Protein Analysis and Purification

Unlocking the Secrets of ADP-Ribosylation with Arg-C Ultra Protease, a Key Enzyme for Studying Ester-Linked Protein Modifications 

Posted on by Michele Arduengo

Post-translational modifications of proteins are critical for proper protein function. Modifications such as phosphorylation/dephosphorylation can act as switches that activate or inactivate proteins in signaling cascades. The addition of specific sugars to membrane proteins on cells are critical for recognition, interaction with the extracellular matrix and other activities. While we know volumes about some types of protein modifications, ADP-ribosylation on aspartate and glutamate residues has been more difficult to study because of the chemical instability of these ester-linked modifications. 

Matić Lab (Eduardo José Longarini and Ivan Matić) recently published a study that explored mono-ADP-ribosylation (ADPr) on aspartate and glutamate residues by the protein PARP1 and its potential reversal by PARG. PARP1 and PARG signaling are central to DNA repair and apoptosis pathways, making them potentially powerful therapeutic targets in cancer or neurodegenerative diseases in which DNA repair processes are often disrupted. 

Continue reading “Unlocking the Secrets of ADP-Ribosylation with Arg-C Ultra Protease, a Key Enzyme for Studying Ester-Linked Protein Modifications “

Fluorescent Ligands in Biological Research: Where We’ve Been, Where We’re Headed

Posted on by Kari Kenefick

Fluorescent tags (fluorophores), have become excellent tools for labeling cells and cellular components. They can be used for imaging large molecules like proteins, on down to cellular components and enzymes such as transcription factors. Once labeled, these molecules can be tracked in tissue or inside a cell, when the right tag is used.

What is the ‘right’ tag? It’s a tag with bright signal, with low background and good photostability. For small cell components like organelles, the tag must be cell-permeable and small enough to not interfere with normal cellular processes such as transcription and metabolism.

Significant advances have been made in fluorescent tags in the past two decades. Here we look at several papers noting these advances.

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Designing Better Therapeutic mAbs: An Assay for Rapid, Parallel Screening of Fc/ FcɣR Interactions

Posted on by Michele Arduengo

The first monoclonal antibody (mAb) was produced in a lab 1975, and the first therapeutic mAb was introduced in the United States to prevent kidney transplant rejection in 1986. The first mAb used in cancer treatment the anti-CD20 mAb, rituximab, was used to treat non-Hodgkin’s lymphoma and chronic lymphocytic leukemia. Today therapeutic mAbs have become a mainstay of cancer, autoimmune disease, and metabolic disease therapies and include HERCEPTIN® used to treat certain forms of breast cancer, Prolia used to treat bone loss in post-menopausal women, and Stelara used to treat autoimmune diseases like psoriatic arthritis and severe Crohn disease, among many others. Therapeutic mAbs bind targets with high specificity and affinity and they can recruit effector cells to drive target elimination through mechanisms such as antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP), making them highly specific, effective therapies.

3D rendering of a Lumit Assay which can be used for plate-based screening assay to measure the affinities of Fc interactions of therapeutic mAbs.
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Use of ProAlanase Digestion Increases Number of Identified Methylglyoxal (MGO)-Modified Proteins in Whole-Cell Lysates

Posted on by Michele Arduengo
space filling structural model methylglyoxal (MGO)
Methylglyoxal is responsible for post translational protein modifications, that result in advanced glycation endproducts (AGEs), which are associated with aging and disease.

Post-translational modifications (PTM) of proteins are essential for the function of many proteins, but aberrant modification of protein residues also can interfere with protein function. PTMs occur in two ways. Proteins may be modified through the activity of enzymes such as kinases, phosphorylases, glycosylases and others that add or remove specific chemical moieties to amino acid residues. PTMs can also result from non-enzymatic reaction between electrophilic compounds and nucleophilic arginine and lysine residues within a protein. Metabolites and metabolic by products produced during glycolysis, especially glyoxal and methylglyoxal (MGO), are examples of such electrophilic compounds. These compounds can react with arginine and lysine to produce advanced glycation end products (AGEs), which are biomarkers associated with aging and degenerative diseases such as Alzheimer disease, diabetes and others. MGO is also elevated in tumors that have switched from oxidative phosphorylation to glycolysis as their main energy production pathway.

Only limited information is available about site-specific MGO PTMs in mammal cells, and most studies have focused on measuring the amount of MGO modifications in a treatment scenario compared to a control. Donnellan and colleagues recently published work to identify specific MGO protein modifications.  They used a “bottom-up” proteomic analysis of WIL2-NS B lymphoblastoid whole-cell lysates to identify specific MGO-modified proteins. In particular, the group was looking for modifications in proteins that might explain how MGO activity contributes to aneuploidy.

For the study, 100µg of cellular protein extract was reduced with dithiothreitol and then alkylated with chloroacetamide. The sample was diluted to reduce urea concentration. Trypsin Gold was added and samples were digested for 8 hours at 37°C. Digestion was terminated by adding formic acid. For ProAlanase digestion, 20µg of protein was reduced, alkylated and diluted to reduce urea concentration before adding digesting with ProAlanase for 4 hours at 37°C.

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The authors identified 519 MGO-modified proteins.  Most of the modifications were identified in the trypsin digestion reactions; however, ProAlanase digestion increased the number of MGO modifications identified by approximately 25% (with less than 4% of the modification sites being detected in both the ProAlanase and trypsin digestion reactions. The authors suggest that ProAlanase increased sequence coverage to reveal sites not detected in the trypsin digestions. Therefore, they conclude that ProAlanase can be used along with trypsin digestion to increase the identification of MGO modifications.

ProAlanase can be used along with trypsin digestion to increase the identification of MGO modifications.

MGO-modified proteins from the WIL2-NS whole cell lysates included proteins involved in glycolysis, translation initiation, protein folding, mRNA splicing, cell-to-cell adhesion, heat response, nucleosome assembly, protein SUMOylation and the G2/M cell cycle transition. More work to further characterize the sites of these modifications and their potential effects on the function of the modified proteins is ongoing.

Read more about ProAlanase, a new site-specific endoprotease from Promega.

Literature Cited

Donnelian, L et al. (2022) Proteomic Analysis of Methylglyoxal Modifications Reveals Susceptibility of Glycolytic Enzymes to Dicarbonyl Stress Int. J. Mol. Sci. 23(7), 3689 doi.org/10.3390/ijms23073689

Detecting Disulfide Bond Shuffling in Biologics Using Trypsin Platinum

Posted on by Michele Arduengo
polypeptide chain showing protease cleavage sites
Learn about the new Trypsin Platinum Protease in this blog.

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

Posted on by Michele Arduengo

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?

Posted on by Gary Kobs
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 differently 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

Posted on by Jordan Villanueva

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!

Posted on by Gary Kobs

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.

Trypsin is typically extracted from bovine or porcine pancreas. In addition to trypsin, both of these sources also contain chymotrypsin. To suppress chymotryptic activity, trypsin is treated with tosyl phenylalanyl chloromethyl ketone, or TPCK, to irreversibly inhibit the chymotrypsin. However, trace amounts of chymotrypsin appear to escape this inhibition and produce non-specific cleavages, as seen in the figure below.

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

Posted on by Riley Bell

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.