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Rising interest in less-invasive sampling using blood, urine or other easily accessible biological fluids for biomarker discovery is expanding analytical methods for many –omics technologies. Chen et al. (2015) explored a new sample-preparation workflow for mass spectrometry–based proteomic analysis of human serum, seeking to facilitate enrichment of low-abundance, low–molecular weight (LMW) proteins.1
Although blood is a good starting material for biomarker discovery, high-abundance proteins (HAPs) such as albumin threaten to mask changes in LMW compounds below 30 kDa. The most valuable biomarkers are often low-abundance LMW proteins, which show variation in terms of quantity, post-translational modification and isoform expression according to disease. Quantitative proteomic analysis using liquid chromatography–mass spectrometry (LC-MS) characterization is thus a valuable tool in biomarker discovery, since it is sensitive enough for both detection and quantitation. However, the necessary removal of HAPs during sample preparation often results in loss of LMW proteins in addition to incomplete removal, masking their quantitative evaluation.
Chen et al. propose a gel filtration enrichment step during sample preparation that passively sieves out the HAPs using a four-layer tracking tricine sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) technique. The technique uses the following four layers:
The researchers obtained serum samples from six healthy volunteers (three male, three female), pooling the volumes to minimize variation. They then took aliquots of the pooled sample and prepared them according to the following workflows:
Once the researchers separated the proteins, they digested the proteins using trypsin prior to LC–tandem mass spectrometry evaluation (LC-MS/MS) using an LTQ Obitrap Velos mass spectrometer (Thermo Scientific).
The team also used a commercial bead enrichment kit to separate proteins prior to trypsin digestion and LC-MS/MS. They conducted recovery assessment for this method in addition to gel filtration enrichment and ODS using a stable isotope labeling in culture absolute quantitation (SILAC-AQUA) approach to label recombinant proteins, which they then spiked into the human serum before processing according to the workflows described above.
On direct examination of the gels, Chen et al. found that the gel filtration enrichment showed better resolution of LMWs than ODS or glycine SDS-PAGE, with no loss of protein bands. The team found that the gel filtration enrichment resulted in a clear demarcation between blocking and separating gel interfaces. Furthermore, band density varied according to sample load, although volumes above 10 µL resulted in saturation with merging into the separation layer. Compared to the glycine and ODS methods, gel filtration enrichment was more consistent and highly efficient, resulting in more proteins, as shown by band intensity.
The team confirmed this using LC-MS/MS, where final analysis yielded more protein identifications from the gel filtration enrichment (1,576) than from the other two workflows (ODS = 1,022; glycine = 1,186) in 10 µL of serum. Of the 1,576 proteins identified, 559 were LMW below 30 kDa. Gel filtration enrichment also gave good recoveries from spiked samples as assessed by SILAC-AQUA. Compared to ODS (18.7%) and the commercial kit (9.6%), gel filtration enrichment resulted in recoveries of 33.1%.
Chen et al. conclude that quantitative LC-MS/MS shows that gel filtration results in good enrichment without protein bias. Furthermore, the workflow efficiently captures the LMW proteins of interest for biomarker discovery without excessive losses during preparation. Removal of HAPs removes interference with quantitative proteomic evaluation, thus increasing the value of this method for biomarker characterization.
1. Chen L., et al. (2015) “Development of gel-filter method for high enrichment of low-molecular weight proteins from serum,” PLoS ONE 10(2): e0115862. doi: 10.1371/journal.pone.0115862
Post Author: Amanda Maxwell. Mixed media artist; blogger and social media communicator; clinical scientist and writer. A digital space explorer, engaging readers by translating complex theories and subjects creatively into everyday language.
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On May 18, 1910, Halley’s comet made its closest recorded contact (0.15 astronomical units,* or approximately 23 million kilometres) with our planet, and the Earth passed through its tail. The event was full of scientific excitement and wonder, since photographic plates and spectroscopy were newly available to researchers. With these new tools, astronomers and the public got a better view of the comet itself and also a first inkling of what it contained.
Exciting times for science!
However, as the comet approached, the spectroscopy results revealed that among other gases, the comet’s tail showed strong band signatures for the toxic gas cyanogen. First synthesized in 1815, cyanogen is toxic, since it reduces rapidly to form cyanide and thus poisons the essential cytochrome c oxidase system to mess with mitochondrial function (not good).
Although many chose to report the existence of the gas in the comet tail scientifically and without alarm (because there was no reason for alarm), some did not let science get in the way of a good story and launched into the general doom-mongering that often accompanies the appearance of celestial bodies.
According to astronomer Camille Flammarion, cyanogen gas would extinguish life on Earth as the planet passed through the comet’s tail. Despite reassurances from more responsible experts who no doubt pointed out the space/vacuum/distance/protective effect of atmospheric abundancy factors, some news outlets, including the New York Times, chose to report this alarming speculation.
