Use of HIC high resolution chromatography and elastase for bottom up proteomics

One of the key applications used to characterize single or complex protein mixtures via bottom up proteomics is liquid chromatography−tandem mass spectrometry (LC−MS/MS).
Recent technical advances allow for identification of >10 000 proteins in a cancer cell line. On the peptide level chromatography methods, like strong cation exchange (SCX)
and hydrophilic interaction chromatography (HILIC), as well as high-pH reversed phase chromatography have been employed successfully. Because of its robustness
and ease of handling, the classical and still widely used approach for protein fractionation prior to LC− MS/MS is gel-based separation under denaturing conditions (SDS-PAGE).
Hydrophobic interaction chromatography (HIC) is a robust standard analytical method to purify proteins while preserving their biological activity. It is widely used
to study post-translational modifications of proteins and drug−protein interactions.  HIC is a high-resolution chromatography mode based on the interaction of
weakly hydrophobic ligands of the stationary phase with hydrophobic patches on the surface of the tertiary structure of proteins. By employment of high concentrations
of structure-promoting (“kosmotropic”) salts, proteins in HIC retain their conform

In a recent publication, HIC was used to separate proteins, followed by bottom up LC−MS/MS experiments (1).  HIC was used to fractionate antibody species
followed by comprehensive peptide mapping as well as to study protein complexes in human cells. The results indicated that HIC−reversed-phase chromatography (RPC)
mass spectrometry (MS) is a powerful alternative to fractionate proteins for bottom-up proteomics experiments making use of their distinct hydrophobic properties.

An additional observation noted that tryptic digests of the antibody used in the study yielded a protein coverage of 56% for the light chain and 63.2% for the
heavy chain. A consecutive proteolytic digestion protocol combing on-filter trypsin and elastase digestion drastically improved sequence coverage of
both light (100%) and heavy chains (99.2%).

1. Rackiewicz, M. et al. (2017) Hydrophobic Interaction Chromatography for Bottom-Up Proteomics Analysis of Single Proteins and Protein Complexes. J.Proteome.Res. 16, 2318–23.

Searching for Secrets in Single Cells

There has been a lot of effort recently to perform whole genome sequencing, for humans and other species. The results yield new frontiers of data analysis that offer a lot of promise for groundbreaking scientific discoveries.

One objective of human genome sequencing has been to identify sources of disease and new therapeutic targets. This movement has opened the door to create personalized medicine for cancer, whereby the genetic makeup of an individual’s tumors can be used to determine the most effective drug intervention to administer.

Interest in studying the characteristics unique to individual cells seems obvious when considering the function of healthy cells versus tumor cells, or brain cells compared to heart cells. What has surprised scientists is the realization that two cells in the same tissue can be more different from each other, genetically, than from a cell in another organ.

For example, a small number of brain cells with a specific mutation can lead to some forms of epilepsy while healthy people may also carry cells with these mutations, but too few to cause disease. The lineage of a cell, where it came from and what events shaped its development, ultimately determines what diseases can exist.

Continue reading

Five Ways to Explain CRISPR Without Delivering a Lecture

Recently a FaceBook friend of mine (who is not a scientist) shared a video from WIRED Science where the concept of CRISPR is explained at 5 Levels of Difficulty— for a 7 year old, a teenager, a college student, a grad student and a CRISPR expert.

First it was pretty amazing to me that my non-scientist friends are interested enough in learning about CRISPR to share this type of information—perhaps showing just how popular and exciting the method has become. People outside the scientific field are hearing a lot about it, and are curious to know more.

This video does a great job of explaining the technique for all its intended audiences. It also is a nice illustration of how to share information in an easily understandable format. Even with the 7 year old and 14 year old, the information is shared in a conversational way, with everyone involved contributing to sharing information about CRISPR.

Really nice. Here’s the WIRED video:

Continue reading

Rapid DNA Act of 2017: What is It?

On May 16, 2017, the U.S House of Representatives and the U.S. Senate passed the Rapid DNA Act of 2017 (H.R.510 and S.139, respectively). The bill was sponsored by Senator Orrin Hatch (R-UT) and Representative James Sensenbrenner (R-Wis) and enjoyed bipartisan support, ending up with seven Republican and five Democratic cosponsors in the Senate, and seventeen Republican and seven Democratic cosponsors in the House. The bill was passed by unanimous consent voice votes in both chambers.

So what is the Rapid DNA Act of 2017 all about?

Simply put, the act will expand the use of rapid DNA technology in law enforcement departments by creating a way for them to use the results they get by connecting them to the FBIs Combined DNA Index System (CODIS). Still curious? Read on and you will learn much more about what the Rapid DNA Act of 2017 does and doesn’t do. Continue reading

Findings May Reveal Earliest Evidence of Selective Dog Breeding

Image showing DeLong chain of islands.

Zhokhov Island is part of the DeLong chain of islands off the north coast of Siberia. Image courtesy of Wikimedia Commons.

A report in the June 2, 2017 edition of Science magazine digs into findings from an ancient archaeological site on the very remote and very, very cold Zhokhov Island, to show that the locals, hardy human hunters, not only lived and worked with dogs, but also quite probably selectively bred the dogs for certain traits.

