sa magnetic beads  (Vazyme Biotech Co)

Journal: bioRxiv

Article Title: Spatial proteomics reveals prefrontal circuit diversity in socioemotional behaviour

doi: 10.64898/2026.02.08.704617

Figure Lengend Snippet: Establishment and validation of ProX-ID for projection-specific presynaptic proteome profiling in mPFC–BLA neural pathways. a, Schematic of the ProX-ID method. The palmitoylation sequence of GAP43 is fused to ultraID to enable proximity-dependent biotinylation of presynaptic proteins in mPFC–BLA and BLA–mPFC projections. b, Experimental design of in vivo ProX-ID labelling. c, Immunoblot analysis of biotinylated proteins in the mPFC–BLA and BLA–mPFC neural pathways. d, Quantification of streptavidin signals in the mPFC–BLA and BLA–mPFC neural pathways. Data are presented as mean ± s.e.m.; n = 4 mice per experimental group. Two-way ANOVA followed by Bonferroni post hoc test. **** P < 0.0001; *** P < 0.001. Results for the selected pairwise comparisons are shown. e – f, Volcano plots for mPFC–BLA ( e ) and BLA–mPFC ( f ) neural pathways. Dashed lines indicate FDR = 0.05 (horizontal) and log₂ fold change = −2 and 2 (vertical). Red dots represent significantly enriched proteins (FDR < 0.05, log₂ fold change > 2). Gene symbols indicate the top 10 proteins with the largest fold changes. g, Heatmap of normalized log₂ biotinylation levels (ProX-ID vs. control) for representative presynaptic proteins. Asterisks indicate proteins significantly enriched in ProX-ID samples (FDR < 0.05, log 2 fold change > 2, ProX-ID vs. control). NA indicates missing values. h, Odds ratios for selected Gene Ontology (GO) Cellular Component terms (see Materials and Methods). i, Proportion of known presynaptic, neurological disease-associated and functionally uncharacterized proteins among the neural pathway-enriched proteins. j, Venn diagram showing the overlap of significantly enriched proteins between the mPFC–BLA and BLA–mPFC neural pathways. Numbers indicate protein numbers. k, Overlap between mPFC–BLA-enriched proteins and transcriptomes from mouse (left) and marmoset (right). Numbers indicate protein numbers. l, Top 20 enriched GO terms for Biological Process and Molecular Function. mPFC–BLA selective and BLA–mPFC selective describe proteins selectively enriched in the mPFC–BLA and BLA–mPFC neural pathway, respectively. m, Protein–term network based on KEGG BRITE annotations (left). Nodes represent proteins and functional terms, with edges indicating associations based on BRITE functional categories. Proteins annotated for the selected category are highlighted (right).

Article Snippet: Biotinylated proteins were captured on a 10 μL slurry of NanoLink streptavidin magnetic beads (Vector) by incubation for 3 h at 4°C.

Techniques: Biomarker Discovery, Sequencing, In Vivo, Western Blot, Control, Functional Assay

Journal: bioRxiv

Article Title: Spatial proteomics reveals prefrontal circuit diversity in socioemotional behaviour

doi: 10.64898/2026.02.08.704617

Figure Lengend Snippet: High-resolution and pathway-specific labelling of presynaptic terminals using ProX-ID. a, Representative images showing biotinylation activity in cultured neurons expressing ProX-ID at 14 days in vitro (DIV), confirming the increased biotinylation compared to control neurons. b, Quantification of streptavidin signals. Data are presented as mean ± s.e.m.; n = 4 per experimental group. Student’s t -test. **** P < 0.0001. c – d, Immunohistochemistry of biotinylated proteins in the mPFC ( c , injection site) and BLA ( d , projecting site) illustrating labelling of the mPFC–BLA neural pathway. e – f, Immunohistochemistry of biotinylated proteins in the BLA ( e , injection site) and mPFC ( f , projecting site) illustrating labelling of the BLA–mPFC neural pathway. g, Super-resolution microscopy images of biotinylated proteins at axonal terminals of the mPFC–BLA (left) and BLA–mPFC (right) projections. h, Super-resolution microscopy images of biotinylated proteins with the excitatory synaptic markers Homer1 and VGLUT1.

Article Snippet: Biotinylated proteins were captured on a 10 μL slurry of NanoLink streptavidin magnetic beads (Vector) by incubation for 3 h at 4°C.

