iTRAQ‐based quantitative proteomic analysis gives insight into sexually different metabolic processes of poplars under nitrogen and phosphorus deficiencies

Male and female poplars (Populus cathayana Rehd.) respond differently to nitrogen (N) and phosphorus (P) deficiencies. In this study, an iTRAQ‐based quantitative proteomic analysis was performed. N and P deficiencies caused 189 and 144 proteins to change in abundance in males and 244 and 464 in females, respectively. Compared to N‐ and P‐deficient males, both N‐ and P‐deficient females showed a wider range of changes in proteins that are involved in amino acid, carbohydrate and protein metabolism, and the sexual differences were significant. When comparing the effects of N‐ and P‐deficiencies, N‐deficient females expressed more changes in proteins that are involved in stress responses and gene expression regulation, while P‐deficient females showed more changes in proteins that are involved in energy and lipid metabolism, stress responses and gene expression regulation. The quantitative RT‐PCR analysis of stress‐related proteins showed that males have a better expression correlation between mRNA and protein levels than do females. This study shows that P. cathayana females are more sensitive and have more rapid metabolic mechanisms when responding to N and P deficiencies than do males, and P deficiency has a wider range of effects on females than does N deficiency.     

Identification of Differentially Expressed Water‐insoluble Proteins in the Encystment Process of Colpoda cucullus by Two‐dimensional Electrophoresis and LC‐MS/MS Analysis

In the encystment process of the ciliate protist Colpoda cucullus, we observed that the cell total protein abundance was reduced at 12 h–1 d after the onset of encystment induction subsequent to the reduction in mRNA abundance. We analyzed the alteration of the expression levels of water‐insoluble proteins by two‐dimensional polyacrylamide gel electrophoresis using polyoxyethylene (20) sorbitan monooleate (Tween‐80), and we identified proteins whose expression levels were altered in the encystment process by a liquid chromatography tandem mass spectrometry analysis. The expression level of a 60‐kDa protein (p60; heat shock protein 60) was temporarily enhanced and that of a 55‐kDa protein (p55; actin) and a 49‐kDa protein (p49; actin) was enhanced in the Colpoda encystment process. In mature cysts, the expression level of p55 and p49 tended to be reduced, whereas the expression level of a 50‐kDa protein (p50d; α‐tubulin), a 25‐kDa protein (p25; α‐tubulin) and a 52‐kDa protein (p52c; β‐tubulin) was enhanced.     

Quantitative analysis of protein interaction network dynamics in yeast

Many cellular functions are mediated by protein–protein interaction networks, which are environment dependent. However, systematic measurement of interactions in diverse environments is required to better understand the relative importance of different mechanisms underlying network dynamics. To investigate environment‐dependent protein complex dynamics, we used a DNA‐barcode‐based multiplexed protein interaction assay in Saccharomyces cerevisiae to measure in vivo abundance of 1,379 binary protein complexes under 14 environments. Many binary complexes (55%) were environment dependent, especially those involving transmembrane transporters. We observed many concerted changes around highly connected proteins, and overall network dynamics suggested that “concerted” protein‐centered changes are prevalent. Under a diauxic shift in carbon source from glucose to ethanol, a mass‐action‐based model using relative mRNA levels explained an estimated 47% of the observed variance in binary complex abundance and predicted the direction of concerted binary complex changes with 88% accuracy. Thus, we provide a resource of yeast protein interaction measurements across diverse environments and illustrate the value of this resource in revealing mechanisms of network dynamics.     

Quantification of mRNA and protein and integration with protein turnover in a bacterium

Biological function and cellular responses to environmental perturbations are regulated by a complex interplay of DNA, RNA, proteins and metabolites inside cells. To understand these central processes in living systems at the molecular level, we integrated experimentally determined abundance data for mRNA, proteins, as well as individual protein half‐lives from the genome‐reduced bacterium Mycoplasma pneumoniae. We provide a fine‐grained, quantitative analysis of basic intracellular processes under various external conditions. Proteome composition changes in response to cellular perturbations reveal specific stress response strategies. The regulation of gene expression is largely decoupled from protein dynamics and translation efficiency has a higher regulatory impact on protein abundance than protein turnover. Stochastic simulations using in vivo data show how low translation efficiency and long protein half‐lives effectively reduce biological noise in gene expression. Protein abundances are regulated in functional units, such as complexes or pathways, and reflect cellular lifestyles. Our study provides a detailed integrative analysis of average cellular protein abundances and the dynamic interplay of mRNA and proteins, the central biomolecules of a cell.