Mass Spec Analysis of PTMs Using Minimal Sample Material

DNA is organized by protein:DNA complexes called nucleosomes in eukaryotes. Nucleosomes are composed of 147 base pairs of DNA wrapped around a histone octamer containing two copies of each core histone protein. Histone proteins play significant roles in many nuclear processes including transcription, DNA damage repair and heterochromatin formation. Histone proteins are extensively and dynamically post-translationally modified, and these post-translational modifications (PTMs) are thought to comprise a specific combinatorial PTM profile of a histone that dictates its specific function.  Abnormal regulations of PTM may lead to developmental disorders and disease development such as cancer.

Antibodies have been widely used to characterize histones and histone PTMs. However, antibody-based techniques have several limitations. Mass spectrometry (MS) has therefore emerged as the most suitable analytical tool to quantify proteomes and protein PTMs.  The most commonly used strategy is still bottom-up MS, and the most widely adopted protocol includes derivatization of lysine residues in histones to allow trypsin to generate Arg-C like peptides (4–20 aa). However, samples such as primary tissues, complex model systems, and biofluids are hard to retrieve in large quantities. Because of this, it is critical to know whether the amount of sample available would lead to an exhaustive analysis if subjected to MS.

In a recent publication, Guo, et al. examined (1) the reproducibility in quantification of histone PTMs using a wide range of starting material: from 50,000 to 5,000,000 cells. They used four different cell lines: HeLa, 293T, human embryonic stem cells (hESCs), and myoblasts. Their results demonstrated that an accurate quantification of abundant histone PTMs can be efficiently obtained by using low-resolution MS and as low as 50,000 cells as starting material Low abundance histone marks showed more variability in quantification when comparing different amounts of starting material, so a larger amount of starting material (at least 500,000 cells) is recommended.

Reference

Guo, Q. et al. (2017) Assessment of Quantification Precision of Histone Post-Translational Modifications by Using an Ion Trap and down To 50,000 Cells as Starting Material. J. Proteome Res. 17, 234–42.

A Virus-like Neural Pathway Hints at the Origins of the Mammalian Brain

The mammalian brain is extremely complex. We know that it processes and stores information through synaptic connections within a complicated neural network. But how exactly do neurons communicate with each other? And how did this neural network come to exist? A recent paper published in Cell may provide some answers. It describes a previously unknown signaling pathway–with surprising origins–that transports RNA between neurons. Continue reading

Two Epigenetic Targets Are More Effective Than One

Lysine-specific histone demethylase 1 (LSD1) via Wikimedia Commons

Epigenetics is a new and exciting territory to explore as we understand more about the role it plays in gene silencing and expression. Because epigenetic regulation of gene expression is caused by specific modification of histone proteins (e.g., methylation) that play a role in disease states like cancer, enzymes like histone deacetylases (HDACs) become viable drug targets. One drawback to inhibiting proteins that modify histones is even when selectively targeting HDACs, the effects can be far ranging with multiple HDAC-containing protein complexes found throughout the cell. These broad effects minimize the effectiveness of an inhibitor, caught between efficacy and toxicity. A recent article in Nature Communications explored how using a single compound to target two epigenetic enzymes was more effective than any individual inhibitor or combination of inhibitors. Continue reading

Top 5 Most Read Promega Papers in 2017

It’s always nice to know that someone is reading your paper. It’s a sign that your research is interesting, useful and actually has an impact on the scientific community. We were thrilled to learn that papers published by Promega scientists made the top 10 most read papers of 2017 in the journal ACS Chemical Biology. In fact, Promega scientists authored five of the top six most read papers! Let’s take a look at what they are.

#5 CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide

Publication Date (Web): September 11, 2017

This 2017 paper introduces our newest star: HiBiT, a tiny 11aa protein tag. To any scientist studying endogenous protein expression, the HiBiT Tagging System is your dream come true. It combines quantitative and highly sensitive luminescence-based measurement with a tiny-sized tag that can be easily inserted into endogenous protein via CRISPR/Cas9 gene editing with little impact on native protein function. The HiBiT Tagging System has been listed as a 2017 Top 10 Innovation by The Scientist, and it will drastically change how we study endogenous protein expression. Continue reading

Luciferase Immunoprecipitation System Assay (LIPS): Expression of Luciferase Antigen using TNT Transcription/Translation Kit

NanoLuc dual reporters

Illustration showing NanoLuc and firefly luciferase reporters.