Techniques: Activity Assay, Cell Culture, Expressing, In Vitro, Control, Immunohistochemistry, Injection, Super-Resolution Microscopy

Journal: bioRxiv

Article Title: Spatial proteomics reveals prefrontal circuit diversity in socioemotional behaviour

doi: 10.64898/2026.02.08.704617

Figure Lengend Snippet: Quantitative profiling of presynaptic proteins across multiple mPFC output pathways. a, Immunoblot analysis of biotinylated proteins in the mPFC–NAc, mPFC–Thal, mPFC–HT and mPFC–CTX neural pathways. b, Quantification of streptavidin signals from NAc, Thal, HT and CTX lysates. Data are presented as mean ± s.e.m.; n = 4 mice per experimental group. Two-way ANOVA followed by Bonferroni post hoc test. *** P < 0.001; ** P < 0.01. Results for the selected pairwise comparisons are shown. c, Heatmap showing the normalized log 2 biotinylation levels in ProX-ID samples relative to control samples for representative presynaptic proteins for mPFC–NAc, mPFC–Thal, mPFC–HT and mPFC–CTX together with mPFC–BLA, and BLA–mPFC. Asterisks indicate proteins significantly enriched in ProX-ID samples (FDR < 0.05, log 2 fold change > 2, ProX-ID vs. control). NA indicates missing values. d – g, Bubble plots summarizing enrichment analysis for the selected Gene Ontology (GO) Cellular Component terms (see Materials and Methods) based on Fisher’s exact test.

Article Snippet: Biotinylated proteins were captured on a 10 μL slurry of NanoLink streptavidin magnetic beads (Vector) by incubation for 3 h at 4°C.

Techniques: Western Blot, Control

Journal: bioRxiv

Article Title: Spatial proteomics reveals prefrontal circuit diversity in socioemotional behaviour

doi: 10.64898/2026.02.08.704617

Figure Lengend Snippet: ProX ID maps projection specific presynaptic proteomes across mPFC centred pathways and links a BLTP2 Neurexin 1 axis to socioemotional behaviours Graphic summary of ProX ID, which enables physiological in vivo proximity labelling of presynaptic terminals with high temporal resolution, followed by streptavidin enrichment and quantitative mass spectrometry to derive pathway enriched presynaptic proteomes. Projection resolved signatures across six mPFC centred pathways (mPFC to BLA, NAc, Thal, HT and CTX, and BLA to mPFC) are integrated with public resources and cross species analyses to decode projection dependent molecular landscapes. The summary highlights BLTP2 enrichment in the mPFC to BLA pathway, its association with memory activated synapses, and a BLTP2 dependent presynaptic program involving Neurexin 1 that shapes synaptic organization and transmission, with consequences for fear memory, anxiety like behaviour and social interaction. Together, these findings reveal projection specific presynaptic molecular diversity and provide a mechanistic framework for circuit level vulnerabilities implicated in neuropsychiatric disorders.

Article Snippet: Biotinylated proteins were captured on a 10 μL slurry of NanoLink streptavidin magnetic beads (Vector) by incubation for 3 h at 4°C.

Techniques: In Vivo, Mass Spectrometry, Transmission Assay

Journal: bioRxiv

Article Title: Cell type-specific proximity labeling of organ secretomes reveals energy balance-dependent proteomic remodeling

doi: 10.64898/2026.01.11.698831

Figure Lengend Snippet: (A) Schematic of LoxP-flanked TurboID-KDEL construct and ER-localized TurboID proximity labeling system. (B) Experimental design for comparison of three tissues under basal conditions. (C) Western blot showing TurboID-catalyzed biotinylation of inguinal adipose tissue and plasma from biotin-treated Adipo-TurboID KDEL mice, as well as V5 tag indicative of TurboID expression. (D) Immunohistochemistry images of inguinal white adipose tissue (iWAT), liver, and spleen tissue in TurboID KDEL mice with and without expression of Adiponectin-Cre, Albumin-Cre or CD19-Cre. Biotinylation is shown in red (streptavidin for iWAT, neutravidin for liver and spleen). Scale bar 50 µm. (E) Workflow for tissue processing, affinity purification, and analysis by Tandem-Mass-Tag (TMT) mass spectrometry. (F) Pathway analysis illustrating predicted subcellular localization of proteins enriched in basal Adipo-TurboID KDEL Cre+ relative to Cre- samples. Dashed line indicates false discovery rate (FDR) of 5%. Gene Ontology term enrichment analysis was performed with GO Slim Cellular Component with the full mouse genome used as the background list. (G) Protein abundance plots highlighting cell-type-specific markers expressed in liver, iWAT, and spleen, respectively.

