Cracking the Epigenetic Code
Mass spectrometry (MS) approaches are increasing our understanding of how DNA is regulated in real life.
Pawel Olszowy |
Everyone knows the genetic code. Not so well known or understood is the epigenetic code, which sits on top (literally and figuratively) of the genetic code. While all the cells of one organism share a genetic code, the epigenetic code, consisting of different types of modifications that impact the genetic code, is unique to every cell.
Within the nucleus, double-stranded DNA helices are wound around proteins called histones to form nucleosomes, which can be compared to a thread wrapped around a spool. Post-translational modifications (PTM) to the N-terminal end of the histone proteins direct the maintenance or remodeling of the nucleosome structure and can have positive (activating) as well as negative (repressing) effects on the regulation of transcription. The profile of histone PTMs is known as the epigenetic histone code for a particular cell, and is a subject of increasing research interest.
MS offers an alternative approach to epigenetic research. The more typical way to detect and identify post-translational modifications of histones is Western Blot. However, this technique requires specific antibodies, which do not exist for new modifications. Using mass spectrometry, all kinds of modifications – known and previously unknown – can be identified. By adding appropriate molecular mass to the precursor ion, post-translational modifications can be identified manually, or the entire process can be determined using proteomic software.
No mass spectrometer stands out as being the best fit for epigenetic research. In fact, it is best to combine fragmentation techniques to gain a comprehensive list of post-translational modifications. As an example, we have used fragmentation data collected from collision-induced dissociation (CID) and electron transfer dissociation (ETD) techniques. Using CID we detected nearly all of the reported acetylation on lysines of the Histone H4 proteins. With ETD, which is a more subtle technique, we identified ubiquitination and phosphorylation modifications, none of which were detected using CID.
Comparison of the LTQ Orbitrap XL (Thermo Scientific) and TripleTOF 5600 (AB Sciex) mass spectrometers showed that one major difference between them is sensitivity. Slightly more modifications were found using TripleTOF but the number of identified modifications using LTQ Orbitrap XL could be increased by applying ETD. If I had to choose a single instrument, I would go for one that allows more than one fragmentation technique. Even though this compromises sensitivity a little, more modifications are identified in comparison to instruments of greater sensitivity that rely on a single fragmentation source.
The MS approach to proteomics does have some weaknesses. One is protein identification. While we can analyze many samples with very good resolution and sensitivity in a short period, the software currently available to study posttranslational modifications and quantitative proteomics is lagging: it takes several times longer to perform a search than to analyze a sample. This computer/software bottleneck will be one of the major problems for companies to deal with.
A second issue is performing quantitative/comparative proteomics. Many methods have been developed, such as stable isotope labeling with amino acids in cell culture (SILAC) and post-labeling during sample preparation for mass spectrometry analyses (that is, isobaric tags for relative and absolute quantitation (iTRAQ) and 18O). Despite the fact that these methods allow quantitative proteomics, there are question marks over their precision and accuracy.
Reports of new post-translational modifications are an almost daily occurrence, so it is unclear when the full epigenetic code will be known. More important, however, is the assignation of functions for these modifications because it is on this knowledge that new targeted therapies can be developed. These will be based on appropriate modifications, on inhibitors and on enzymes, such as histone deacetylases. A complication is that this new range of therapeutic options will have to be delivered to the nucleus.
Shortly after receiving his PhD in analytical chemistry from the Nicolaus Copernicus University in Torun, Poland, Pawel Olszowy joined the Proteomics Laboratory at the University of Nebraska Medical Center in Omaha. “Here, I began studying proteomics using mass spectrometry. I am trying to crack the epigenetic code of histones by identifying post-translational modifications and their functions.” This includes measuring changes in histones after exposure to drugs of abuse and following viral infections. The answers to these questions, Pawel believes, will point to targeted therapies.
