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Metabolomics and exposomics studies have been growing rapidly during the past decade and providing insights into cellular metabolism and how a system responds to stimuli. Ion mobility spectrometry-mass spectrometry (IMS-MS) has become an appealing tool for small molecule studies as it enables the analysis and separation of the many isomeric species. However, to facilitate the identification of endogenous metabolites and xenobiotics from complex samples, a library with accurate collision cross sections (CCS) is needed. In this work, we generated a CCS library for >1000 small molecule standards using triplicate analyses in positive and negative polarity. This library was then coupled with ultrafast small molecule analyses using a sub-minute online SPE-IMS-MS method for characterization of different metabolic and xenobiotic conditions.IMS-MS is capable of separating molecules that have the same mass-to-charge (m/z) ratios, but different sizes, shapes or conformations. In this work an Agilent drift tube-IMS-QTOF MS platform was used to characterize over one thousand endogenous metabolites and xenobiotics. The CCS values were measured using seven stepped fields in both positive and negative polarity. For small molecule studies, the samples were analyzed by coupling Rapidfire, an online SPE system to the IMS-MS platform which enables sub-minute ultrafast analyses. Different SPE cartridges such as C18, graphitic carbon, HILIC and phenyl cartridges were explored to effectively extract different classes of molecules. In addition, different ionization sources including ESI, APCI and APPI were evaluated for efficient small molecule studies. A large scale ion mobility CCS library including over 800 endogenous metabolites and over 200 xenobiotics was generated using drift tube IMS-MS. Different classes of metabolites displayed different trend lines for their CCS values. For instance, fatty acids show larger CCS values than carbohydrates or amino acids. We were also able to map the CCS values for key metabolites and intermediates in important metabolite pathways such as glycolysis/gluconeogenesis, pentose phosphate pathway and citrate cycle, etc. These results are essential for future studies aimed at identification of metabolites within different structure classes and pathways. To evaluate the use of this CCS library for metabolite identification, we analyzed how small molecules change in patients with and without exercises, as well as those with and without ethanol use, and compared the results from the SPE-IMS-MS method with those from LC-MS method. Xenobiotics that are important for the evaluation of environmental exposure such as polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) exposures were also studied. Different ionization sources including ESI, APCI and APPI, were evaluated for efficient characterization and detection of the PAHs and PCBs and their metabolites. Preliminary data showed that we were able to detect PAHs in [M∙]+ and [M + H]+ forms and PCBs in the [M-Cl+O]- form using APCI/APPI methods. We also showed that the pre-separation, clean-up and enrichment steps by Rapidfire SPE greatly improved the sensitivity, enabling ultrafast and simultaneous analyses of PAHs and PCBs and their metabolites in complex biological and environmental samples.

Microglia, the innate immune cells of the central nervous system, have been genetically implicated in multiple neurodegenerative diseases. We previously mapped the genetic regulation of gene expression and mRNA splicing in human microglia, identifying several loci where common genetic variants in microglia-specific regulatory elements explain disease risk loci identified by GWAS. However, identifying genetic effects on splicing has been challenging due to the use of short sequencing reads to identify causal isoforms. Here we present the isoform-centric microglia genomic atlas (isoMiGA) which leverages the power of long-read RNA-seq to identify 35,879 novel microglia isoforms. We show that the novel microglia isoforms are involved in stimulation response and brain region specificity. We then quantified the expression of both known and novel isoforms in a multi-ethnic meta-analysis of 555 human microglia short-read RNA-seq samples from 391 donors, the largest to date, and found associations with genetic risk loci in Alzheimer's disease and Parkinson's disease. We nominate several loci that may act through complex changes in isoform and splice site usage.

Building mechanistic models of kinase-driven signaling pathways requires quantitative measurements of protein phosphorylation across physiologically relevant conditions, but this is rarely done because of the insensitivity of traditional technologies. By using a multiplexed deep phosphoproteome profiling workflow, we were able to generate a deep phosphoproteomics dataset of the EGFR-MAPK pathway in non-transformed MCF10A cells across physiological ligand concentrations with a time resolution of <12 min and in the presence and absence of multiple kinase inhibitors. An improved phosphosite mapping technique allowed us to reliably identify >46,000 phosphorylation sites on >6600 proteins, of which >4500 sites from 2110 proteins displayed a >2-fold increase in phosphorylation in response to EGF. This data was then placed into a cellular context by linking it to 15 previously published protein databases. We found that our results were consistent with much, but not all previously reported data regarding the activation and negative feedback phosphorylation of core EGFR-ERK pathway proteins. We also found that EGFR signaling is biphasic with substrates downstream of RAS/MAPK activation showing a maximum response at <3ng/ml EGF while direct substrates, such as HGS and STAT5B, showing no saturation. We found that RAS activation is mediated by at least 3 parallel pathways, two of which depend on PTPN11. There appears to be an approximately 4-minute delay in pathway activation at the step between RAS and RAF, but subsequent pathway phosphorylation was extremely rapid. Approximately 80 proteins showed a >2-fold increase in phosphorylation across all experiments and these proteins had a significantly higher median number of phosphorylation sites (~18) relative to total cellular phosphoproteins (~4). Over 60% of EGF-stimulated phosphoproteins were downstream of MAPK and included mediators of cellular processes such as gene transcription, transport, signal transduction and cytoskeletal arrangement. Their phosphorylation was either linear with respect to MAPK activation or biphasic, corresponding to the biphasic signaling seen at the level of the EGFR. This deep, integrated phosphoproteomics data resource should be useful in building mechanistic models of EGFR and MAPK signaling and for understanding how downstream responses are regulated., Competing Interests: DECLARATION OF INTERESTS The authors declare no competing interests.