To monitor the secreted signaling proteome in plasma, we manufactured glass-based microarrays with commercially available antibodies to measure the relative levels of close to 600 distinct secreted signaling proteins. Using these arrays, we obtained quantifiable results for 582 signaling proteins (Additional file 1: Figure S1A to D and Additional file 2) in archived blood plasma from 47 sporadic, cognitively impaired AD patients and 52 non-demented, closely age- and sex-matched controls obtained from two clinical centers (Additional file 1: Table S1). While these proteins do not encompass all secreted signaling proteins, they do provide a strong representation of all major signaling pathways and represent the largest dataset of this kind available today (Additional file 1: Figure S1A). Raw data were processed, normalized (Additional file 1: Figure S2), and then subjected to three parallel analyses, aimed at integrating both molecular and clinical data, followed by external and internal validation steps (Fig. 1a).
Fig. 1 The circulatory AD signaling proteome reveals changes in cellular communication. a Overview of the experimental and analysis workflow. Plasma samples were collected at clinical centers, relative protein abundance was determined by antibody microarray and three types of analyses were performed: Protein level, MMSE correlation (cognitive performance), and protein co-secretion analysis. The analyses results were then integrated in a network and pathway enrichment framework and finally subjected to internal and external validation. b Heat map representation of the protein level analysis showing the top 50 most different proteins after unsupervised clustering (q < 0.05), separating samples into AD (pink, right) and controls (blue, left) and proteins into higher in control (blue, top) and higher in AD (pink, bottom). c Volcano-plot showing the distribution of all proteins and naming those significantly different between AD and control subjects (p corr < 0.01). d A network representation of the most significantly changed proteins (p corr < 0.015; un-connected proteins omitted) after integration with known pathway and physical interaction data reveals many densely connected hits in pathways related to TGFβ/GDF/BMP, angiogenesis, and apoptosis signaling. e Example scatter plots of the six top changed proteins (see dashed box in e, mean ± s.e.m; all p-values are corrected for multiple hypothesis testing)