Archaeologist Vladmir Pitulko with the Russian Academy of Sciences has been excavating on Zhokhov Island since 1989, where he has found dog bones as well as remnants of wooden sleds. With archaezoologist Aleksey Kasparov, also of the RAS, they’ve compared two of the most complete dog skulls found to those of contemporary Siberian Huskies and wolves.

Pitulko and Kasparov wanted to first determine if the skulls were those of dogs or wolves. They first employed two key skull ratios: snout height to skull length and cranium height to skull length. Using these ratios, they were able to reliably distinguish between skulls of a modern wolf and husky. Continue reading

Creating ART from 3D Printed Ovaries

It is remarkable to me how quickly in vitro fertilization has gone from an experimental, controversial and prohibitively expensive procedure to becoming a mainstream option for those struggling with fertility issues. What was unheard of in my parents’ generation is nothing extraordinary among my friends who are having children.

My personal observations are supported by the CDC, which reported that 1.6% of all infants born in the U.S. in 2015 were the result of assisted reproductive technology (ART). This is a 33% increase since 2006, which can be attributed to rapid advances and refinements of the various technologies available to those seeking reproductive assistance.

It challenges the mind to imagine what reproductive technologies might be widespread when my children and their friends are adults. When experts speculate about the future of human reproduction, there always seems to be a lot of focus on provocative scenarios that portend a dystopian future, such as designer babies. What gets lost are some of the more general scientific advances that are being applied to ART in fascinating ways.

While improvements in reproductive technologies serve many, one group that remains underserved are pediatric cancer patients. As a result of treatment, these patients are often faced with impaired ovarian function that can prevent puberty and result in infertility. In vitro fertilization and ovarian transplants are currently used, but do not provide lasting solutions for all individuals.

In response to this need, researchers are working to develop an organ replacement that can provide long-term hormone function and fertility for all patients.  A recent study in Nature Communications presented encouraging results in mice using bioprosthetic ovaries that may further revolutionize the field of ART. Continue reading

Real-Time Analysis for Cell Viability, Cytotoxicity and Apoptosis: What Would You Do with More Data from One Sample?

You are studying the effects of a compound(s) on your cells. You want to know how the compound affects cell health over a period of hours, or even days. Real-time assays allow you to monitor cell viability, cytotoxicity and apoptosis continuously, to detect changes over time.

Why use a real-time assay?
A real-time assay enables you to repeatedly measure specific events or conditions over time from the same sample or plate well. Repeated measurement is possible because the cells are not harmed by real-time assay reagents. Real-time assays allow you to collect data without lysing the cells.

Advantages of  Real-Time Measurement
Real-time assays allow you to: Continue reading

In Healthy Eating Less is More: The Science Behind Intermittent Fasting

Mix a love of eating with a desire to live a long, healthy life what do you get? Probably the average 21st century person looking for a way to continue enjoying food despite insufficient exercise and/or an age-related decline in caloric needs.

Enter intermittent fasting, a topic that has found it’s way into most news sources, from National Institutes of Health (NIH) and Proceedings of the National Academy of Sciences publications to WebMD and even the popular press. For instance, National Public Radio’s “The Salt” writers have tried and written about their experiences with dietary restriction.

While fasting has enjoyed fad-like popularity the past several years, it is not new. Fasting, whether purposely not eating or eating a restricted diet, has been practiced for 1,000s of years. What is new is research studies from which we are learning the physiologic effects of fasting and other forms of decreased nutrient intake.

You may have heard the claims that fasting makes people smarter, more focused and thinner? Researchers today are using cell and animal models, and even human subjects, to measure biochemical responses at the cellular level to restricted nutrient intake and meal timing, in part to prove/disprove such claims (1,2). Continue reading

Knots: Friend or Foe?

Knots affect our lives in perplexing ways. They can perform life-saving assistance, such as during rock climbing, or provide Sisyphean puzzles of entanglement. Often, knots seem to have the contrarian personality of an adolescent. They loosen and unwind when you want them to stay fastened, and inevitably form tangles of confounding complexity when you seek to avoid them. These puzzling characteristics of knots were brought to mind when I read two recent articles about the scientific investigation of knots.37190697-May-5-Darcia---Option-2

Why Knots Fail

The explanation of how shoelaces come untied, published in Proceedings of the Royal Society A, was quite prevalent in the news cycle recently. After observing slow-motion video footage of the shoelaces of a runner on a treadmill, researchers were able to explain how motion affects knots and results in untied shoelaces.

First, they observed that the failure of a knot is not a gradual process, but happens abruptly over the course of only one or two strides. This is possible due to the surprising amount of force generated by the impact of one step, which this study calculated to be an average of 7 g—more than twice the g-force experienced by the Space Shuttle upon reentry into the Earth’s atmosphere. Continue reading

Don’t Let These Three Common Issues Hurt Your Luminescent Assay Results

4621CAThere is a lot riding on your luminescent assay results. Each plate represents precious time, effort and resources. Did you know that there are three things about your detection instrument that can impact how much useful information you get from each plate?  Instruments with poor sensitivity may cause you to miss low-level samples that could be the “hit” you are looking for.  Instruments with a narrow detection range limit the accuracy or reproducibility you needed to repeat your work.  Finally, instruments that let the signal from bright wells spill into adjacent wells allow crosstalk to occur and skew experimental results, costing you time and leading to failed or repeated experiments. Continue reading