molecular interactome

SMI-seq

Single-molecule counting of proteins in both free and assembled states in complex mixtures (like where proteins are found in nature) is the holy grail of biochemistry. Mathematically speaking, the measure of possible interactions in a protein mixture is exponentially more complex than detecting protein species in it—for example, N proteins could form N^m possible assemblies comprising ≤ m subunits. Direct counting protein assemblies without labeling them is still an unsolved problem. Even for high-resolution methods such as electron microscopy, the single particle analysis requires to average images of many identical protein molecules. However, we can indirectly count proteins using an in situ protein interaction sequencing technology, SMI-seq, via tagging proteins with DNAs and in situ sequencing DNA tags:

SMI-seq relies on highly efficient bridge amplification of DNA tags (or barcoding DNAs) of proteins embedded in crosslinked polyacrylamide (polony gel). As shown below, DNA tags of a tetrameric protein presented in mixed states (monomeric, partly and fully assembled) in the gel were amplified into polonies. Different DNA tags in a protein complex were in situ amplified into polyclonal polonies, showing mixed colors in the below image. Some in pure green are monoclonal polonies but others show mixed colors in different sequencing cycles are also polyclonal polonies. If you are interested, you can locate them by comparing the header images of the same gel region acquired in different sequencing cycles.

Reference:

Multiplex Single-Molecule Interaction Profiling of DNA Barcoded Proteins. Liangcai Gu#, Chao Li, John Aach, David E. Hill, Marc Vidal & George M. Church#; Nature, 2014, 515(7528):554-7