offer even more research, with LifeTein keeping up as well and ready to assist any team that may need their new CPPs developed.

Park H, Park J-H, Kim MS, Cho K, Shin J-M. In Silico Screening and Optimization of Cell-Penetrating Peptides Using Deep Learning Methods. Biomolecules. 2023; 13(3):522. https://doi.org/10.3390/biom13030522

Fibrin and Short Peptides Co-assembled into Hydrogels

peptide hydrogel
Schematic drawing of the co-assembled peptide hydrogels for regenerative medicine developed in the work from the cited paper.

Fibrin Hydrogels are a widely used material in regenerative medicine, often made with fibrinogen obtained from human plasma. However, this process can be very costly and have problems when used in other applications due to their limited mechanical properties. Researchers were interested in the prospect of composite hydrogels, made from the self-assembly of fibrinogen together with Fmoc-FF and Fmoc-RGD. Their results saw that these hydrogels co-assembled from short peptides and fibrin display biocompatibility, as well as the enhanced mechanical properties they were looking for.

Fibrin hydrogel enhanced with short peptide co-assembly

LifeTein provided the group with the essential peptides, Fmoc-FF and Fmoc-RGD, where the team tested different ratios of these peptides in combination with the fibrin precursor. Even from the naked eye alone, they could immediately tell the difference when the peptides were added in the presence of CaCl2. The resulting mixture displayed very favorable and biocompatible characteristics, even similar to the fibrin hydrogels it was replicating. The mixture could even jellify in situ, potentially allowing the pregel to be administered via injection

The protocol the group explored holds great potential for the future of regenerative medicine. Not only do they not cause any inflammatory response, but the possibilities still exist to modify these fibrin hydrogels for even further medicinal usage. The Fmoc-FF and Fmoc-RGD peptides used for the co-assembly of this hydrogel are commercially available, and companies like LifeTein are always ready to supply vital peptides like these to any groups researching to better our future.

Cristina Gila-Vilchez, Mari Carmen Mañas-Torres, Óscar Darío García-García, Alfredo Escribano-Huesca, Laura Rodríguez-Arco, Víctor Carriel, Ismael Rodriguez, Miguel Alaminos, Modesto Torcuato Lopez-Lopez, and Luis Álvarez de Cienfuegos
ACS Applied Polymer Materials 2023 5 (3), 2154-2165
DOI: 10.1021/acsapm.2c02164


Coxsackievirus A21 Viral Proteins Quantified with Capillary Western and Peptides

Simple Western Identity of the capsid peptides, from the cited paper

Oncolytic viruses are a novel type of virus that specifically targets and eliminates tumor cells, and have been an emerging immunotherapy agent. One such is Coxsackievirus A21 (CVA21), which has been demonstrating a healthy safety profile as well as desired targeting of the cancerous cells. While producing drugs that can properly utilize this virus, there rises a need to characterize the ratio of total particles to empty ones, thus researchers developed a method to see Coxsackievirus A21 viral proteins quantified with capillary Western and peptides made up of the components of the capsid proteins.

CVA21 characterized with capillary Western and capsid peptides

LifeTein provided the group with the capsid peptides, VP1, VP2, VP3, and VP4, as well as the antibodies necessary for the testing. Results utilizing these peptides and antibodies included total particle concentration, empty particle concentration, as well as the empty to full ratio. Being able to characterize this kind of information in this downstream process will prove to be immensely valuable in future studies.

The Simple Western method displayed by the researchers proved itself to be an invaluable tool for optimizing decisions for these types of oncolytic virus studies. Consistency in these tests will surely lead to massive amounts of time being saved in future drug development and assessments. LifeTein will always be ready to lend a hand to any work needing peptides or antibodies to further their research in the broad field of drug application.

Paul F. Gillespie, Richard R. Rustandi, Andrew R. Swartz, Liang Shang, Jessica Raffaele, Ashley Prout, Nicholas Cunningham, Mohamed Dawod, James Z. Deng, Shiyi Wang, Jessica Olson, Yvonne Shieh, and John W. Loughney.
Quantitation of Coxsackievirus A21 Viral Proteins in Mixtures of Empty and Full Capsids Using Capillary Western.
Human Gene Therapy.Jan 2023.68-77.http://doi.org/10.1089/hum.2022.147