Physics has dark matter, a form of matter that makes up about 85% of the universe and is composed of as-yet undiscovered particles. Similarly, biology has dark molecules, the unidentified members of the metabolome, which is the totality of small molecules in living organisms.

In a recent review paper in the Annual Review of Analytical Chemistry, Facundo Fernandez and others describe the challenges of identifying the dark molecules of life. Fernandez is a professor and metabolomics expert in the School of Chemistry and Biochemistry. We asked Fernandez to tell us more about the dark side of the metabolome.

What are the dark molecules of life? Why should we care?

The dark molecules of biology are products of enzymatic reactions. They include the molecules of primary metabolism – the network of reactions that enable organisms to live, grow, and reproduce. They also include secondary metabolites, which are compounds not involved in primary metabolic pathways but are produced for defense and interactions with other organisms through chemical signals.

Tens of thousands of molecules can be detected in living organisms, but we can only tell what a fraction of them are – typically less than 10%. Identifying them is key to understanding their roles because they determine how living organisms function and how disease affects them. Our overall well-being depends partly on how the environment and our lifestyle choices progressively expose us to chemicals, beneficial or not.

What are the most important dark molecules?

We don’t know yet! They are still in the “dark.”

But we know that families of molecules, such as lipids, play a crucial role in many physiological processes. Because of the chemical complexity of lipids, the complement of all lipid molecules – the lipidome – is one of the darkest parts of the metabolome “universe.”

Our current research efforts are geared toward better mapping of these unknown regions of biochemical space. For example, we have been working with the Molecular Transducers of Physical Activity (MoTrPAC) NIH consortium to understand the beneficial effects of exercise by mapping the known and unknown regions of the lipidome in mice and humans.  

What is the biggest challenge in identifying dark molecules?

The number-one challenge is that we do not yet have a bombproof method for identifying any metabolite in a high-throughput fashion at the concentration levels that are observed in living organisms.

Although chemists have become really good at identifying molecules, this is typically done after extensive purification that could take months, if not years. When we are talking about tens of thousands of molecules, this “purify and identify” approach does not really work, as it is  resource-intensive and time-consuming.

What we need is the magic tricorder that will give us the identity of any interesting molecule in real time and even at trace levels.  

What are scientists doing to overcome this challenge?

We are heavily invested in using mass spectrometry, a technique that is equivalent to a molecular weight scale, to pursue this task.

Mass spectrometry has been improving very rapidly and is becoming the tool of choice for both metabolomics and lipidomics. We can now detect molecules at the zeptomole (10^-21 mole) levels routinely. And we can identify a large fraction of them without purification.

But challenges remain, particularly in deconvoluting the huge amounts of information generated by mass spectrometry data. We are currently in data-overload mode, similar to what happened in genomics and proteomics decades ago.