Mass spectrometry has evolved into a powerful tool to study proteins in an unbiased and global manner. The current improvements in identification accuracy, sample throughput and data analysis allow us to answer biological questions at an unprecedented speed and comprehensiveness. The introduction of quantitative approaches to mass spectrometry enables direct comparison of thousands of proteins in complex mixtures.
In one such approach, termed stable isotope labeling of amino acids in cell culture (SILAC), cells are metabolically labeled with isotope enriched amino acids to differentiate the origin of the proteins by a specific shift in the mass spectrum. While this technique can be applied to study the difference in expression levels of thousands of proteins in a single experiment, it has also improved the identification of specific interactions within a vast number of background binders by providing a quantitative filter to identify the interacting proteins.
We described the application of SILAC to identify sequence specific transcription factors by a single affinity purification step. This technique is generic and can even be applied to identify differentially binding transcription factors at single nucleotide polymorphisms (SNPs). The development of these technologies resulted in the identification of a not previously annotated transposable element (MGR/Zbed6), which is recruited as a transcription factor in placental mammals and is the long-sought repressor of the Igf2 G3072A SNP.
We also developed a similar strategy to identify RNA-protein interactions by quantitative mass spectrometry. In contrast to protein-centered approaches like, for example, RIP, CLIP and PAR-CLIP, our method is able to identify proteins binding to an RNA of interest. Recently, we applied the quantitative RNA-protein interaction screen to identify proteins binding to long non-coding RNA (lncRNA). For example, we identified proteins binding to the telomeric repeat containing RNA (TERRA). We have already conducted knock-down experiments against 20 candidates and observed changes in TERRA levels and localization. This kind of systematic analysis will serve as a basis for the further investigation of TERRA regulation.
We will use a combination of quantitative mass spectrometry, next generation sequencing and classical assays to gain insight into gene regulation, development and evolution at the mechanistic and systems biology level.