Article Snippet: To enrich biotinylated proteins, 100 μL of streptavidin (SA) magnetic beads (Pierce, 88816) per sample was washed twice with RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 7.5) with protease and phosphatase inhibitors.

Techniques: Construct, Labeling, Comparison, Western Blot, Clinical Proteomics, Expressing, Immunohistochemistry, Affinity Purification, Mass Spectrometry, Quantitative Proteomics

Journal: bioRxiv

Article Title: Cell type-specific proximity labeling of organ secretomes reveals energy balance-dependent proteomic remodeling

doi: 10.64898/2026.01.11.698831

Figure Lengend Snippet: (A) Experimental design to capture the secretory pathway proteome in inguinal (iWAT) and epididymal white adipocytes (eWAT) of obese Adipo-TurboID KDEL mice after 6 and 15 weeks of high-fat diet (HFD) feeding. (B) Body weights of mice across 15 weeks of HFD feeding. Biotin (1.5 mg/mL) was administered in drinking water for 7 days on the last week of feeding. Conditions were compared to basal by two-way ANOVA with Tukey’s multiple comparisons test (ns, p > 0.05, not shown; *, p < 0.05; **, p < 0.01). Error bars show mean ± SEM; n = 4-7 mice per condition. (C) Total biotin intake in mice after 7 days. Conditions were compared to basal ordinary one-way ANOVA with Šidák multiple comparisons test (ns, p > 0.05; *** p < 0.001). Error bars show mean ± SEM; n = 3-7 mice per condition. (D) Fasted glucose in mice fed chow or HFD for 15 weeks. Conditions were compared to basal ordinary one-way ANOVA with Šidák multiple comparisons test (ns, p > 0.05; **, p < 0.01; *** p < 0.001). Error bars show mean ± SEM; n = 3-7 mice per condition. (E) Glucose tolerance in mice fed chow or HFD for 15 weeks and area under curve (AUC). Conditions were compared to basal by two-way ANOVA with Tukey’s multiple comparisons test (ns, p > 0.05, not shown; ***, p < 0.001). Error bars show mean ± SEM; n = 3-7 mice per condition. AUC was compared using ordinary one-way ANOVA with Šidák multiple comparisons test (ns, p > 0.05; **** p < 0.0001). Error bars show mean ± SEM; n = 3-7 mice per condition. (F) Fat and lean mass of Adipo-TurboID KDEL mice after 15 weeks of HFD feeding. Conditions were compared to basal by two-way ANOVA with Tukey’s multiple comparisons test (ns, p > 0.05; ****, p < 0.0001). Error bars show mean ± SEM; n = 3-7 mice per condition. ( G-H ) Western blot validation of protein biotinylation using streptavidin-HRP, tissue-specific V5 expression, and levels of known adipokines including complement factor D (CFD), adiponectin (ADIPOQ), leucine-rich glycoprotein 1 (LRG1), fatty acid-binding protein 4 (FABP4), and retinol-binding protein 4 (RBP4) in bulk iWAT (G) and eWAT (H) from Adipo-TurboID KDEL mice after 15 weeks of HFD feeding. Vinculin (VINC) was used as a loading control. (I) Representative TMT channel assignment for a 16-plex LC-MS/MS/MS experiment comparing proteomic changes induced by 15-week HFD vs. chow in iWAT or eWAT adipocytes from Adipo-TurboID KDEL Cre+ and Cre- mice (technical replicates not shown). The number of proteins passing the ROC cutoff in each experiment is also shown. ( J-K ) Volcano plots showing log 2 fold changes in secretory pathway protein expression in iWAT (J) and eWAT (K) samples following HFD feeding for 15 weeks relative to basal. Dashed lines represent cutoffs of p-value < 0.05 and fold-change > 1.5. ( L ) Gene set enrichment analysis of biological processes differentially impacted by HFD and adipocyte source depot. Red color is enriched in upregulated genes and blue is enriched in downregulated genes (p-value < 0.05, FC > 1.5). ( M ) Gene expression of HFD-regulated proteins in iWAT and eWAT from Adipo-TurboID KDEL mice after 15 weeks of HFD feeding. Conditions were compared to chow by t-test (ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). Error bars show mean ± SEM; n = 4-7 mice per condition.

