Pressure-assisted sample preparation for proteomic analysis.

Pressure-assisted digestion of proteins, also known as pressure cycling technology (PCT), using a Barocycler NEP 2320 was compared with the conventional method using atmospheric pressure. Our objective was to demonstrate that PCT provides more controlled enzymatic digestion of proteins than prolonged digestion at atmospheric pressure ranging from 18 to 24 h. More controlled digestion would be beneficial for studies of highly posttranslationally modified protein such as histones. For the comparison of these two techniques, recombinant and native histone H4 were used as model proteins. PCT was optimized for pressure and time, and it was found to be most effective at 15 kpsi for 120 min of incubation. In conclusion, the PCT method was found to be much faster than using atmospheric pressure. PCT was also found to allow for unambiguous control of digestion parameters and to provide a high yield of sequence coverage compared with atmospheric pressure.

Plasma proteomic analysis of simian immunodeficiency virus infection of rhesus macaques.

Lentiviral replication in its target cells affects a delicate balance between cellular cofactors required for virus propagation and immunoregulation for host defense. To better elucidate cellular proteins linked to viral infection, we tested plasma from rhesus macaques infected with the simian immunodeficiency viral strain SIVsmm9, prior to, 10 days (acute), and 49 weeks (chronic) after viral infection. Changes in plasma protein content were measured by quantitative mass spectrometry by isobaric tags for absolute and relative quantitation (iTRAQ) methods. An 81 and 232% increase in SERPINA1 was seen during acute and chronic infection, respectively. Interestingly, gelsolin, vitamin D binding protein and histidine rich glycoprotein were decreased by 45% in acute conditions but returned to baseline during chronic infection. When compared to uninfected controls, a 48-103% increase in leucine rich alpha 2-glycoprotein, vitronectin, and ceruloplasmin was observed during chronic viral infection. Observed changes in plasma proteins expression likely represent a compensatory host response to persistent viral infection.

Methamphetamine Increases the Proportion of SIV-Infected Microglia/Macrophages, Alters Metabolic Pathways, and Elevates Cell Death Pathways: A Single-Cell Analysis.

Both substance use disorder and HIV infection continue to affect many individuals. Both have untoward effects on the brain, and the two conditions often co-exist. In the brain, macrophages and microglia are infectable by HIV, and these cells are also targets for the effects of drugs of abuse, such as the psychostimulant methamphetamine. To determine the interaction of HIV and methamphetamine, we isolated microglia and brain macrophages from SIV-infected rhesus monkeys that were treated with or without methamphetamine. Cells were subjected to single-cell RNA sequencing and results were analyzed by statistical and bioinformatic analysis. In the animals treated with methamphetamine, a significantly increased proportion of the microglia and/or macrophages were infected by SIV. In addition, gene encoding functions in cell death pathways were increased, and the brain-derived neurotropic factor pathway was inhibited. The gene expression patterns in infected cells did not cluster separately from uninfected cells, but clusters comprised of microglia and/or macrophages from methamphetamine-treated animals differed in neuroinflammatory and metabolic pathways from those comprised of cells from untreated animals. Methamphetamine increases CNS infection by SIV and has adverse effects on both infected and uninfected microglia and brain macrophages, highlighting the dual and interacting harms of HIV infection and drug abuse on the brain.

Genomic and proteomic microglial profiling: pathways for neuroprotective inflammatory responses following nerve fragment clearance and activation.

Microglia, a primary immune effector cell of the central nervous system (CNS) affects homeostatic, neuroprotective, regenerative and degenerative outcomes in health and disease. Despite these broad neuroimmune activities linked to specific environmental cues, a precise cellular genetic profile for microglia in the context of disease and repair has not been elucidated. To this end we used nucleic acid microarrays, proteomics, immunochemical and histochemical tests to profile microglia in neuroprotective immune responses. Optic and sciatic nerve (ON and SN) fragments were used to stimulate microglia in order to reflect immune consequences of nervous system injury. Lipopolysaccharide and latex beads-induced microglial activation served as positive controls. Cytosolic and secreted proteins were profiled by surface enhanced laser desorption ionization-time of flight (SELDI-TOF) ProteinChip, 1D and 2D difference gel electrophoresis. Proteins were identified by peptide sequencing with tandem mass spectrometry, ELISA and western blot tests. Temporal expression of pro-inflammatory cytokines, antioxidants, neurotrophins, and lysosomal enzyme expression provided, for the first time, a unique profile of secreted microglia proteins with neuroregulatory functions. Most importantly, this molecular and biochemical signature supports a broad range of microglial functions for debris clearance and promotion of neural repair after injury.

Proteomic fingerprints distinguish microglia, bone marrow, and spleen macrophage populations.

Mononuclear phagocytes (MP; dendritic cells, monocytes, tissue macrophages, and microglia) maintain tissue homeostasis and provide a first line of defense against invading pathogens. In specific circumstances, MPs also induce inflammatory responses and as such affect disease onset and progression. Despite intensive research into MP biology, little is known of the functional and molecular properties of individual MP subtypes. Using a novel proteomics platform, unique protein patterns and protein identities were observed among populations of spleen and bone marrow macrophages and microglia. Cells were obtained from C57BL/6 mice and were cultivated in macrophage colony-stimulating factor. MP subtypes were indistinguishable by morphological or antigenic criteria. Protein profiling by Surface Enhanced Laser Desorption Ionization-Time of Flight (SELDI-TOF) ProteinChip assays with weak cationic exchange chips showed unique MP spectral profiles. Corresponding protein fractions were recovered by high performance liquid chromatography and identified by liquid chromatography tandem mass spectrometry. The results provide a unique means to distinguish microglia from other MP subtypes.