Journal: Molecular & Cellular Proteomics : MCP

Article Title: Improved T Cell Surfaceomics by Depleting Intracellularly Labelled Dead Cells

doi: 10.1016/j.mcpro.2025.101503

Figure Lengend Snippet: NonViable Cells Drive Intracellular Protein Contamination in Amine-Reactive Surface Biotinylation . A , jurkat cells were labeled with sulfo-NHS-biotin and stained with streptavidin-FITC, 7-AAD-PerCP, and Annexin V-PE to assess biotinylation levels and cell viability by flow cytometry. Dot plots gated on biotin high ( right ) and biotin mod ( middle ) are shown. B , jurkat cells were biotinylated using one of four NHS-biotin variants: sulfo-NHS-biotin (sNb), sulfo-NHS-SS-biotin (ssNb), sulfo-NHS-LC-biotin (LC), or sulfo-NHS-LCLC-biotin (LCLC). Cells were then stained with streptavidin-AF647, and mean fluorescence intensities (MFI) of biotin mod and biotin high populations were quantified by flow cytometry. C , Representative images of Jurkat cells labeled with sulfo-NHS-biotin, stained with streptavidin-AF647, Annexin V, and DAPI, and acquired using the ImageStreamX Mark II imaging flow cytometer to visualize biotin localization and cell viability. ( D ) Cells were gated based on biotin signal intensity, and nuclear colocalization of the biotin signal ( top ) and nuclear streptavidin staining intensity ( bottom ) were quantified using IDEAS software ( https://cytekbio.com/pages/imagestream ). E , human T cells, HeLa, and HEK293 cells were biotinylated with sulfo-NHS-biotin, stained with streptavidin-AF647, and analyzed by flow cytometry to examine biotin high populations across cell types. ∗∗∗ p < 0.001, ∗∗∗∗ p -value <0.0001 by unpaired two-tailed t test with Welch’s correction.

Article Snippet: Streptavidin-DyLight 649 and Streptavidin-FITC were used for detecting biotinylated proteins (Vector Laboratories).

Techniques: Labeling, Staining, Flow Cytometry, Fluorescence, Imaging, Software, Two Tailed Test

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Improved T Cell Surfaceomics by Depleting Intracellularly Labelled Dead Cells

doi: 10.1016/j.mcpro.2025.101503

Figure Lengend Snippet: Optimization of Dead Cell Removal Strategies to Minimize Intracellular Protein Contamination Following Amine-Reactive Biotinylation . A , schematic illustration of the experimental workflow, depicting NHS-biotin surface labeling followed by dead cell depletion. B , Annexin V–positive cells remaining after NHS-biotin surface labeling were depleted using various methods as indicated. The efficiency of depletion and cell recovery rates were quantified by flow cytometry. C , jurkat cells containing Annexin V–positive cells were either left undepleted ( top panel ), depleted prior to surface labeling ( middle panel ), or depleted after surface labeling ( bottom panel ). Cells were stained with streptavidin to assess the efficiency of removing the Biotin high population by flow cytometry. D , surface-labeled cells, with or without Annexin V–positive cell removal, was lysed, and biotinylated proteins were enriched using streptavidin-coated agarose beads at 4 °C overnight. Postenrichment, proteins were eluted and loaded onto SDS-PAGE gels at varying volumes (10 μl, 20 μl, and 40 μl), alongside input and flow-through fractions. Western blotting was performed using primary antibodies against Actin, Histone H3, PTPRC (CD45), and CD7, followed by HRP-conjugated secondary antibodies. Band intensities were quantified using ImageJ, and immunoprecipitated signals were normalized to input to calculate the percentage of enrichment. NS, not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001 by unpaired two-tailed t test with Welch’s correction.

Article Snippet: Streptavidin-DyLight 649 and Streptavidin-FITC were used for detecting biotinylated proteins (Vector Laboratories).

