Detecting Disulfide Bond Shuffling in Biologics Using Trypsin Platinum

Biologic therapeutics such as monoclonal antibodies and biosimilars are complex proteins that are susceptible to post-translational modifications (PTMs). These chemical modifications can affect the performance and activity of the biologic, potentially resulting in decreased potency and increased immunogenicity. Such modifications include glycosylation, deamidation, oxidation and disulfide bond shuffling. These PTMs can be signs of protein degradation, manufacturing issues or improper storage. Several of these modifications are well characterized, and methods exist for detecting them during biologic manufacture. However, disulfide shuffling is not particularly well characterized for biologics, and no methods exist to easily detect and quantify disulfide bond shuffling in biologics.

Disulfide bond shuffling occurs when the S-S linkage is not between a Cys and its normal partner
Disulfide bonds are important for protein conformation and function

Normally the cysteines in a protein will pair with a predictable or “normal” partner residue either within a polypeptide chain or between two polypeptide chains when they form disulfide bonds. These normal disulfide bonds are important for final protein conformation and stability. Indeed, disulfide bonds are considered an important quality indicator for biologics.

In a recently published study, Coghlan and colleagues designed a semi-automated method for characterizing disulfide bond shuffling on two IgG1 biologics: rituximab (originator drug Rituxan® and biosimilar Acellbia®) and bevacizumab (originator Avastin® and biosimilar Avegra®).

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Run to Remember: A Mouse-Model Study Investigating the Mechanism of Exercise-Induced Neuroprotection

Research in animal models shows physical exercise can induce changes in the brain. In humans, studies also revealed changes in brain physiology and function resulting from physical exercise, including increased hippocampal and cognitive performance (1). Several studies in mice and rats also demonstrated that exercise can improve learning and memory and decrease neuroinflammation in models of Alzheimer’s disease and other neurodegenerative pathologies (2); these benefits are tied to increased plasticity and decreased inflammation in the hippocampus in mice (2). If regular time pounding the pavement does improve brain function, what is the underlying molecular biology of exercise-induced neuroprotection? Can we identify the cellular pathways and components involved? Can we detect important components in blood plasma? And, is the benefit of these components transferrable between organisms? De Miguel and colleagues set out to answer these questions and describe their results in a recent study published in Nature.

A recent study investigates the underlying molecular mechanisms of exercise-induced neuroprotection in a mouse model.
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How Can You Improve Protein Digests for Mass Spectrometry Analysis?

Can predigestion with trypisin (ribbon structure shown) improve protein digests for mass spectrometry analysis?
Can pre-digestion with trypsin improve mass spec analysis?

The trypsin protease cleaves proteins on the carboxyterminus of Arginine (Arg) and Lysine (Lys). This cleavage reaction leaves a positive charge on the C-terminus of the resulting peptide, which enhances mass spectrometry analysis (1,2). Because of this advantage, trypsin has become the most commonly used protease for mass spectrometry analysis. Other proteases which cleave diffrently from trypsin, yielding complementary data are also used in mass spec analysis: these include Asp-N and Glu-C , which cleave acidic residues, and chymotrypsin which cleaves at aromatic residues. The broad spectrum protease, proteinase K is also used for some proteomic analyses. In a recent study, Dau and colleagues investigated whether sequential digestion with trypsin followed by the complementary proteases could improve protein digests for mass spectrometry analysis.

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RNA-Protein Interactions: A New Frontier for Drug Discovery

Almost 90% of the human genome is transcribed into RNA, but only 3% is ultimately translated into a protein. Some non-translated RNA is thought to be useless, while some play a significant yet often mysterious role in cancer and other diseases. Despite its abundance and biological significance, RNA is rarely the target of therapeutics.

“We say it’s undruggable, but I would say that ‘not-yet-drugged’ is a better way to put it,” says Amanda Garner, Associate Professor of Medicinal Chemistry at the University of Michigan. “We know that RNA biology is important, but we don’t yet know how to target it.”

Amanda’s lab develops systems to study RNA biology. She employs a variety of approaches to analyze the functions of different RNAs and study their interactions with proteins. Her lab recently published a paper describing a novel method for studying RNA-protein interactions (RPI) in live cells. Amanda says that with the right tools, RPI could become a critical target for drug discovery.

