Advances in protein turnover analysis at the global level and biological insights

The concept of a dynamic state of body constituents, a precursor of the modern term of proteome dynamics, was conceived over a century ago. But, not until recently can we examine the dynamics of individual ‘constituents’ e.g., proteins at a truly global level. The path of advancement in our understanding of protein turnover at the global level is marked by the introduction of some key technological innovations. These methods include the isotopic tracer technique in the 1930s, the two-dimensional gel electrophoresis technique in the 1970s, the sector mass spectrometer that could analyze isotopomers of peptides in the early 1990s, the 2D gel/MALDI-TOF proteomics technology in the late 1990s, the booming liquid chromatography/mass spectrometry proteomics technology in this decade, and the recently emerging protein-tagging approaches that offer single-cell resolution for protein turnover measurements. The long-standing inquiry raised in the 1950s about the existence of a dynamic state in different organisms at different physiological conditions can now be answered with an individual ‘constituent’ resolution on a truly global scale. Now it appears that protein degradation is not necessarily an end to the protein function. Rather, it can be the start of a new function because protein degradation clears the way for the action of other proteins. Protein turnover participates in a multi-layer complex regulatory network and shares equal importance with gene transcription and protein translation. The advances in technologies for protein turnover analysis and the improved understanding of the biological role of protein turnover will likely help to solve some long-standing biomedical problems such as the tuberculosis disease that at the present day still affects one-third of the world population

Determination of Global Protein Turnover in Stressed Mycobacterium Cells Using Hybrid-Linear Ion Trap-Fourier Transform Mass Spectrometry

We determined the global protein turnover profiles for Mycobacterium smegmatis under acid shock and iron starvation conditions using a simple 15N isotope doping technique and a complete medium replacement method for chasing. We used a high-resolution hybrid-linear ion trap-Fourier transform mass spectrometer coupled with nanoliquid chromatography separation to measure protein turnover values for 151 proteins over a dynamic range of 3 orders of magnitude ranging from about 0.2 to 500. Of these 151 proteins, 31 had significant protein turnover changes (p <0.05) at both stress conditions and had protein turnover values increased or decreased by more than 2-fold under at least one stress condition. Protein turnover increased under acid shock for 28 of the 31 proteins but decreased under iron starvation for all the 31 proteins. Only two proteins had protein turnover lowered by more than 2-fold (p <0.05) under both stress conditions, including an ATP synthase F1 β subunit (MSMEG4921; AtpD) and a catalase/peroxidase (MSMEG6346; KatG). KatG is required for in vivo activation of isoniazid to be bacterialcidal. Decrease of KatG protein turnover under both stress conditions supports the view that isoniazid may induce a dormancy program in mycobacteria, which in turn limits the efficacy of this drug against dormant subpopulation of mycobacteria. Thus, measuring protein turnover in stressed Mycobacterium cells has implications in understanding drug action and resistance mechanisms

Protein Dynamics in Iron-Starved <i>Mycobacterium tuberculosis</i> Revealed by Turnover and Abundance Measurement Using Hybrid-Linear Ion Trap-Fourier Transform Mass Spectrometry

To study the proteome response of Mycobacterium tuberculosis H37Rv to a change in iron level, iron-starved late-log-phase cells were diluted in fresh low- and high-iron media containing [15N]-labeled asparagine as the sole nitrogen source for labeling the proteins synthesized upon dilution. We determined the relative protein abundance and protein turnover in M. tuberculosis H37Rv under these two conditions. For measurements, we used a high-resolution hybrid-linear ion trap-Fourier transform mass spectrometer coupled with nanoliquid chromatography separation. While relative protein abundance analysis shows that only 5 proteins were upregulated by high iron, 24 proteins had elevated protein turnover for the cells in the high-iron medium. This suggests that protein turnover is a sensitive parameter to assess the proteome dynamics. Cluster analysis was used to explore the interconnection of protein abundance and turnover, revealing coordination of the cellular processes of protein synthesis, degradation, and secretion that determine the abundance and allocation of a protein in the cytosol and the extracellular matrix of the cells. Further potential utility of the approach is discussed

Additional file 5 of The expression level of chicken telomerase reverse transcriptase in tumors induced by ALV-J is positively correlated with methylation and mutation of its promoter region

Additional file 5. Analysis of DNA sequence mutations in the promoter region of chTERT. Green box: normal tissues; blue box: tumor-adjacent tissues; red box: tumor tissues; orange box: mutant tumors. T: tumor tissues; TA: tumor-adjacent tissues; N: normal tissues

Additional file 2 of The expression level of chicken telomerase reverse transcriptase in tumors induced by ALV-J is positively correlated with methylation and mutation of its promoter region

Additional file 2. Matrix of methylation levels of chTERT amplicon CG sites in LMH cells and DF-1 cells

sj-docx-1-asp-10.1177_00037028231206191 - Supplemental material for Dynamic Multivariate Outlier Detection Algorithm Using Ultraviolet Visible Spectroscopy for Monitoring Surface Water Contamination With Hydrological Fluctuation in Real-Time

Supplemental material, sj-docx-1-asp-10.1177_00037028231206191 for Dynamic Multivariate Outlier Detection Algorithm Using Ultraviolet Visible Spectroscopy for Monitoring Surface Water Contamination With Hydrological Fluctuation in Real-Time by Qingbo Li, Xupeng Shao, Houxin Cui, Yuan Wei and Yongchang Shang in Applied Spectroscopy</p

Additional file 1 of The expression level of chicken telomerase reverse transcriptase in tumors induced by ALV-J is positively correlated with methylation and mutation of its promoter region

Additional file 1. ALV-J Tumor tissues and their sources and quantities

Additional file 4 of The expression level of chicken telomerase reverse transcriptase in tumors induced by ALV-J is positively correlated with methylation and mutation of its promoter region

Additional file 4. Agarose gel electrophoresis of PCR amplification of the chTERT promoter region. Lanes 1-8: different tissue samples; Lane 9: negative control

Additional file 3 of The expression level of chicken telomerase reverse transcriptase in tumors induced by ALV-J is positively correlated with methylation and mutation of its promoter region

Additional file 3. Matrix of methylation levels of chTERT amplicon CG sites in ALV-J tumor, tumor-adjacent and normal tissues

Label-free Proteomics and Systems Biology Analysis of Mycobacterial Phagosomes in Dendritic Cells and Macrophages

Proteomics has been applied to study intracellular bacteria and phagocytic vacuoles in different host cell lines, especially macrophages (Mφs). For mycobacterial phagosomes, few studies have identified over several hundred proteins for systems assessment of the phagosome maturation and antigen presentation pathways. More importantly, there has been a scarcity in publication on proteomic characterization of mycobacterial phagosomes in dendritic cells (DCs). In this work, we report a global proteomic analysis of Mφ and DC phagosomes infected with a virulent, an attenuated, and a vaccine strain of mycobacteria. We used label-free quantitative proteomics and bioinformatics tools to decipher the regulation of phagosome maturation and antigen presentation pathways in Mφs and DCs. We found that the phagosomal antigen presentation pathways are repressed more in DCs than in Mφs. The results suggest that virulent mycobacteria might co-opt the host immune system to stimulate granuloma formation for persistence while minimizing the antimicrobial immune response to enhance mycobacterial survival. The studies on phagosomal proteomes have also shown promise in discovering new antigen presentation mechanisms that a professional antigen presentation cell might use to overcome the mycobacterial blockade of conventional antigen presentation pathways