Journal: Communications Medicine

Article Title: Preclinical characterization of an active immunotherapy targeting calcitonin gene-related peptide

doi: 10.1038/s43856-025-00870-2

Figure Lengend Snippet: Proteome and peptide screens were used to evaluate potential off-target binding of affinity-purified p4796kb antibodies. a Manhattan plot displays results from the HuProt Human Proteome Microarray screen of antibody binding. Colors represent each block from the array, containing approximately 1000 proteins each. Spots represent individual protein intensity score from array. Hits are annotated with protein names. b Manhattan plot from peptide counter screen of 21 putative hits from proteome screen. Overlapping 15mer peptides were synthesized and printed on arrays. Spots represent individual peptide intensity scores from array. Peptide sequences of hits are displayed. Sera from guinea pigs immunized with p4796kb were tested for binding to putative hits from screens ( c ) and calcitonin family ( d ) measured by ELISA. Pre-immune sera were used as controls. Data are presented as means +/− SEM; n = 3. *** p < 0.0001.

Article Snippet: Binding of p4796kb-derived sera to 3 hits from the HuProt™ microarray screen (HSP90 beta protein, StressMarq Bioscience, SPR-102B; AKR1B10 protein, MyBioSource, MBS203315; PTPRD protein, Acro, PTD-H52H9) and to other propeptides that belong to the calcitonin/CGRP peptide family, including recombinant adrenomedullin (ADM, MyBioSource, MBS2012013), recombinant adrenomedullin 2 (ADM2, MyBioSource, MBS2123890), synthetic amylin (Abcam, ab142398), and recombinant calcitonin (Abcam, ab153793), were further evaluated.

Techniques: Binding Assay, Affinity Purification, Microarray, Blocking Assay, Synthesized, Enzyme-linked Immunosorbent Assay

Journal: Cell

Article Title: Mapping Systemic Inflammation and Antibody Responses in Multisystem Inflammatory Syndrome in Children (MIS-C)

doi: 10.1016/j.cell.2020.09.034

Figure Lengend Snippet: Autoantibody Detection Unveils an Autoreactive Repertoire Enriched in MIS-C Patients (A) Upset plots delineating the number of shared autoantibodies between MIS-C patients, which were at least two-fold enriched when compared with controls for IgG autoantigens in HuProt protein microarray analysis. Upset plots were anchored on autoantibodies that were present in at least one IVIG-treatment-naïve sample (MIS-C 3 and MIS-C 9). Only intersections of 6 or more patients are visualized. (B) Corresponding upset plots for IgA autoantigens. (C) Heatmap of all IgG autoantigens with at least 4-fold enrichment in MIS-C compared to controls, in addition to the selection criteria above. Color intensity corresponds to the log 2 FC expression value relative to the mean of healthy pediatric controls (N=4). Flagged autoantigens were enriched in 5 patients and at least one treatment naive IVIG (MIS-C 3 or MIS-C 9) sample. (D) Corresponding heatmap for IgA autoantigens. (E) Top: validation of protein microarray hits identified by phage immunoprecipitation sequencing (PhIP-seq) for IgG autoantigens. The purple circle and corresponding number indicate the number of autoantigens enriched in the HuProt protein microarray that were also validated by PhIP-seq. Autoantigen peptides were collapsed at the gene level for overlap analyses. Bottom: corresponding overlap for IgA autoantigens. (F) Standard ELISA for CD244 auto-reactivity in MIS-C and healthy control plasma. (G) GSEA (gene set enrichment analysis) analysis of IgG autoantigens in treatment naïve MIS-C patients (N = 2; MIS-C 3 and MIS-C 9) versus age matched healthy controls (N = 4) ranked by t statistic. Dot color intensity corresponds to adjusted p value (FDR) and dot size represents the number of autoantigens found to be enriched in the associated gene set. (H) Corresponding enrichment scores for significantly (FDR<0.05) enriched biological pathways for IgG (regulation of immune response) and IgA (lymphocyte mediated immunity). Benjamini-Hochberg method was used to correct for multiple comparisons. See also Figure S5 .

Article Snippet: Raw HuProt protein microarray matrices , This paper; Mendeley data , https://doi.org/10.17632/9kcv4fdy3s.1.