The luciferase immunoprecipitation system (LIPS) assay is a liquid phase immunoassay allowing high-throughput serological screening of antigen-specific antibodies. The immunoassay involves quantitating serum antibodies by measuring luminescence emitted by the reporter enzyme Renilla luciferase (Rluc) fused to an antigen of interest. The Rluc-antigen fusion protein is recognized by antigen-specific antibodies, and antigen-antibody complexes are captured by protein A/G beads that recognize the Fc region of the IgG antibody (1).

In a recent publication (2), this assay was used to assess the presence of autoantibodies against ATP4A and ATP4B subunits of parietal cells H+, K+-ATPase in patients with atrophic body gastritis and in controls. Continue reading

They’re Eating WHAT? Understanding Ecosystems Through Weird Meals

A few days ago, while taking an unplanned distraction break on Facebook, I came across a video of an enormous coconut crab attacking a red-footed booby. The footage was captured by a biologist studying crab behavior in the Chagos Archipelago in the middle of the Indian Ocean. On this trip he had already confirmed that the monstrous crustaceans snacked on large rats, but he never expected to watch one devour a full bird.
This video sent me on a research journey into other interesting meals discovered by animal researchers. Besides providing sensational headlines about what’s eating what, these studies help us understand everything from nutrient exchange to learned behavior. I’ve compiled a short list of observations and discoveries made in the past few months where researchers have used weird meals to understand complex phenomena. Warning: this might get gruesome! Continue reading

Ancient Images of Dogs Include Restraints?

This dog is wearing a leash.

You, like me, may occasionally find youself in need of a canine control device. While I’m not a fan of the dog tie out, I do walk dogs on leash—as is required by our county and city government here in Madison, WI.

If you have read Temple Grandin’s books about dogs, you might feel a tug at your heartstrings while enduring a tug on the leash. Aren’t dogs meant to run freely? Don’t we love to watch them run? Are leashes humane?

When walking dogs I feel the need to protect them, but also a desire to let them live like dogs, sniffing, marking and other behaviors that are all limited when the dog is leashed.

When a report in Science last week showed evidence that our ancient ancestors were using leashes 8,000-9,000 years ago I was: 1) surprised; and 2) felt vindicated from self-imposed dog owner guilt.

Continue reading

Using CellTiter-Glo® Luminescent Cell Viability Assay to Assess Cell Viability in Cancer Cells Treated with Silver Nanoparticles and DNA-PKcs Inhibitor

Silver nanoparticles (Ag-np) are commonly used in many consumer products, including cosmetics, textiles, electronics and medicine, largely due to their antimicrobial properties. More recently, Ag-np are being used to target and kill cancer cells. It has been known for years that silver nanoparticles (Ag-np) can induce cell death and DNA damage. Studies have also shown that Ag-np inhibit cell proliferation and induce apoptosis in cancer cells. However, cancer cells are able to fight back with DNA repair mechanisms such as non-homologous end joining repair (NHEJ). The NHEJ pathway requires the activation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), thus DNA-PKcs may protect against the Ag-np-induced DNA damage in cancer cells.

Could inhibition of DNA-PKcs increase the ability of Ag-np to kill cancer cells? In a 2017 study, Lim et al. wanted to test whether inhibition of DNA-PKcs can increase the cytotoxic effect of Ag-np in breast cancer and glioblastoma cell lines. To effectively determine cell viability in these cancer cell lines, the authors used the CellTiter-Glo® Luminescent Cell Viability Assay. The CellTiter-Glo® Assay determines the number of viable cells in culture based on quantitation of ATP, an indicator of metabolically active cells. A major advantage of this assay is its simplicity. This plate-based assay involves adding the single reagent (CellTiter-Glo® Reagent) directly to cells cultured in serum-supplemented medium. This generates a luminescent signal proportional to the amount of ATP present, which is detected using a luminometer. Cell washing, removal of medium and multiple pipetting steps are not required. Another advantage of the CellTiter-Glo® Assay is its high sensitivity. The system detects as few as 15 cells/well in a 384-well format in 10 minutes after adding reagent and mixing, making it ideal for automated high-throughput screening, cell proliferation and cytotoxicity assays.