Article Snippet: To enrich biotinylated proteins, 100 μL of streptavidin (SA) magnetic beads (Pierce, 88816) per sample was washed twice with RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 7.5) with protease and phosphatase inhibitors.

Techniques: Western Blot, Biomarker Discovery, Expressing, Binding Assay, Control, Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy, Gene Expression

Journal: bioRxiv

Article Title: Cell type-specific proximity labeling of organ secretomes reveals energy balance-dependent proteomic remodeling

doi: 10.64898/2026.01.11.698831

Figure Lengend Snippet: (A) Body weights of Adipo-TurboID KDEL mice fed HFD or chow for 6 weeks. Biotin (1.5 mg/mL) was administrated in drinking water for 7 days on the last week of feeding. Conditions were compared to basal by two-way ANOVA with Tukey’s multiple comparisons test (ns, p > 0.05, not shown; *, p < 0.05; **, p < 0.01). Error bars show mean ± SEM; n = 4-6 mice per condition. ( B ) Total biotin intake in mice after 7 days. Conditions were compared to basal ordinary one-way ANOVA with Šidák multiple comparisons test (ns, p > 0.05). Error bars show mean ± SEM; n = 4-7 mice per condition. ( C ) Fasted glucose in mice fed chow or HFD for 6 weeks. Conditions were compared to basal ordinary one-way ANOVA with Šidák multiple comparisons test (ns, p > 0.05; *, p < 0.05; **, p < 0.01; ****, p < 0.0001). Error bars show mean ± SEM; n = 4-7 mice per condition. ( D ) Glucose tolerance in mice fed chow or HFD for 6 weeks and area under curve (AUC). Conditions were compared to basal by two-way ANOVA with Tukey’s multiple comparisons test (ns, p > 0.05, not shown; *, p < 0.05; **, p < 0.01; ***, p < 0.001). Error bars show mean ± SEM; n = 4-7 mice per condition. AUC was compared using ordinary one-way ANOVA with Šidák multiple comparisons test (ns, p > 0.05; ****, p < 0.0001). Error bars show mean ± SEM; n = 4-7 mice per condition. ( E-F ) Western blot validation of protein biotinylation using streptavidin-HRP, tissue-specific V5 expression, and levels of known adipokines including CFD, LRG1, FABP4, and RBP4 in bulk iWAT (E) and eWAT (F) from Adipo-TurboID KDEL mice after 6 weeks of HFD feeding. Vinculin (VINC) was used as a loading control. ( G ) Representative TMT channel assignment for a 16-plex LC-MS/MS/MS experiment comparing proteomic changes induced by 6-week HFD vs. chow in iWAT or eWAT adipocytes from Adipo-TurboID KDEL Cre+ and Cre- mice (technical replicates not shown). The number of proteins passing the ROC cutoff in each experiment is also shown. (H-I) Volcano plots showing log 2 fold changes in secretory pathway protein expression in iWAT (H) and eWAT (I) samples following HFD feeding for 6 weeks relative to basal. Dashed lines represent cutoffs of p-value < 0.05 and fold-change > 1.5. ( J ) Biological process over-represented in gene set enrichment affected by HFD feeding in iWAT-derived adipocytes at 6 weeks of HFD. ( K-L ) Heat maps showing differentially expressed proteins contributing to protein folding GO-term enrichment in iWAT (K) and eWAT (L) samples following HFD feeding for 6 weeks. Data is presented as log 2 (fold-change) compared to the chow condition.

Article Snippet: To enrich biotinylated proteins, 100 μL of streptavidin (SA) magnetic beads (Pierce, 88816) per sample was washed twice with RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 7.5) with protease and phosphatase inhibitors.