Techniques: Immunopeptidomics, Microarray, Selection, Expressing, Biomarker Discovery, Immunoprecipitation, Sequencing, Enzyme-linked Immunosorbent Assay, Control, Clinical Proteomics

Journal: Cell

Article Title: Mapping Systemic Inflammation and Antibody Responses in Multisystem Inflammatory Syndrome in Children (MIS-C)

doi: 10.1016/j.cell.2020.09.034

Figure Lengend Snippet:

Article Snippet: Raw HuProt protein microarray matrices , This paper; Mendeley data , https://doi.org/10.17632/9kcv4fdy3s.1.

Techniques: Virus, Recombinant, Saline, Blocking Assay, Staining, Microarray, Software, Immunoprecipitation

Journal: bioRxiv

Article Title: PAK6 rescues pathogenic LRRK2-mediated ciliogenesis and centrosomal cohesion defects in a mutation-specific manner

doi: 10.1101/2024.04.11.589075

Figure Lengend Snippet: (a) Distribution of PAK6 candidate interactors according to their Z-score retrieved from a Human Proteome Microarray probed with recombinant full-length human PAK6. (b) A GO:BP analysis using gProfiler g:GOSt ( https://biit.cs.ut.ee/gprofiler/gost ) was performed for PAK6 candidate interactors with Z score >2.5 (left) and for PAK6 interactors annotated in PPI web-based tools PINOT, HIPPIE and MIST (PHM) (right). GO:BP terms with 2000 (array) and 1000 (PHM) term size were grouped into semantic categories. (c) Venn diagrams showing overlaps between the primary cilium proteome (GO:0005929, 640 genes) and the experimental (array) PAK6 interactome (left) or the literature-based (PHM) PAK6 interactome (right). (d) Protein network of overlapping PAK6 interactors with the primary cilium proteome (c) (including PAK6) obtained with STRING ( https://string-db.org/cgi/input?sessionId=b1S4T5BW27rz&input_page_show_search=on ); number of nodes: 11, number of edges: 11, average node degree: 2, average local clustering coefficient: 0.591, expected number of edges: 3, PPI enrichment P -value: 0.000502. Blue nodes are ciliary proteins present in the experimental PAK6 interactome (array) and grey nodes are those found in the literature-based PAK6 interactome.

Article Snippet: HuProt TM Human Proteome Microarray v4.0 was purchased from Cambridge Protein Arrays (Babraham Research Campus, Cambridge, UK) and employed to screen PAK6 interactor candidates following manufacturer’s instructions.

Techniques: Microarray, Recombinant

Journal: Nature Communications

Article Title: Capture of RNA-binding proteins across mouse tissues using HARD-AP

doi: 10.1038/s41467-024-52765-w

Figure Lengend Snippet: a Schematic of generating a pool of Cy5-labeled RNAs and hybridizing this pool with the human protein microarray. b Scatter plot showing the correlation of the fold change (Cy5 foreground signals over local background) of the HARD-AP identified RBPs ( n = 1447) in HEK293 cells. The fold change was calculated from the average of four independent protein spots on the two independent protein arrays (the same for c ). The Pearson correlation coefficient is given. c Distributions of the fold change and signal-noise-ratio (SNR) of indicated sets of proteins on the protein microarray (GO RBP n = 1365; HARD-AP RBP n = 1447; HARD-AP specific RBP n = 643). The buffer ( n = 320), BSA ( n = 80), GST ( n = 320), Ig A/G ( n = 720) at different concentrations were used as negative controls, and the Alexa 647 labeled IgG ( n = 80) was used as the positive control. d Pie chart showing the distribution of fold change (Cy5 signals over local background) of the HARD-AP identified RBPs ( n = 1447) and HARD-AP specific RBPs (n = 643) in HEK293 cells available on the protein microarray. e Top GO terms (molecular function and biological process) over-represent in HARD-AP RBPs with FC = 1-1.5 ( n = 745) or FC ≥ 1.5 ( n = 620) on two independent protein microarrays. The GO enrichment analysis used the two-sided Fisher’s exact test with the p -value adjusted using the Bonferroni correction for multiple testing. The number of proteins is labeled on each bubble. The color scale indicates the enrichment. f Images of Cy5-RNA incubation signal of selected proteins on the protein microarray. g Fold change of subunits of the Integrator, Exosome, Mediator, and 26S proteasome complex. Subunits with fold change over 1.5 were selected for plotting. Data are from four protein spots on two independent protein arrays and shown as the Mean ± SD. Source data for ( b – d , g ) are provided as a Source Data file.

