text: Unlike conventional evolutionary and biochemical approaches to studying proteins, i.e. the so-called horizontal comparison of related protein homologues from different branch ends of the tree of life; ASR probes the statistically inferred ancestral proteins within the nodes of the tree – in a vertical manner (see diagram, right). This approach gives access to protein properties that may have transiently arisen over evolutionary time and has recently been used as a way to infer the potential selection pressures that resulted in present-day sequences. ASR has been used to probe the causative mutation that resulted in a proteins neofunctionalization after duplication by first determining that said mutation was located between ancestors 5 and 4 on the diagram (illustratively) using functional assays. In the field of protein biophysics, ASR has also been used to study the development of a proteins thermodynamic and kinetic landscapes over evolutionary time as well as protein folding pathways by combining many modern day analytical techniques such as HX/MS. These sort of insights are typically inferred from several ancestors reconstructed along a phylogeny – referring to the previous analogy, by studying nodes higher and higher (further and further back in evolutionary time) within the tree of life.
  Most ASR studies are conducted in vitro, and have revealed ancestral protein properties that seem to be evolutionarily desirable traits – such as increased thermostability, catalytic activity and catalytic promiscuity. These data have been accredited to artifacts of the ASR algorithms, as well as indicative illustrations of ancient Earths environment – often, ASR research must be complemented with extensive controls (usually alternate ASR experiments) to mitigate algorithmic error. Not all studied ASR proteins exhibit this so-called ancestral superiority. The nascent field of evolutionary biochemistry has been bolstered by the recent increase in ASR studies using the ancestors as ways to probe organismal fitness within certain cellular contexts – effectively testing ancestral proteins in vivo. Due to inherent limitations in these sorts of studies – primarily being the lack of suitably ancient genomes to fit these ancestors in to, the small repertoire of well categorised laboratory model systems, and the inability to mimic ancient cellular environments; very few ASR studies in vivo have been conducted. Despite the above mentioned obstacles, preliminary insights into this avenue of research from a 2015 paper, have revealed that observed ancestral superiority in vitro were not recapitulated in vivo of a given protein. ASR presents one of a few mechanisms to study biochemistry of the Precambrian era of life (>541Ma) and is hence often used in paleogenetics; indeed Zuckerkandl and Pauling originally intended ASR to be the starting point of a field they termed Paleobiochemistry'.