Techniques: Labeling, Cell Recovery, Flow Cytometry, Staining, SDS Page, Western Blot, Immunoprecipitation, Two Tailed Test

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Improved T Cell Surfaceomics by Depleting Intracellularly Labelled Dead Cells

doi: 10.1016/j.mcpro.2025.101503

Figure Lengend Snippet: Annexin V–Based Dead Cell Depletion Enhances Specificity and Sensitivity of Plasma Membrane Proteomics by Mass Spectrometry . Peptide ( A ) and protein ( B ) overlaps between control and dead cell depletion (DCD) runs are shown. C , quantification of plasma membrane (PM) proteins in control and DCD samples was performed based on SURFY annotations. Peptide coverage percentages for PM and non-PM ( D ) and iBAQ intensities ( E ) are compared between control and DCD samples in ( D ). iBAQ intensities. The percentage intensity of PM proteins ( F ) and Non-PM proteins ( G ) was calculated relative to the total protein intensity within each sample. Comparisons were made between control and DCD groups to assess distribution differences. Bar plots illustrate the top 10 highest-abundance proteins based on raw intensity in control ( H ) and DCD ( I ) samples, with red bars indicating SURFY-annotated PM proteins and asterisks denoting endogenous biotinylated proteins. J and K , scatter plots depict proteins identified in control ( J ) and DCD ( K ) datasets, where PM proteins are shown as large , dark circles ( red in control, blue in DCD) and non-PM proteins as smaller , lighter circles . Protein features are annotated as purple rectangles for type I membrane proteins, cyan triangles for multi-pass proteins, and green diamonds for GPI-anchored proteins. The x-axis represents summed intensities across control and DCD datasets, and the y-axis indicates intensities in either control or DCD samples. L and M , subcellular enrichment analyses were performed using SubcellulaRVis with the Jurkat genome as background, with red bars indicating significantly enriched compartments and bubble sizes reflecting the number of proteins identified in control ( L ) and DCD ( M ) samples. N , comparison of Human Protein Atlas–derived expression levels is shown for PM and non-PM proteins identified in control and DCD samples. NS, not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001 by unpaired two-tailed t test with Welch’s correction. iBAQ, intensity-based absolute quantification; PM, plasma membrane.

Article Snippet: Streptavidin-DyLight 649 and Streptavidin-FITC were used for detecting biotinylated proteins (Vector Laboratories).

Techniques: Clinical Proteomics, Membrane, Mass Spectrometry, Control, Comparison, Derivative Assay, Expressing, Two Tailed Test, Quantitative Proteomics

Journal: bioRxiv

Article Title: Differential Assembly of Native ENaC Complexes Across Mouse Epithelial Tissues

doi: 10.64898/2026.01.23.701393

Figure Lengend Snippet: a . Schematic workflow illustrating the experimental pipeline. Tissues (lung, kidney, colon) were harvested from ENaCγ-VF mice, homogenized in TRIS-based buffer, and followed by solubilization. Cleared supernatants were injected onto a Superose 6 Increase column for FSEC analysis, and fractions corresponding to high-molecular-weight complexes were subsequently analyzed by SiMPull. b . Representative FSEC traces showing mVenus fluorescence from lung, kidney, and colon lysates. Fluorescent peaks indicate the presence of γ-containing ENaC complexes in each tissue. c . Representative TIRF images of SiMPull experiments from lung lysates. mVenus-tagged ENaC complexes were captured using a biotinylated anti-GFP/mVenus nanobody. A background control image was collected from a flow chamber lacking the nanobody to demonstrate specificity. d . Quantification of SiMPull signal from lung, kidney, and colon tissues. Each condition represents data pooled from 40 TIRF images per sample, analyzed using the ComDet plugin in ImageJ. Statistical analysis was performed in GraphPad Prism.

Article Snippet: Biotinylated proteins bound to the Strep-Tactin resin were subsequently eluted using excess biotin (10x Buffer BXT, IBA Lifesciences), and 7B1-mScarlet was added to the eluted plasma membrane fraction.

Techniques: Injection, High Molecular Weight, Fluorescence, Control