The authors first confirmed that Ag-np treatment reduced proliferation and induced cell death/DNA damage in two breast cancer cell lines and two glioblastoma cell lines. The cytotoxic effect of Ag-np is specific to cancer cells, as minimal cytotoxicity was observed in non-cancerous human lung fibroblasts used as control. Next, they pre-treated the cancer cells with a DNA-PKcs inhibitor for 1 hour before adding Ag-np. Inhibition of DNA-PKcs increased Ag-np-mediated cell death in all four cancer cell lines. This suggests that DNA-PKcs may be protecting the cells from Ag-np cytotoxicity. The authors further showed that DNA-PKcs may repair Ag-np induced DNA damage by NHEJ and JNK1 pathways. In addition, DNA-PKcs may help recruit DNA repair machinery to damaged telomeres.

This study suggests that a combination of Ag-np treatment and DNA-PKcs inhibition may be a potential strategy to enhance the anticancer effect of Ag-np.

Reference: Hande M.P., et.al. (2017) DNA-dependent protein kinase modulates the anti-cancer properties of silver nanoparticles in human cancer cells. Mutat Res Gen Tox En. 824, 32

Determination of Antibody Mechanism of Action Using IdeS

Monoclonal antibodies (mAbs) have been widely used to eliminate undesired cells via various mechanisms, including antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and programmed cell death (PCD). Unlike the Fc-dependent mechanism of ADCC and CDC, certain antibody–antigen interactions can evoke direct PCD via apoptosis or oncosis. Previously, researchers have reported the specific killing of undifferentiated human embryonic stem cells (hESC) by mAb84 (IgM) via oncosis (1)

In a recent publication (2), a monoclonal antibody (mAb), TAG-A1 (A1), was generated to selectively kill residual undifferentiated human embryonic stem cells (hESC). One of the many experimental tools used to characterize the mechanism of oncosis was the fragmention of the A1 antibody with IdeS and papain.

Papain digestion of IgG produces Fab fragments in the presence of reducing agent. F(ab)2 fragments of A1 were produced using IdeS Protease.

The results indicate that both Fab_A1 and F(ab)2_A1 bind to hESC but only F(ab)2_A1 retained hESC killing. Hence bivalency, but not Fc-domain, is essential for A1 killing on hESC.

  1. Choo, A.B. et al. (2008) Selection against undifferentiated human embryonic stem cells by a cytotoxic antibody recognizing podocalyxin-like protein-1. Stem Cells  26, 1454.
  2. Zheng, J.Y. et al. (2017) Excess reactive oxygen species production mediates monoclonal antibody-induced human embryonic stem cell death via oncosis. Cell Death and Differentiation 24, 546–58.

Further reading about IdeS Protease is available here.

Hot Wings and Snow Birds: A Study of Genetic Selection in Chickens

African chicken breed Boschvelder. Image copyright ICBH GROUP.

This past summer, I visited the county fair and stopped by the animal barn to look at some of the poultry on display. Specifically, I wanted to see examples of the breeds of chickens available that I may be interested in adding to my flock. Rather than each chicken in their display cage being labeled with a bird’s breed, each cage listed the geographic origin of the chicken within such as Asiatic, Continental or American. This did not benefit my search for potential new members of my flock, but intrigued me enough that I wanted to find out how my flock of 19 hens and pullets would be characterized. Using the classes delineated by the Wisconsin State Fair, my feathered ladies break down to 12 American, 4 English and 3 Continental chickens. There are also classes for Mediterranean and Asiatic (and Other). I live in a part of the United States that gets cold, snowy weather for what seems like six months out of the year, weather that my chickens seem to take in stride. But in other places in the world, heat is the name of the game for the poultry strutting there. In a Genes, Genomics, Genetics publication, Fleming et al. wanted to know if there were genetic differences in Northern European and African chickens that might be caused by their environment. Continue reading