Article Snippet: The HuProt Human proteome microarray contains over 21,000 GST-purified unique recombinant human proteins in yeast, including >81% of the canonically expressed proteins as defined by the Human Protein Atlas , .

Techniques: Labeling, Microarray, Positive Control, Incubation

Journal: Nature Communications

Article Title: Capture of RNA-binding proteins across mouse tissues using HARD-AP

doi: 10.1038/s41467-024-52765-w

Figure Lengend Snippet: a Volcano plot showing distributions of proteins captured by HARD beads compared to EGFP beads in indicated samples. The fold changes were calculated from means of the ion intensities of three independent biological samples. The significance (p) was determined using the two-tailed Student’s t-test and further adjusted using the Benjamini-Hochberg correction for multiple testing (p-adjust). b UpSet plot comparing RBPs identified in mESC and different mouse organs by HARD-AP. c Venn diagram comparing RBPs identified in different mouse samples by HARD-AP. d Matrix bubble plot showing enrichments of molecular function GO terms of in RBPs of indicated samples. The GO enrichment analysis used the two-sided Fisher’s exact test with the p-value adjusted using the Bonferroni correction for multiple testing. e Venn diagram comparing the mouse RBPome identified using HARD-AP (mouse RBPs_HARD) and all human RBPome (human RBPs_All) to their indicated orthologs. f Heatmap of the hierarchical clustering analysis using normalized ion intensities from indicated samples. g Top GO terms over-represent in tissue- and cell-enriched RBPs identified by HARD-AP. The GO enrichment analysis used the two-sided Fisher’s exact test with the p -value adjusted using the Bonferroni correction for multiple testing. h Relative levels of indicated proteins in different samples, which are calculated from the ion intensities of three independent biological samples. Data are means ± SD. HARD and EGFP represent proteins isolated by the HARD beads and EGFP beads respectively. i Western blot analysis showing the endogenous protein levels of Bcr, Prkar1a and Mylk3 in different mouse organs and mESC. This experiment was repeated once with similar results. j Western blot analysis for indicated proteins in indicated organ lysates after capture under indicated conditions. This experiment was repeated once with similar results. k Images of Cy5-RNA incubation signal of the human orthologs of Bcr, Prkar1a and Mylk3 on the protein microarray. Source data for ( a – c ) are provided as a Source Data file.

Article Snippet: The HuProt Human proteome microarray contains over 21,000 GST-purified unique recombinant human proteins in yeast, including >81% of the canonically expressed proteins as defined by the Human Protein Atlas , .

Techniques: Two Tailed Test, Isolation, Western Blot, Incubation, Microarray

Journal: Nature Communications

Article Title: Capture of RNA-binding proteins across mouse tissues using HARD-AP

doi: 10.1038/s41467-024-52765-w

Figure Lengend Snippet: a Left: Color matrix showing LIM domain protein on the protein microarray. Color scales represent the FC (Cy5 foreground signals over local background). Right: Image of Cy5-RNA incubation signal of the human CSRP1 on the protein microarray. b Relative levels of Csrp1 isolated from indicated mouse lysate using HARD beads and EGFP beads. Means ± SD are plotted; three independent biological samples were used for the analysis ( n = 3). The p -values were calculated using a two-tailed Student’s t -test. c Western blot analysis showing levels of Csrp1 isolated from the mouse brain and lung lysate using HARD beads and EGFP beads. This experiment was repeated once with similar results. d Heatmap of high-confidence CLIP-seq signals ± 2 kb around the center of peaks ( n = 10,028). Csrp1 IP: EBs expressing Csrp1-V5; Control: WT EBs. Two biologically independent replicates are shown. e Distribution of Csrp1 CLIP-seq signals ( n = 10,028) in different gene features. The average of the two biologically independent replicates is shown. f The top five GO terms associated with genes identified by CLIP-seq. The GO enrichment analysis used the two-sided Fisher’s exact test with the p -value adjusted using the Bonferroni correction for multiple testing. g Representative genome browser tracks showing normalized CLIP-seq and RNA-seq signals. h The top five enriched Csrp1-binding motifs on all target RNAs (target sequences = 6071, background sequences = 39,667). Motif enrichment significance was computed by HOMER (binomial test without adjustment). i Volcano plot showing distributions of genes differentially expressed in Csrp1 KO and WT embryoid bodies ( n = 22,625). The significance (p) was determined using the two-tailed Student’s t-test and further adjusted using the Benjamini-Hochberg correction for multiple testing (p-adjust). j Gene Set Enrichment Analysis of genes down-regulated in Csrp1 KO embryoid bodies compared to WT embryoid bodies. NES, normalized enrichment score. k The most enriched GO terms in significantly expressed genes in the Csrp1 KO embryoid bodies. The GO enrichment analysis used the two-sided Fisher’s exact test with the p -value adjusted using the Bonferroni correction for multiple testing. Source data for (b-c) are provided as a Source Data file.