“It’s amazing that current drugs ever work, because they’re all based on really old approaches,” Amanda says. “This isn’t going to be like developing a small molecule kinase inhibitor. It’s a whole new world.”

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The Path is Clear: Trypsin Platinum is Here!

Mass spectrometry depends on the successful digestion of proteins using proteases. Many commercially available proteomic-grade trypsins contain natural contaminants that produce non-specific cleavages. Trypsin Platinum, a new protease from Promega provides maximum specificity, giving you cleaner and more conclusive data from mass spec.

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Paving New Ways for Drug Discovery & Development: Targeted Protein Degradation

The Dana-Farber Targeted Protein Degradation Webinar Series discusses new discoveries and modalities in protein degradation.

In this webinar, Senior Research Scientist, Dr. Danette Daniels, focuses primarily on proteolysis-targeting chimeras, or PROTACs. A variety of topics are covered including the design, potency, and efficacy of PROTACs in targeted protein degradation. Watch the video below to learn more about how PROTACs are shifting perspectives through fascinating research and discoveries in targeted protein degradation.

Learn more about targeted protein degradation and PROTACS here.

A Bioluminescent Biosensor for Detection of Mycotoxins in Food

3D artistic rendering of a NanoBiT assay, the system used in this study for detection of mycotoxins in food

Food contamination is a serious global health issue. According to the WHO, an estimated 600 million, almost 1 in 10 people globally, suffer from illness after eating contaminated food—and 420,000 die. Developing new technologies for more effective testing of food contaminants can help reduce that number and improve public health.

A recent application of bioluminescent technology could change the way we test for mycotoxins in the future. Dr. Jae-Hyuk Yu, Professor of Bacteriology at the University of Wisconsin-Madison, and his then graduate student, Dr. Tawfiq Alsulami, collaborated with Promega to develop a bioluminescent biosensor that enables simple and rapid detection of mycotoxins in food samples.

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Characterizing DNA Repair Proteins with Cell-Free Protein Expression

Cell-free protein expression helped researchers take a closer look at DNA double-strand breaks.

A new article in Nature Scientific Reports answers open questions about TOPBP1, a protein involved in repairing DNA double-strand breaks (DSBs). The study used cell-free protein expression and a unique DSB system to identify domains that were important for activation of a protein kinase.

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Mass Spec for Glycosylation Analysis of SARS-CoV-2 Proteins Implicated in Host-Cell Entry

The spike protein of the SARS-CoV-2 virus is a very commonly researched target in COVID-19 vaccine and therapeutic studies because it is an integral part of host cell entry through interactions between the S1 subunit of the spike protein with the ACE2 protein on the target cell surface. Viral proteins important in host cell entry are typically highly glycosylated. Looking at the sequence of the SARS-CoV-2 virus, researchers predict that the spike protein is highly glycosylated. In a recent study, researchers conducted a glycosylation analysis of SARS-CoV-2 proteins using mass spec analysis to determine the N-glycosylation profile of the subunits that make up the spike protein.

3d model of coronavirus covid-19 showing the spike protein. A recent study performed a glycosylation analysis of SARS-CoV-2 protein.

Glycans assist in protein folding and help the virus avoid immune recognition by the host. Glycosylation can also have an impact on the antigenicity of the virus, as well as potential effects on vaccine safety and efficacy. Mass spectrometry is widely used for viral characterization studies of influenza viruses. Specifically, mass spec has been used to study influenza protein glycosylation, antigen quantification, and determination of vaccine potency.

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More muscle from eggs? Proteo-lipid complex may help prevent age-associated loss of muscle-mass

In older people, low muscle mass is strongly associated with reduced functional capacity and an increased risk of disability. Myostatin is a negative regulator of muscle growth and has become an important target for pharmaceutical companies designing therapeutics to address age-associated muscle loss.

Anti-myostatin drugs increase muscle size and strength in preclinical studies. Fortetropin is a proteo-lipid complex made from fertilized egg yolk and shows anti-myostatin activity. When Fortetropin is provided as a supplement, lowered circulating myostatin levels are observed both in rodents and in young men. Fortetropin in combination with resistance exercise also lowers myostatin and increased lean body mass.

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