Date of Award
Master of Science
Biochemistry and Cellular and Molecular Biology
Albrecht von Arnim
Tessa Burch-Smith, Daniel Roberts
Translation is a biological function present in all forms of life that allows for the production of proteins from strands of messenger RNA (mRNA). In plants, such as Arabidopsis thaliana or Pisum sativum (garden pea), translation is tightly regulated because of the plants’ sessile nature and need to adapt to a variety of stresses. PUNCH-P is a proteomic technique that offers a snapshot of the translational status of cells in both a noninvasive and time sensitive manner. It was previously developed for mammalian tissues but had not been adapted to plants. PUNCH-P delivers an estimate of the rate of protein production using a technique complementary to ribosome foot printing or polysome loading. This method circumvents the in-vivo labeling needed for isotope-based mass spectrometric methods and avoids high sequencing costs associated with ribosome foot-printing methods. Nascent peptides attached to ribosomes are isolated from a frozen tissue sample by centrifugation through a sucrose cushion. These peptides are then C-terminally labelled with biotin-puromycin. The incorporation of biotin allows for downstream peptide visualization on a western blot using streptavidin-horseradish peroxidase as a detection agent. Peptides may also be captured with streptavidin coated beads and analyzed via mass spectrometry (LC-MS/MS). Here, I validate the PUNCH-P method for Arabidopsis and peas. It is well known that heat shock causes an overall decrease in the translational activity of plants due to mechanisms such as the sequestering of cytoplasmic mRNAs into stress granules and the shift to translating heat shock proteins (HSPs). This work presents methods to efficiently label, capture and detect biotin-labeled nascent peptides from plants. With heat treatment as a proof of concept, we show that puromycylation recapitulates the shift in the patterns of translation expected from polysome profiling. In addition, I show preliminary data on the identity of the nascent proteins as identified by mass spectrometry. These advances suggest that PUNCH-P is a promising technique that can complement other proteomic and RNA-based methods to measure protein synthesis rates in plants.
Noras, Aleksandra Izabela, "APPLICATION OF PUROMYCIN ASSOCIATED NASCENT CHAIN PROTEOMICS (PUNCH-P) TO PLANT SAMPLES. " Master's Thesis, University of Tennessee, 2020.