Article Snippet: The HuProt Human proteome microarray contains over 21,000 GST-purified unique recombinant human proteins in yeast, including >81% of the canonically expressed proteins as defined by the Human Protein Atlas , .

Techniques: Microarray, Incubation, Isolation, Two Tailed Test, Western Blot, Expressing, Control, RNA Sequencing, Binding Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Human CPPED1 belongs to calcineurin‐like metallophosphoesterase superfamily and dephosphorylates PI3K‐AKT pathway component PAK4

doi: 10.1111/jcmm.16607

Figure Lengend Snippet: CPPED1‐interacting proteins obtained by human proteome microarray. Above‐threshold interactions are listed in order of highest to lowest binding affinity with CPPED1

Article Snippet: The HuProt TM v3.1‐Human Proteome Microarray (Cambridge Protein Arrays Ltd.) was used to identify protein interactions on an immobilized array.

Techniques: Microarray, Binding Assay, Derivative Assay, RNA Binding Assay, Ubiquitin Proteomics, Transduction

Journal: Journal of Cellular and Molecular Medicine

Article Title: Human CPPED1 belongs to calcineurin‐like metallophosphoesterase superfamily and dephosphorylates PI3K‐AKT pathway component PAK4

doi: 10.1111/jcmm.16607

Figure Lengend Snippet: KEGG pathway analysis of CPPED1 binding partners identified by human proteome microarray. Significant terms ( P <.05) are shown

Article Snippet: The HuProt TM v3.1‐Human Proteome Microarray (Cambridge Protein Arrays Ltd.) was used to identify protein interactions on an immobilized array.

Techniques: Binding Assay, Microarray

Journal: Frontiers in Medicine

Article Title: Follicular dendritic cell differentiation is associated with distinct synovial pathotype signatures in rheumatoid arthritis

doi: 10.3389/fmed.2022.1013660

Figure Lengend Snippet: The FDC mAb CNA.42 recognizes FBXO2. (A) Immunoprecipitation (IP) and characterization of the CNA-42 binding protein. (A-I) Western blotting of total cell lysate (a), negative control (agarose beads only) and the CNA.42-immunoprecipitated proteins (c and d, 1.5 and 6 uls/lane respectively) from tonsillar single cell suspension probed with CNA-42. A single band is detectable at 120 Kd. (A-II) the reactivity of the CNA.42 mAb on the HuProt™ human proteome microarray showing subarray 9-1 of array 1300017931 (used for the CNA.42) with fluorescence detection at 633 nm excitation (a) and 543 nm excitation (b). (a) Staining with biotinylated anti-GST and Streptavidin-647. Rows 1-28 show generic staining of the GST-tagged immobilized human proteins, among them FBXO2 in row 11. (b) Probing with CNA.42 and Cy3 labeled anti-mouse IgM shows one hit, the human protein FBXO2 in the subarray. (A-III) Western blotting of tonsillar lysates with FBXO2 and CNA-42-specific antibodies recognize 120 Kd bands in the lysates [CNA.42 BP = CNA.42 binding protein]. (B) In situ hybridization of FBXO2 mRNA (green) showing intracellular signal in tonsillar CD21 + FDC reticula (red). (C) Western blotting of lysates from the CAN.42 expressing CEM cell line using mAb CAN.42 and anti FBXO2. CEM were untreated or treated either with Accell human FBXO2 siRNA (1 uM), or non-targeting control (NTC). GAPDH is used as a loading control. Compared to untreated cells, densitometric analysis with Image J indicates that FBXO2 siRNA-treated cells expressed 50% (*) and 35% (**) less FBXO2 and CNA.42, respectively.

Article Snippet: CNA.42 specificity was assessed in Cambridge Protein Arrays Ltd., Babraham Research Campus, Cambridge, UK using human proteome arrays (HuProt Ver2).

Techniques: Immunoprecipitation, Binding Assay, Western Blot, Negative Control, Suspension, Microarray, Fluorescence, Staining, Labeling, In Situ Hybridization, Expressing, Control