Stardust was okay, but the public was not happy with the thought of being bathed in cyanogen; in the panic, sales of gas masks and comet pills increased.
May 19 dawned; Earth passed safely through the comet tail. Mark Twain, the only “casualty,” had died a month earlier (just sayin’).
For more information on spotting clickbait, check out these blog posts:
* One astronomical unit (AU) is equivalent to the mean distance between the Sun and the Earth, according to this blog post from the Jet Propulsion Laboratory at the California Institute of Technology.
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One of the reasons for highlighting upcoming science conferences in Vancouver in my Around Town series is that it gives me a push to find out more on a subject I may have little exposure to. It’s also a great inspiration for a regular series of blog posts!
Last month, press releases ahead of one of these conferences, the 68th American Academy of Neurology Annual Meeting, caught my eye. As is common in the promotion of meetings for large organizations, the preceding month saw a few press releases announcing some of the research coming to the Vancouver Convention Centre. The first reported a preliminary study on the effect of daylight saving time (DST) on incidence of ischemic stroke, and the second investigated engagement in various mentally stimulating activities in older age and the effect on development of thinking and memory problems.
When I blog about upcoming events, I usually write about something that fascinates me from the program, or brush up on terminology, or even wax lyrical about proteins. Press releases are a handy inspiration. All of this takes careful research, and it’s equally true for the press releases.
At around the same time as I was exploring the American Academy of Neurology conference, Talk Science Mastermind Eve and I took part in a workshop for health sciences students at Kwantlen University, where we explained a little about the science communications work Talk Science to Me does and about science writing in general. A few of the questions from the students focused on how we find subjects to write about and how we work out whether they are valid.
— Talk Science To Me (@TalkScienceToMe) March 30, 2016
So, here’s a brief summary of the workflow I go through when choosing a topic.
In the first study I read about, Finnish researchers found an association between the incidence of stroke in the first two days following the switch to DST. In the second, a team from the Mayo Clinic found that a group of seniors 70 years or older regularly taking part in late-life mentally stimulating activities were less likely to develop mild cognitive impairment over four years.
Why did the press releases catch my eye?
Apart from the fact that they turned up in my feed reader’s daily aggregation, the topics seemed highly relevant to me in the moment. Number one: for the first time ever, I think, I missed the dreaded Spring Forward by virtue of travel and location, being in the United Kingdom for the North American transition, then missing European DST a couple of weeks later when I flew back home. And number two: my reason for travel was to visit my parents, both in the older age group.
How did I decide if the topics were appropriate for the Talk Science blog?
Or, in other words, am I falling for clickbait? Old age, Alzheimer’s disease, DST, stroke, debility…these are all newsworthy subjects, so how do I know that I’m not falling under the spell?
First, what are my trusted sources saying about the subject? My handy feed reader turned up nothing critical of the reports. Now this in itself is not perfect, but it has in the past been a good indicator of questionable veracity. There are many gifted scientific writers out there to learn from, and they are much sharper at digging into the data than I am. No early alarm bells rang for either of these press releases. Note, though, that if I were writing a feature piece for a bigger organization, independent fact-checking would be required; phoning an expert on the subject for comment is a good first step here. At Talk Science, we have a number of experts we can turn to for this kind of verification.
I also turned to two other critical resources, Health News Review and the United Kingdom’s National Health Services Behind the Headlines blog, to see if there was any discussion. I found none. Both are pretty sharp at picking up and dissecting bogus hype.
Then I reread the press releases, in addition to looking at the two conference abstracts supplied and noting that neither had been through journal peer review for publication. In terms of study numbers, looking for sufficient sample size for veracity, data for the first study on stroke and DST came from ten years’ worth of health records. This encompassed over 11,000 individuals for the control group and more than 3,000 patients. The second study followed almost 2,000 seniors over four years.
One of the key areas for clickbait generation is the confusion between association and causation. For example, while rising costs for a bag of potato chips are statistically associated with an increase in reported deaths for wheelchair users (r = 0.971706), they are unlikely to be a root cause even if the data suggest otherwise. In both the neurology studies, neither the research team nor the press release stated absolute causation for the observed results, stating only that x was associated with y.
The DST/stroke study press release quoted one of the study authors as saying, “Further studies must now be done to better understand the relationship between these transitions and stroke risk…”
Janina Krell-Roesch, co-author of the mental activity and cognitive decline study, described the study as showing only an association, not cause and effect; however, she also suggested in the press release that “…as people age, they may want to consider participating in activities like these because they may keep a mind healthier, longer.”
I am the first to admit that my critical thinking skills and grasp of study design/statistical analysis are rusty. Applying my own critical eye in addition to scoping out other informed reactions is a part of my process for avoiding clickbait. While none of the steps described above is by itself foolproof, the results obtained are usually enough for me to decide whether or not to mention studies on the Talk Science blog.
In conclusion: nothing sensational here, but interesting studies that I felt were worth sharing.
If you have any more tips on checking out possible science stories, please leave a comment below.