Fuzzier Logic

At the heart of Systems Biology is a vast hunger for measurements. mRNA abundance, metabolite concentration, reactions rates, degradation rates, protein abundance. This last measurement has long been problematic for researchers, mass spectrometers get increasingly accurate and powerful, but are still hindered by the simple fact that observed signal intensity does not necessarily correlate directly with the abundance of that peptide in the sample. Factors such as peptide ionisation efficiencies, dominant neighbour effects, and missing observations all give rise to erroneous estimates of peptide quantities. Until recently, the best way to get close to measures of protein abundance was to use a peptide tagging methodology, but these are typically expensive, and provide only relative quantification (useful for expression proteomics studies, less useful if you need to know the absolute levels of a protein for a Systems Biology study).

Recently, a three step method has been proposed for determining the absolute quantities of proteins in the cell, on a proteome scale. Step one is isoelectric focussing of tryptic digests of whole cell extracts. Step two, calculating the absolute abundance of a small group of proteins by Selective Reaction Monitoring (SRM). SRM uses spike in, isotopically labelled peptides of known concentration as references to calculate the actual abundance of peptides of interest. Finally, step three uses these abundances as reference points to calculate the abundance of all proteins in the sample, using the median intensities from the 3 most intense peptides for each protein.

Leptospira interrogans (Wikimedia Commons)

Using this methodology, the abundances of >50% of the proteome of a human parasite (Leptospira interrogans) have been determined to an accuracy of ~2-fold. These abundance measurements were confirmed by almost literally counting the number of flagellar proteins present in a cell by cryo-electron tomography.

Although current hardware probably limits this technique to a few thousand proteins, that is still a big step forward on what was previously possible. If whole proteome scale absolute abundance measurements become an achievable reality, maybe proteomics can finally take on microarrays as the dominant technique in the post genomics world.

Malmström, J., Beck, M., Schmidt, A., Lange, V., Deutsch, E., & Aebersold, R. (2009). Proteome-wide cellular protein concentrations of the human pathogen Leptospira interrogans Nature, 460 (7256), 762-765 DOI: 10.1038/nature08184

I am still catching up on my podcast backlog after my 2 week holiday in August. The excellent ‘More or Less’ provided the gem of a quote in the title during a discussion about meta-analyses.

Professor Stephen Senn was explaining why careless mathematics can distort the results of a meta-analysis (things like including a prior meta-analysis amongst your data sets can lead to double-counting – see this paper). The presenter, Tim Harford, suggested that surely this is a problem easily fixed. A reader spots an error in a published meta-analysis, contacts the journal and a correction ensues. A suggestion that was quickly knocked back by Prof Senn. The problem, as he sees it, is that we have no culture of correction; that peer reviewed results are considered irreproachable.

Doesn’t peer review offer some guarantee of quality?, suggests Harford. “Peer review is of minimal value” is the response to this, “…checkability is what really guarantees quality”. Senn goes on to suggest that scientists sign an undertaking to provide raw original data to anyone who requests it.

This was the clearest argument I’ve heard, not against peer review, but for the availability of raw data, and for post-publication quality control on a grand scale.

This multi-eyes approach to quality checking, post-publication, is familiar from somewhere…

Charles Minard's 1869 chart showing the losses in men, their movements, and the temperature of Napoleon's 1812 Russian campaign.

The same edition of the show had a section on data visualisation, and bought the ‘Napoleon’s March’ graphic to my attention. I had not previously been aware of this ‘infographic’, produced in the mid-19th century.

Eczema and asthma often co-occur, indeed, I suffer from both (albeit mildly). What I wasn’t aware of was that eczema often comes first. Though eczema often precedes asthma (asthma has an underlying rate of 4-8% in the general population, but 70% in individuals with a history of chronic severe eczema), the underlying mechanism for this so called ‘atopic march’ isn’t known, though work published today in PLoS Biology elucidates a possible mechanism.

Researchers genetically engineered mice with chronic skin barrier defects (mice lacking Notch signalling in the skin, leading to impairment of epidermal differentiation), who exhibit an eczema like skin condition. They then used these mice to demonstrate the predisposition of such affected individuals to allergic asthma. Occurance of allergic asthma was 7-fold higher in the mutant mouse population, compared to a wild-type population.

The authors then went on to demonstrate that a cytokine called thymic stromal lymphopoietin (TSLP), which is secreted by the damaged skin into the circulation, is required for atopic march in the mutant mice. They show that by knocking out the TSLP receptor in these mice, they can prevent atopic march. They also show that over-production of TSLP in the skin is sufficient to cause allergic asthma, regardless of the cause of that over-production.

This is a paper a little outside my areas of expertise, which is why this is much more of a skim overview than normal. However, there is clearly good work being done here elucidating the molecular mechanisms of a very common disease process. There are also clear implications in this paper on the future management and treatment of eczema and asthma patients. Even though this is unlikely to improve my own experiences of these conditions, I’m very happy this kind of work is being done.

Demehri, S., Morimoto, M., Holtzman, M., & Kopan, R. (2009). Skin-Derived TSLP Triggers Progression from Epidermal-Barrier Defects to Asthma PLoS Biology, 7 (5) DOI: 10.1371/journal.pbio.1000067

I love my free Nature Methods subscription. It allows me to get my hands on a paper journal, which I rarely get to do these days, and the content is actually pretty marvellous.

This month there’s a new technique for enzymatic assembly of DNA molecules from the Venter Institute, a standardised methodology for proteomics sample preparation, and a great technology feature from Nathan Blow about new proteomics techniques, including surface plasmon resonance (about which I knew nothing before today). Not to mention cool pictures of mice having light shone on their brains.

You can still apply for a free subscription, and if you are eligible to do so (individuals in North America and Europe involved in research within the life sciences or chemistry), I would urge you to.