Proteomic studies often employ multi-dimensional analytical methods such as nano-LC-ESI-MS/MS to simplify the sample complexity. The time and expertise required to implement LCMS methods can be a barrier to integrating targeted proteomics within translational research programs. We present a robust method that combines multiplexed immuno-affinity capture with MALDI MS, called Affi-BAMS. This platform enables development of highly specific assays for simultaneous profiling of multiple analytes. The workflow utilizes enrichment on single beads that contain one antibody, having enough binding capacity to quantify within 3 orders of magnitude. Multiplexing is achieved by combining assay beads with different specificities. Assay beads are spatially arrayed and captured peptides are eluted into individual micro-wells. The resulting array of micro-spots contain concentrated analytes for direct measurement by MALDI MS. While both intact proteins and protein fragments can be monitored by Affi-BAMS, we focused this work for bottom-up and middle-down proteomics. We illustrate Affi-BAMS assays to several protein targets associated with progression of Alzheimer's disease. Examples include a multiplexed assay for beta-amyloid to monitor fifteen C-terminal fragments, localized within the region of aa672 - aa711 (including AB1-38, AB1-40, AB1-42 & APP669-711). We have also configured assays to monitor twelve different regions of tau (MAPT), spanning from the N-terminus to the C-terminus, including assays for both total and known phosphorylation sites. Lastly, we demonstrate how Affi-BAMS can be used to monitor epigenetic marks on Histone H3 in normal and disease brain. The unique features of this technology include multiplexing capacity that exceeds the current limit of bead-based sandwich immunoassay platforms and the ability to independently screen multiple sites within a single protein. This platform should aid protein panel profiling across a wide range of research applications.
