A methodology for discovering novel brain-relevant peptides: Combination of ribosome profiling and peptidomics.

Brain derived peptides function as signaling molecules in the brain and regulate various physiological and behavioral processes. The low abundance and atypical fragmentation of these brain derived peptides makes detection using traditional proteomic methods challenging. In this study, we introduce and validate a new methodology for the discovery of novel peptides derived from mammalian brain. This methodology combines ribosome profiling and mass spectrometry-based peptidomics. Using this framework, we have identified a novel peptide in mouse whole brain whose expression is highest in the basal ganglia, hypothalamus and amygdala. Although its functional role is unknown, it has been previously detected in peripheral tissue as a component of the mRNA decapping complex. Continued discovery and studies of novel regulating peptides in mammalian brain may also provide insight into brain disorders.

The Human Skeletal Muscle Proteome Project: a reappraisal of the current literature.

Skeletal muscle is a large organ that accounts for up to half the total mass of the human body. A progressive decline in muscle mass and strength occurs with ageing and in some individuals configures the syndrome of 'sarcopenia', a condition that impairs mobility, challenges autonomy, and is a risk factor for mortality. The mechanisms leading to sarcopenia as well as myopathies are still little understood. The Human Skeletal Muscle Proteome Project was initiated with the aim to characterize muscle proteins and how they change with ageing and disease. We conducted an extensive review of the literature and analysed publically available protein databases. A systematic search of peer-reviewed studies was performed using PubMed. Search terms included 'human', 'skeletal muscle', 'proteome', 'proteomic(s)', and 'mass spectrometry', 'liquid chromatography-mass spectrometry (LC-MS/MS)'. A catalogue of 5431 non-redundant muscle proteins identified by mass spectrometry-based proteomics from 38 peer-reviewed scientific publications from 2002 to November 2015 was created. We also developed a nosology system for the classification of muscle proteins based on localization and function. Such inventory of proteins should serve as a useful background reference for future research on changes in muscle proteome assessed by quantitative mass spectrometry-based proteomic approaches that occur with ageing and diseases. This classification and compilation of the human skeletal muscle proteome can be used for the identification and quantification of proteins in skeletal muscle to discover new mechanisms for sarcopenia and specific muscle diseases that can be targeted for the prevention and treatment.

Bioorthogonal mimetics of palmitoyl-CoA and myristoyl-CoA and their subsequent isolation by click chemistry and characterization by mass spectrometry reveal novel acylated host-proteins modified by HIV-1 infection.

Protein acylation plays a critical role in protein localization and function. Acylation is essential for human immunodeficiency virus 1 (HIV-1) assembly and budding of HIV-1 from the plasma membrane in lipid raft microdomains and is mediated by myristoylation of the Gag polyprotein and the copackaging of the envelope protein is facilitated by colocalization mediated by palmitoylation. Since the viral accessory protein NEF has been shown to alter the substrate specificity of myristoyl transferases, and alter cargo trafficking lipid rafts, we hypothesized that HIV-1 infection may alter protein acylation globally. To test this hypothesis, we labeled HIV-1 infected cells with biomimetics of acyl azides, which are incorporated in a manner analogous to natural acyl-Co-A. A terminal azide group allowed us to use a copper catalyzed click chemistry to conjugate the incorporated modifications to a number of substrates to carry out SDS-PAGE, fluorescence microscopy, and enrichment for LC-MS/MS. Using LC-MS/MS, we identified 103 and 174 proteins from the myristic and palmitic azide enrichments, with 27 and 45 proteins respectively that differentiated HIV-1 infected from uninfected cells. This approach has provided us with important insights into HIV-1 biology and is widely applicable to many virological systems.

Integrated microfluidic chip and online SCX separation allows untargeted nanoscale metabolomic and peptidomic profiling.

Metabolomics and peptidomics are systems biology approaches in which broad populations of molecular species produced in a cell or tissue sample are identified and quantified. These two molecular populations, metabolites and peptides, can be extracted from tissues in a similar fashion, and we therefore have here developed an integrated platform for their extraction and characterization. This was accomplished by liquid-liquid extraction of peptides and metabolites from tissue samples and online strong cation exchange (SCX) separation to allow characterization of each population individually. The platform was validated both by a mixed set of purified standards and by an analysis of splenic tissue from SIV-infected macaques, showing both good reproducibility in chromatography, with relative standard deviation (RSD) of hold time less than 0.4%, and clear separation of charge state, with ∼ 95% of molecular features in SCX separated runs at charge states of +1 or +2. Finally, we used this platform to analyze the physiological response to infection in the spleen, showing that the spleen contains an abundance of hemoglobin-derived peptides, which do not appear to change in response to infection, and that there appears to be a large and variable metabolic response to infection. We therefore present a method for peptidomic and metabolomic profiling which is simple, robust, and easy to implement.

Elevated brain monoamine oxidase activity in SIV- and HIV-associated neurological disease.

We recently demonstrated direct evidence of increased monoamine oxidase (MAO) activity in the brain of a simian immunodeficiency virus (SIV) model of human immunodeficiency virus (HIV)-associated central nervous system (CNS) disease, consistent with previously reported dopamine deficits in both SIV and HIV infection. In this study, we explored potential mechanisms behind this elevated activity. MAO B messenger RNA was highest in macaques with the most severe SIV-associated CNS lesions and was positively correlated with levels of CD68 and GFAP transcripts in the striatum. MAO B messenger RNA also correlated with viral loads in the CNS of SIV-infected macaques and with oxidative stress. Furthermore, in humans, striatal MAO activity was elevated in individuals with HIV encephalitis, compared with activity in HIV-seronegative controls. These data suggest that the neuroinflammation and oxidative stress caused by SIV infection in the CNS may provide the impetus for increased transcription of MAO B and that MAO, and more broadly, oxidative stress, have significant potential as therapeutic targets in CNS disease due to HIV.

MASPECTRAS 2: An integration and analysis platform for proteomic data.

MASPECTRAS 2 is a freely available platform for integrating MS protein identifications with information from the major bioinformatics databases (ontologies, domains, literature, etc.). It assists researchers in understanding their data and publishing through sample comparisons, targeted queries, summaries, and exports in multiple formats such as PRIDE XML (Jones et al., Nucleic Acids Res. 2008, 36, D878-D883). MASPECTRAS 2 also comprises mechanisms to facilitate its integration in a high-throughput infrastructure. We illustrate application of MASPECTRAS 2 with unpublished tyrosine kinase inhibitor drug proteomics profiles in cancerous cells.