Systematic Optimization of Long Gradient Chromatography Mass Spectrometry for Deep Analysis of Brain Proteome

Wang, Hong; Yang, Yi-Yan; Li, Yuxin; Bai, Bing; Wang, Xusheng; Tan, Haidong; Liu, Tao; Beach, Thomas G.; Peng, Junmin; Wu, Zhiping

doi:10.1021/pr500882h

Cited by 79 publications

(77 citation statements)

References 49 publications

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“…The analysis was performed based on our optimized platform 35 . For whole proteome analysis, the dried peptides were reconstituted in 5% formic acid, loaded on a reverse phase column (75 μm × 30 cm, 1.9 μm C18 resin (Dr. Maisch GmbH, Germany)) interfaced with a Q-Exactive HF mass spectrometer (ThermoFisher Scientific).…”

Section: Methodsmentioning

confidence: 99%

Hippo/Mst signalling couples metabolic state and immune function of CD8α+ dendritic cells

Wen

Wang

et al. 2018

Nature

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166

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Dendritic cells (DCs) orchestrate the crosstalk between innate and adaptive immunity. CD8α+ DCs present antigens to CD8+ T cells and elicit cytotoxic T-cell responses to viruses, bacteria and tumors1. Although lineage-specific transcriptional regulators of CD8α+ DC development have been identified2, the molecular pathways that selectively orchestrate CD8α+ DC function remain elusive. Moreover, metabolic reprogramming is important for DC development and activation3,4, but metabolic dependence and regulation of DC subsets are unknown. Here, we describe a data-driven systems biology algorithm (NetBID) and an unexpected role of Hippo pathway kinases, Mst1 and Mst2 (Mst1/2), in selectively programming CD8α+ DC function and metabolism. Our NetBID analysis reveals a marked enrichment of the activities of Hippo pathway kinases in CD8α+ DCs relative to CD8α− DCs. DC-specific deletion of Mst1/2, but not Lats1/2 or Yap/Taz that mediate canonical Hippo signaling, disrupts homeostasis and function of CD8+ T cells and anti-tumor immunity. Mst1/2-deficient CD8α+ DCs are impaired in presenting extracellular proteins and cognate peptides to prime CD8+ T cells, while CD8α− DCs lacking Mst1/2 have largely normal function. Mechanistically, compared with CD8α− DCs, CD8α+ DCs show much stronger oxidative metabolism and critically depend upon Mst1/2 signaling to maintain bioenergetic activities and mitochondrial dynamics for functional capacities. Further, CD8α+ DCs selectively express IL-12 that depends upon Mst1/2 and the crosstalk with non-canonical NF-κB signaling. Our findings identify Mst1/2 as selective drivers of CD8α+ DC function by integrating metabolic activity and cytokine signaling, and highlight that the interplay between immune signaling and metabolic reprogramming underlies the unique function of DC subsets.

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Section: Methodsmentioning

confidence: 99%

Hippo/Mst signalling couples metabolic state and immune function of CD8α+ dendritic cells

Wen

Wang

et al. 2018

Nature

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166

145

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“…Fresh iodoacetamide was added to 20 mM for 15 min alkylation at room temperature in the dark. The samples were run on an SDS gel until the dye front was 3 mm past the stacking gel border to ensure complete stacking but little separation (29 (30). TMT labeled peptides were loaded on a long C18 column (ϳ1 m ϫ 75 m) packed with 1.9 m resin (Dr. Amish GmbH, Germany), and eluted during a 9 h gradient (ϳ0.15 l/min; 20 -55%; buffer A: 0.2% formic acid, 5% DMSO; buffer B: 0.2% formic acid, 5% DMSO, and 65% ACN).…”

Section: Methodsmentioning

confidence: 99%

Sequential Elution Interactome Analysis of the Mind Bomb 1 Ubiquitin Ligase Reveals a Novel Role in Dendritic Spine Outgrowth

