Here we show that graphene oxide greatly enhances the imaging ability of a peptide probe that selectively targets microtubules of the cytoskeleton, thus enabling the dynamic tracking of mitosis in live cells.

•Established a dimethyl-SRM method for Salmonella protein analysis•Analyzed activation dynamics of Salmonella PhoP/PhoQ network•Discovered distinct patterns in PhoP/PhoQ activation•Found new protein groups and pathways regulated by PhoP/PhoQSRM (selected reaction monitoring), a tandem mass spectrometry-based method characterized by high repeatability and accuracy, is an effective tool for the quantification of predetermined proteins. In this study, we built a time-scheduled dimethyl-SRM method that can provide the precise relative quantification of 92 proteins in one run. By applying this method to the Salmonella PhoP/PhoQ two-component system, we found that the expression of selected PhoP/PhoQ-activated proteins in response to Mg2 + concentrations could be divided into two distinct patterns. For the time-course SRM experiment, we found that the dynamics of the selected PhoP/PhoQ-activated proteins could be divided into three distinct patterns, providing a new clue regarding PhoP/PhoQ activation and regulation. Moreover, the results for iron homeostasis proteins in response to Mg2 + concentrations revealed that the PhoP/PhoQ two-component system may serve as a repressor for iron uptake proteins. And ribosomal protein levels clearly showed a response to different Mg2 + concentrations and to time.

•Proteomic analysis revealed the coordinated regulation of RstA/RstB and PhoP/PhoQ.•Discovered RstA/RstB's involvement in pyrimidine metabolism and iron acquisition•Functional assay revealed the roles of RstA/RstB in cellular motility and invasion.In pathogenic bacteria, the two-component regulatory systems (TCSs) play important roles in signal transduction and regulation of their pathogenesis. Here, we used quantitative proteomic methods to comparatively analyze functional networks under the control of the RstA/RstB system versus the PhoP/PhoQ system in Salmonella typhimurium. By comparing the proteomic profile from a wild-type strain to that from a ΔrstB strain or a ΔphoPQ strain under a condition known to activate these TCSs, we found that the levels of 159 proteins representing 6.92% of the 2297 proteins identified from the ΔrstB strain and 341 proteins representing 14.9% of the 2288 proteins identified from the ΔphoPQ strain were significantly changed, respectively. Bioinformatics analysis revealed that the RstA/RstB system and the PhoP/PhoQ system coordinated with regard to the regulation of specific proteins as well as metabolic processes. Our observations suggested that the regulatory networks controlled by the PhoP/PhoQ system were much more extensive than those by the RstA/RstB system, whereas the RstA/RstB system specifically regulated expression of the constituents participating in pyrimidine metabolism and iron acquisition. Additional results also suggested that the RstA/RstB system was required for regulation of Salmonella motility and invasion.

•Odor deprivation and odor exposure lead to different alterations in OB proteome.•Many regulated proteins could be mapped to interconnected metabolic networks.•Neurotransmitter synthesis and release were regulated in odor deprived mice.•Metabolic rates of glutamate and GABA were reduced in odor deprived mice.Olfactory sensory information is processed and integrated by circuits within the olfactory bulb (OB) before being sent to the olfactory cortex. In the mammalian OB, neural activity driven by external stimuli can lead to experience-dependent changes in structures and functions. In this study, quantitative proteomics techniques were employed to study proteome-wide changes in the OB under four levels of neural activity (from low to high): devoid of peripheral input (using a transgenic model), wild-type control, and short-term and long-term odor exposures. Our results revealed that proteins related to various processes were altered in the OBs of odor-deprived and odor-stimulated mice compared to the wild-type controls. These changes induced by odor stimulation were quite different from those induced by a deficit in peripheral olfactory inputs. Detailed analysis demonstrated that metabolic process and synaptic transmission were the most commonly altered pathways and that the effects of peripheral deprivation were more profound. Our comparative proteomics analysis indicated that olfactory deprivation and odor exposure lead to different alterations in the OB proteome, which provides new clues about the mechanisms underlying the olfactory deprivation- or odor stimulation-induced plasticity of OB function and organization.Biological significanceBy combining quantitative proteomics, bioinformatics and WB/IHC analysis, this study reports the results of the first comparative study on proteome-wide changes in the olfactory bulb under different levels of olfactory input. Odor deprivation and stimulation induced proteomic changes clearly demonstrate significant metabolic shifts and alterations on synaptic transmission. This quantitative system biology study leads to a new level of understanding in the development of olfactory bulb plasticity induced by odor deprivation or stimulation, and provides many new clues for the olfactory bulb related functional studies.

