Multidimensional Normalization to Minimize Plate Effects of Suspension Bead Array Data

Enhanced by the growing number of biobanks, biomarker studies can now be performed with reasonable statistical power by using large sets of samples. Antibody-based proteomics by means of suspension bead arrays offers one attractive approach to analyze serum, plasma, or CSF samples for such studies in microtiter plates. To expand measurements beyond single batches, with either 96 or 384 samples per plate, suitable normalization methods are required to minimize the variation between plates. Here we propose two normalization approaches utilizing <i>MA</i> coordinates. The multidimensional <i>MA</i> (<i>multi-MA</i>) and <i>MA-loess</i> both consider all samples of a microtiter plate per suspension bead array assay and thus do not require any external reference samples. We demonstrate the performance of the two <i>MA</i> normalization methods with data obtained from the analysis of 384 samples including both serum and plasma. Samples were randomized across 96-well sample plates, processed, and analyzed in assay plates, respectively. Using principal component analysis (PCA), we could show that plate-wise clusters found in the first two components were eliminated by <i>multi-MA</i> normalization as compared with other normalization methods. Furthermore, we studied the correlation profiles between random pairs of antibodies and found that both <i>MA</i> normalization methods substantially reduced the inflated correlation introduced by plate effects. Normalization approaches using <i>multi-MA</i> and <i>MA-loess</i> minimized batch effects arising from the analysis of several assay plates with antibody suspension bead arrays. In a simulated biomarker study, <i>multi-MA</i> restored associations lost due to plate effects. Our normalization approaches, which are available as R package MDimNormn, could also be useful in studies using other types of high-throughput assay data

Additional file 2: of Affinity proteomic profiling of plasma for proteins associated to area-based mammographic breast density

Tables S3-S6, S8, S9 and Figures S1-S7 Additional material and methods description, results and detailed discussion. (PDF 2026 kb

Additional file 1: of Affinity proteomic profiling of plasma for proteins associated to area-based mammographic breast density

Tables S1-S2 Complete list of antibodies included in serum bead array (SBA) 1 and 2 (sheet 1 and 2). Table S7 Summary of antibody validation (sheet 3). (XLSX 71 kb

Additional file 1 of Mitochondrial heteroplasmic shifts reveal a positive selection of breast cancer

Additional file 1. Information of all mtDNA mutations detected in this study after the quanlity control steps

Affinity Proteomic Profiling of Plasma, Cerebrospinal Fluid, and Brain Tissue within Multiple Sclerosis

The brain is a vital organ and because it is well shielded from the outside environment, possibilities for noninvasive analysis are often limited. Instead, fluids taken from the spinal cord or circulatory system are preferred sources for the discovery of candidate markers within neurological diseases. In the context of multiple sclerosis (MS), we applied an affinity proteomic strategy and screened 22 plasma samples with 4595 antibodies (3450 genes) on bead arrays, then defined 375 antibodies (334 genes) for targeted analysis in a set of 172 samples and finally used 101 antibodies (43 genes) on 443 plasma as well as 573 cerebrospinal spinal fluid (CSF) samples. This revealed alteration of protein profiles in relation to MS subtypes for IRF8, IL7, METTL14, SLC30A7, and GAP43. Respective antibodies were subsequently used for immunofluorescence on human post-mortem brain tissue with MS pathology for expression and association analysis. There, antibodies for IRF8, IL7, and METTL14 stained neurons in proximity of lesions, which highlighted these candidate protein targets for further studies within MS and brain tissue. The affinity proteomic translation of profiles discovered by profiling human body fluids and tissue provides a powerful strategy to suggest additional candidates to studies of neurological disorders