Mertz¹,

Tan

Pagala

et al. 2015

Molecular & Cellular Proteomics

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The mind bomb 1 (Mib1) ubiquitin ligase is essential for controlling metazoan development by Notch signaling and possibly the Wnt pathway. It is also expressed in postmitotic neurons and regulates neuronal morphogenesis and synaptic activity by mechanisms that are largely unknown. We sought to comprehensively characterize the Mib1 interactome and study its potential function in neuron development utilizing a novel sequential elution strategy for affinity purification, in which Mib1 binding proteins were eluted under different stringency and then quantified by the isobaric labeling method. The strategy identified the Mib1 interactome with both deep coverage and the ability to distinguish high-affinity partners from low-affinity partners. A total of 817 proteins were identified during the Mib1 affinity purification, including 56 high-affinity partners and 335 low-affinity partners, whereas the remaining 426 proteins are likely copurified contaminants or extremely weak binding proteins. The analysis detected all previously known Mib1-interacting proteins and revealed a large number of novel components involved in Notch and Wnt pathways, endocytosis and vesicle transport, the ubiquitin-proteasome system, cellular morphogenesis, and synaptic activities. Immunofluorescence studies further showed colocalization of Mib1 with five selected proteins: the Usp9x (FAM) deubiquitinating enzyme, alpha-, beta-, and delta-catenins, and CDKL5. Mutations of CDKL5 are associated with early infantile epileptic encephalopathy-2 (EIEE2), a severe form of mental retardation. We found that the expression of Mib1 downregulated the protein level of CDKL5 by ubiquitination, and antagonized CDKL5 function during the formation of dendritic spines. Thus, the sequential elution strategy enables biochemical characterization of protein interac-

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“…52 The second dataset was obtained from a multiple myeloma cell line (ANBL6), 53 including tandem mass tag (TMT, Thermo Fisher Scientific) labeled raw data (6.2 million MS/MS scans) and RNA-seq data (242 million reads).…”

Section: Methodsmentioning

confidence: 99%

JUMPg: An Integrative Proteogenomics Pipeline Identifying Unannotated Proteins in Human Brain and Cancer Cells

Li¹,

Wang²,

Cho³

et al. 2016

J. Proteome Res.

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Proteogenomics is an emerging approach to improve gene annotation and interpretation of proteomics data. Here we present JUMPg, an integrative proteogenomics pipeline including customized database construction, tag-based database search, peptide-spectrum match filtering, and data visualization. JUMPg creates multiple databases of DNA polymorphisms, mutations, splice junctions, partially trypticity, as well as protein fragments translated from the whole transcriptome in all six frames upon RNA-seq de novo assembly. We use a multistage strategy to search these databases sequentially, in which the performance is optimized by researching only unmatched high quality spectra, and re-using amino acid tags generated by the JUMP search engine. The identified peptides/proteins are displayed with gene loci using the UCSC genome browser. Then the JUMPg program is applied to process a label-free mass spectrometry dataset of Alzheimer’s disease postmortem brain, uncovering 496 new peptides of amino acid substitutions, alternative splicing, frame shift, and “non-coding gene” translation. The novel protein PNMA6BL specifically expressed in the brain is highlighted. We also tested JUMPg to analyze a stable-isotope labeled dataset of multiple myeloma cells, revealing 991 sample-specific peptides that include protein sequences in the immunoglobulin light chain variable region. Thus, the JUMPg program is an effective proteogenomics tool for multi-omics data integration.

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Systematic Optimization of Long Gradient Chromatography Mass Spectrometry for Deep Analysis of Brain Proteome

Cited by 79 publications

References 49 publications

Hippo/Mst signalling couples metabolic state and immune function of CD8α+ dendritic cells

Hippo/Mst signalling couples metabolic state and immune function of CD8α+ dendritic cells

Sequential Elution Interactome Analysis of the Mind Bomb 1 Ubiquitin Ligase Reveals a Novel Role in Dendritic Spine Outgrowth

JUMPg: An Integrative Proteogenomics Pipeline Identifying Unannotated Proteins in Human Brain and Cancer Cells

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