Techniques: Western Blot, Biomarker Discovery, Expressing, Control, Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy, Derivative Assay

Journal: bioRxiv

Article Title: Cell type-specific proximity labeling of organ secretomes reveals energy balance-dependent proteomic remodeling

doi: 10.64898/2026.01.11.698831

Figure Lengend Snippet: (A) Experimental design for the discovery of circulating adipokines in obese Adipo-TurboID KDEL mice after 6 and 15 weeks of HFD feeding; n = 5 mice per condition. ( B ) Representative TMT channel assignments for a 16-plex LC-MS/MS/MS experiment comparing adipo-plasma proteomes of Adipo-TurboID KDEL Cre+ and Cre- mice fed chow or HFD for 6 or 15 weeks (technical replicates not shown). The number of proteins passing the enrichment cutoff in each experiment is also shown. ( C ) Bar plot of proteins quantified in Adipo-TurboID KDEL plasma proteome at 6 and 15 weeks of DIO, with protein class labeled based on SignalP and OutCyte predictions. (D-E) Heatmaps showing Adipo-TurboID KDEL plasma proteome affected by HFD feeding in early (D) and late (E) stage of obesity. Data is presented as log 2 (fold-change) compared to chow condition. ( F ) Scatterplot of log 2 fold changes in adipo-plasma protein expression following HFD feeding for 6 and 15 weeks compared to chow. Proteins quantified for only one of the HFD timepoints are shown on margins. ( G ) Scatterplot of log 2 fold changes in protein expression in iWAT vs. plasma proteomes of Adipo-TurboID KDEL mice following HFD feeding for 15 weeks compared to chow. Proteins quantified for only one of the conditions are shown on margins. ( H ) Western blot validation of protein biotinylation using streptavidin-HRP and levels of known adipokines including CFD, ADIPOQ, LRG1, and RBP4 in bulk plasma samples from Adipo-TurboID KDEL mice after 15 weeks of HFD feeding. Albumin (ALB) was used as a loading control. ( I ) Rank plot of mean signal intensity values of proteins quantified by TurboID-TMT in Adipo-TurboID KDEL plasma proteome following HFD feeding for 15 weeks colored by known basal plasma concentrations.

Article Snippet: To enrich biotinylated proteins, 100 μL of streptavidin (SA) magnetic beads (Pierce, 88816) per sample was washed twice with RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 7.5) with protease and phosphatase inhibitors.

Techniques: Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy, Clinical Proteomics, Labeling, Expressing, Western Blot, Biomarker Discovery, Control

Journal: bioRxiv

Article Title: Cell type-specific proximity labeling of organ secretomes reveals energy balance-dependent proteomic remodeling

doi: 10.64898/2026.01.11.698831

Figure Lengend Snippet: (A) Bar plot of proteins quantified in Adipo-TurboID KDEL iWAT and plasma proteomes at 6 and 15 weeks of DIO, with protein class labeled based on SignalP and OutCyte predictions. (B) Scatterplot of log 2 fold changes in protein expression in eWAT vs. plasma proteomes of Adipo-TurboID KDEL mice following HFD feeding for 15 weeks compared to chow. Proteins quantified for only one of the conditions are shown on margins. (C) Western blot validation of protein biotinylation using streptavidin-HRP and levels of known adipokines including CFD, ADIPOQ, LRG1, and RBP4 in bulk plasma samples from Adipo-TurboID KDEL mice after 6 weeks of HFD feeding. Albumin (ALB) was used as a loading control. (D) Rank plot of mean signal intensity values of proteins quantified by TurboID-TMT in Adipo-TurboID KDEL plasma proteome following HFD feeding for 6 weeks colored by known basal plasma concentrations. ( E-F ) Scatterplots of log 2 fold changes in protein expression in plasma proteomes of Adipo-TurboID KDEL mice following 48-hour fasting and HFD feeding for 6 weeks (E) or 15 weeks (F) compared to basal or chow. Proteins quantified for only one of the conditions are shown on margins. ( G ) Dot plot of log 2 fold changes and p-values of selected proteins detected in adipocytes, hepatocytes, and plasma across negative and positive energy balance conditions. Data are shown relative to own control group (e.g. fasting vs. basal, LPS vs. basal, HFD vs. chow).

Article Snippet: To enrich biotinylated proteins, 100 μL of streptavidin (SA) magnetic beads (Pierce, 88816) per sample was washed twice with RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 7.5) with protease and phosphatase inhibitors.

Techniques: Clinical Proteomics, Labeling, Expressing, Western Blot, Biomarker Discovery, Control