Affinity-Bead Assisted Mass Spectrometry (Affi-BAMS): A Multiplexed Microarray Platform for Targeted Proteomics

Abstract

The ability to quantitatively probe diverse panels of proteins and their post-translational modifications (PTMs) across multiple samples would aid a broad spectrum of biological, biochemical and pharmacological studies. We report a novel, microarray analytical technology that combines immuno-affinity capture with Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI MS), which is capable of supporting highly multiplexed, targeted proteomic assays. Termed “Affinity-Bead Assisted Mass Spectrometry” (Affi-BAMS), this LC-free technology enables development of highly specific and customizable assay panels for simultaneous profiling of multiple proteins and PTMs. While affinity beads have been used previously in combination with MS, the Affi-BAMS workflow uses enrichment on a single bead that contains one type of antibody, generally capturing a single analyte (protein or PTM) while having enough binding capacity to enable quantification within approximately 3 orders of magnitude. The multiplexing capability is achieved by combining Affi-BAMS beads with different protein specificities. To enable screening of bead-captured analytes by MS, we further developed a novel method of performing spatially localized elution of targets from individual beads arrayed on a microscope slide. The resulting arrays of micro spots contain highly concentrated analytes localized within 0.5 mm diameter spots that can be directly measured using MALDI MS. While both intact proteins and protein fragments can be monitored by Affi-BAMS, we initially focused on applying this technology for bottom-up proteomics to enable screening of hundreds of samples per day by combining the robust magnetic bead-based workflow with the high throughput nature of MALDI MS acquisition. To demonstrate the variety of applications and robustness of Affi-BAMS, several studies are presented that focus on the response of 4EBP1, RPS6, ERK1/ERK2, mTOR, Histone H3 and C-MET to stimuli including rapamycin, H2O2, EPO, SU11274, Staurosporine and Vorinostat.

Integrated -omics approach reveals persistent DNA damage rewires lipid metabolism and histone hyperacetylation via MYS-1/Tip60

“…4A). To probe multiple histone acetylation marks, we used bead-assisted mass spectrometry (BAMS) approach ( 37 ). In this method, protease-digested histone peptides were isolated using antigen-bound immuno-affinity beads and analyzed through mass spectrometry.…”

Recent advances in microfluidics for single-cell functional proteomics

“…In targeted proteomics techniques, proteins are analyzed by using the affinity probes with sensing labels (e.g., conjugated fluorophores, isotope tags, or quantum dots), which emit or transduce signals. 16,[75][76][77][78] To achieve the high sensitivity and facilitate targeted proteomics identification at the single-cell level, microfluidics have been increasingly used, primarily because they offer advantages including higher effective concentration during labeling, controllable cell manipulation, substantially reduced reagent consumption, and design flexibility to increase multiplexity and throughput. 10,79,80 In the following, we review targeted-based SCP methods that are enabled by microfluidics systems, with particular focus on their developments in the last 5 years, as their earlier developments had been discussed in the previous reviews.…”

Food allergen analysis: A review of current gaps and the potential to fill them by matrix‐assisted laser desorption/ionization

“…Specificity issues have plagued the development of new allergen testing technologies, to overcome these issues, IMS assays for MALDI have been developed, both within laboratories and by at least one commercial entity (Hamza et al., 2020; Zhao et al., 2021). Affinity bead‐assisted mass spectrometry (Affi‐BAMS) is the name given to the technique by a recent start‐up in the United States (Adeptrix®), whilst Zhao et al.…”

“…Affinity bead‐assisted mass spectrometry (Affi‐BAMS) is the name given to the technique by a recent start‐up in the United States (Adeptrix®), whilst Zhao et al. from Shanghai Ocean University in China refer more generally to the technique as using “Magnetic Beads.” IMS was previously described in several papers around 20 years ago, primarily for the detection of pathogens in samples as diverse as ground beef and body fluids; it then appears sporadically over the intervening two decades but showed no evidence of having been developed commercially until more recently (Hasan et al., 2016; Madonna et al., 2001, 2003; Schlosser et al., 2007) and there are no reports of IMS for MALDI allergen detection until very recently (Hamza et al., 2020).…”

“…Groups working on magnetic beads and IMS for allergen detection show promising results, on a par or better than existing ELISAs (Hamza et al., 2020; Zhao et al., 2021). This is not entirely unsurprising as previous work using MALDI with two‐dimensional electrophoresis and immunoblotting for allergen detection produced useful results (Sancho & Mills, 2010).…”