We present a detailed quantitative map of single and coexisting histone post-translational modifications (PTMs) in rat retinas affected by ischemia and reperfusion (I/R) injury. Retinal I/R injury contributes to serious ocular diseases, which can lead to vision loss and blindness. We applied linear ion trap–orbitrap hybrid tandem mass spectrometry (MS/MS) to quantify 131 single histone marks and 143 combinations of multiple histone marks in noninjured and injured retinas. We observed 34 histone PTMs that exhibited significantly (p < 0.05) different abundance between healthy and I/R injured eyes, of which we confirmed three H4 histone marks by Western blotting. H4K20me2 was up to 4-fold change up-regulated after the injury and is associated with the response to DNA damage as demonstrated by an increase in the phosphorylation of p53 and Chk1. This study demonstrates that quantitative MS provides a sensitive and accurate way to dissect the changes in the histone code after retinal injury. Specifically, DNA damage associated histone PTMs may contribute to neurovascular degeneration during the process of ischemia/reperfusion injury.

Japanese encephalitis virus (JEV) enters host cells via receptor-mediated endocytosis and replicates in the cytoplasm of infected cells. To study virus–host cell interactions, we performed a SILAC-based quantitative proteomics study of JEV-infected HeLa cells using a subcellular fractionation strategy. We identified 158 host proteins as differentially regulated by JEV (defined as exhibiting a greater than 1.5-fold change in protein abundance upon JEV infection). The mass spectrometry quantitation data for selected proteins were validated by Western blot and immunofluorescence confocal microscopy. Bioinformatics analyses were used to generate JEV-regulated host response networks consisting of regulated proteins, which included 35 proteins that were newly added based on the results of this study. The JEV infection-induced host response was found to be coordinated primarily through the immune response process, the ubiquitin-proteasome system (UPS), the intracellular membrane system, and lipid metabolism-related proteins. Protein functional studies of selected host proteins using RNA interference-based techniques were carried out in HeLa cells infected with an attenuated or a highly virulent strain of JEV. We demonstrated that the knockdown of interferon-induced transmembrane protein 3 (IFITM3), Ran-binding protein 2 (RANBP2), sterile alpha motif domain-containing protein 9 (SAMD9) and vesicle-associated membrane protein 8 (VAMP8) significantly increased JEV replication. The results presented here not only promote a better understanding of the host response to JEV infection but also highlight multiple potential targets for the development of antiviral agents.

Lipopolysaccharide (LPS) from Gram-negative bacteria and cell wall components from Gram-positive bacteria are pathogenic inducers of host cell innate immune systems. In this study, we adapted stable isotope labeling with amino acid in cell culture (SILAC) and Fe3 +-IMAC phosphopeptide enrichment method to study phosphoproteomic changes in bacterial virulence factors induced macrophage RAW 264.7 cells. In total, we quantified 2657 phosphopeptides and 1990 phosphopeptides in LPS treated and heat-killed Staphylococcus aureus (HKSA) treated macrophage samples respectively. Functional bioinformatics analysis was followed to show differences between LPS and HKSA stimulated macrophage signaling pathways. We identified differences in immune-related signaling networks, including Erk1/2 signaling pathway, Jak/Stat signaling pathway, Jnk signaling pathway, and revealed differences in cytoskeleton reorganization, GTPase regulators and in phosphorylation motifs.Highlights► Protein phosphorylation changes in LPS- and HKSA-stimulated macrophages were analyzed. ► Functional bioinformatics analysis was followed to show differences between LPS and HKSA stimulated macrophage. ► We identified protein phosphorylation differences in Erk1/2, Jak/Stat, and Jnk signaling pathway. ► Differences in cytoskeleton reorganization network, GTPase regulators and phosphorylation motifs were also found.

