An effective plasma membrane proteomics approach for small tissue samples

Smolders, Katrien; Lombaert, Nathalie; Valkenborg, Dirk; Baggerman, Geert; Arckens, Lutgarde

doi:10.1038/srep10917

Cited by 30 publications

(21 citation statements)

References 32 publications

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“…For cell-surface biotin labeling of membrane proteins 75 , retinas were dissected out of the eyecup into ice-cold HBSS. Retinas were washed with HBSS followed by incubation in HBSS supplemented with EZ-Link Sulfo_NHS-SS-Biotin (0.5 mg/ml in HBSS) for 45 min on ice.…”

Section: Isoform Clusteringmentioning

confidence: 99%

Comprehensive identification of mRNA isoforms reveals the diversity of neural cell-surface molecules with roles in retinal development and disease

et al. 2020

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Genes encoding cell-surface proteins control nervous system development and are implicated in neurological disorders. These genes produce alternative mRNA isoforms which remain poorly characterized, impeding understanding of how disease-associated mutations cause pathology. Here we introduce a strategy to define complete portfolios of full-length isoforms encoded by individual genes. Applying this approach to neural cell-surface molecules, we identify thousands of unannotated isoforms expressed in retina and brain. By mass spectrometry we confirm expression of newly-discovered proteins on the cell surface in vivo. Remarkably, we discover that the major isoform of a retinal degeneration gene, CRB1, was previously overlooked. This CRB1 isoform is the only one expressed by photoreceptors, the affected cells in CRB1 disease. Using mouse mutants, we identify a function for this isoform at photoreceptor-glial junctions and demonstrate that loss of this isoform accelerates photoreceptor death. Therefore, our isoform identification strategy enables discovery of new gene functions relevant to disease.

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Section: Isoform Clusteringmentioning

confidence: 99%

Comprehensive identification of mRNA isoforms reveals the diversity of neural cell-surface molecules with roles in retinal development and disease

et al. 2020

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“…After centrifugation at 4°C for 2.5 h at 180,000 g (44,200 rpm), each sample was divided into 12 fractions (0.5 ml each) and the first two fractions were used for further analyses. Cell surface proteins were isolated as previously [31] by using pierce cell surface protein isolation kit.…”

Section: Western Blottingmentioning

confidence: 99%

Coupling of terminal differentiation deficit with neurodegenerative pathology in Vps35-deficient pyramidal neurons

Tang

Zhao

et al. 2020

Cell Death Differ

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Vps35 (vacuolar protein sorting 35) is a key component of retromer that regulates transmembrane protein trafficking. Dysfunctional Vps35 is a risk factor for neurodegenerative diseases, including Parkinson's and Alzheimer's diseases. Vps35 is highly expressed in developing pyramidal neurons, and its physiological role in developing neurons remains to be explored. Here, we provide evidence that Vps35 in embryonic neurons is necessary for axonal and dendritic terminal differentiation. Loss of Vps35 in embryonic neurons results in not only terminal differentiation deficits, but also neurodegenerative pathology, such as cortical brain atrophy and reactive glial responses. The atrophy of neocortex appears to be in association with increases in neuronal death, autophagosome proteins (LC3-II and P62), and neurodegeneration associated proteins (TDP43 and ubiquitin-conjugated proteins). Further studies reveal an increase of retromer cargo protein, sortilin1 (Sort1), in lysosomes of Vps35-KO neurons, and lysosomal dysfunction. Suppression of Sort1 diminishes Vps35-KO-induced dendritic defects. Expression of lysosomal Sort1 recapitulates Vps35-KO-induced phenotypes. Together, these results demonstrate embryonic neuronal Vps35's function in terminal axonal and dendritic differentiation, reveal an association of terminal differentiation deficit with neurodegenerative pathology, and uncover an important lysosomal contribution to both events.

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“…Tube-gel (TG) principle relies on the polymerization of polyacrylamide gel directly in the sample in a Laemmli-like solution. It was first introduced in 2005 by Lu X. et al and consequently applied and adapted by others, in particular for the analysis of membrane proteins, lipid rafts proteins and the charge derivatization of peptides [1][2][3][4][5][6][7][8]. We have recently demonstrated its compatibility and repeatability for label-free quantitative proteomics by comparing it with a stacking gel and a standard urea-based liquid digestion protocol using well-calibrated samples [9].…”

Section: Introductionmentioning

confidence: 99%

Tube-Gel: A Fast and Effective Sample Preparation Method for High-Throughput Quantitative Proteomics

Muller¹,

Fornecker²,

Cianférani³

et al. 2019

Methods in Molecular Biology

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Sample preparation is a key step in proteomics workflows. Tube-gel (TG) is a fast and repeatable sample preparation method that consists in the instantaneous trapping of the sample in a polyacrylamide gel matrix. It takes advantage of in-gel sample preparations by allowing the use of high concentrations of sodium-dodecyl sulfate but avoids the timeconsuming step of electrophoresis. Therefore, TG limits the sample handling and is thus particularly suitable for high-throughput quantitative proteomics when large sample numbers have to be processed, as it is often the case in biomarker research and clinical proteomics projects.

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An effective plasma membrane proteomics approach for small tissue samples

Cited by 30 publications

References 32 publications

Comprehensive identification of mRNA isoforms reveals the diversity of neural cell-surface molecules with roles in retinal development and disease

Comprehensive identification of mRNA isoforms reveals the diversity of neural cell-surface molecules with roles in retinal development and disease

Coupling of terminal differentiation deficit with neurodegenerative pathology in Vps35-deficient pyramidal neurons

Tube-Gel: A Fast and Effective Sample Preparation Method for High-Throughput Quantitative Proteomics

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