A team led by researchers at the Swiss Federal Institute of Technology Zurich and University College London has completed a global proteomic and transcriptomic analysis measuring levels of proteins and RNAs in fission yeast cells in both quiescent and proliferating states.
The study, which was published in Cell, is one of the most comprehensive of its kind performed to date and raises a number of interesting questions regarding the mechanisms of cell function, Ruedi Aebersold, an ETH Zurich researcher and study leader, says.
For the project, Aebersold and his lab collaborated with University College researcher Jurg Bahler, whose team brought its transcriptomics expertise to the effort. While proteomics has typically been seen as more technically challenging than nucleic acid-based work, in this case the transcriptomic work was as or more demanding than the protein portion of the study, Aebersold says.
"Absolute quantification of transcripts actually is non-trivial," he notes. "We always hear about genomics being so fast and so powerful, but if you really want quantitatively accurate data there are a lot of hoops to go through to calibrate the data from the high-throughput sequencing runs."
For the proteomic portion of the study, the researchers used mass spec analysis on a Thermo Fisher Scientific Orbitrap Velos instrument, applying a workflow based on a method described by Aebersold's group in a 2011 Molecular Systems Biology paper.
The researchers determined the absolute abundances for 39 proteins via spiked-in reference peptides and then used that data to do proteome-wide quantitation by translating the mass spec intensities of all peptides present to an absolute copy number per cell. In this way, they quantified roughly 3,397 proteins in proliferating cells and 2,500 in quiescent cells, correlating these expression levels with measurements of their corresponding transcripts.
The study, Aebersold says, offers a number of insights into cell function and highlights a variety of potential avenues for future research. For instance, he notes, the very small number of transcripts — typically fewer than 10 per cell — compared to the much larger number of proteins — averaging in the thousands of copies per cell — suggests two different modes of regulation at these different levels.
The lower number of transcripts present "means that the transcript regulation is a stochastic domain," Aebersold says. "And this of course has implications for how processes are controlled at the transcriptional level. The protein levels, by contrast, are much higher. The mean is in the range of thousands [per cell], so they are clearly the non-stochastic domain. So the means of regulation are probably completely different."
Despite this likely difference in regulatory mechanisms, Aebersold notes, the study found evidence of great coordination between the two levels. For instance, using their proteomic and transcriptomic data independently, the researchers were able to arrive at roughly matching estimates of the per-cell number of ribosomes — complexes made up of single copies of various proteins and transcripts.