The PhoP/PhoQ two-component system plays a central regulatory role in the pathogenesis of Salmonella enterica serovar Typhimurium (S. Typhimurium), and it can be activated by low Mg2+ concentrations and sublethal concentrations of cationic antimicrobial peptides (CAMP). Therefore, these two PhoP/PhoQ activation signals are considered as in vivo environmental cues sensed by S. Typhimurium for adaptation and survival. In this work, we conducted a SILAC (stable isotope labeling by amino acids in cell culture)-based quantitative proteomic study to survey the proteomic changes of S. Typhimurium in response to low Mg2+ concentrations or CAMP. We discovered that CAMP activated a portion of the PhoP/PhoQ regulatory network, whereas low Mg2+ concentrations upregulated nearly all known members of this network, a number of previously unknown proteins, and some proteins regulated by IHF and RpoS. Systematic analysis following metabolic pathways revealed that low Mg2+ concentrations selectively influenced proteins of certain metabolic functions while CAMP did not. Our study indicates that the low Mg2+-concentration condition may lead S. Typhimurium into a growth-control lifestyle, which provides new perspectives about Salmonella’s adaptation to the host environment.

Protein phosphorylation activates or deactivates many other proteins especially protein enzymes, and plays a significant role in a wide range of cellular processes. Recent advances in phosphopeptide enrichment procedures and mass spectrometry-based peptide sequencing techniques have enabled us to identify large number of protein phosphorylation sites. In this study, we combined three different HPLC techniques in fractionating enriched phosphopeptides before RPLC–MS/MS analysis, and found that although between 4000–5000 unique phosphopeptides could be identified following any of the HPLC fraction method, different HPLC method yielded a considerable amount of non-overlapping unique phosphopeptides. Combining data from all the HPLC methods, we were able to identify 9069 unique phosphopeptides and 3260 phosphoproteins covering 9463 unique phosphorylation sites, indicating that different HPLC methods are complementary to each other, and can be used together in order to increase the phosphoproteome coverage. A number of new phosphorylation sites and novel phosphorylation motifs were also discovered from our study.

Silica-zirconia composite (SiO2-ZrO2) was synthesized by sol-gel method, and its physical-chemical properties were investigated using several techniques. The enrichment capacity of silica-zirconia composite and zirconia was compared using tryptic digest of a-casein as probe. The results showed that the selectivity of SiO2-ZrO2 was better than that of ZrO2 when the pH of the loading solution was below 2. The enrichment capacity of SiO2-ZrO2 for multiple phosphorylated peptides was better than that of ZrO2. In combination with matrix-assisted laser desorption ionization-time of flight (MALDI-TOF)-based peptide mass finger printing technique, SiO2-ZrO2 was used to enrich the phosphopeptides from nonfat milk successfully.

Analysis of phosphopeptides from complex mixtures derived from proteolytic digestion of biological samples is a challenging yet highly important task. Since phosphopeptides are usually present in small amounts, enrichment is often necessary prior to their characterization by mass spectrometry. In this study, a thin layer of titanium dioxide (TiO2) nanoparticles (NPs) was deposited onto the surface of capillary column by liquid phase deposition (LPD) technique and applied to selectively concentrate phosphopeptides from protein digest products. This is, to our knowledge, the first demonstration of using liquid phase deposition to construct in-tube solid phase microextraction devices for biological analysis. By coupling the device off-line or on-line with mass spectrometry analysis, experiments for systematic optimization of loading and washing conditions were carried out, and good trapping selectivity of TiO2 NP-deposited capillary columns towards phosphopeptides was demonstrated.

Here we show that graphene oxide greatly enhances the imaging ability of a peptide probe that selectively targets microtubules of the cytoskeleton, thus enabling the dynamic tracking of mitosis in live cells.