Discovery Sciences, BioPharmaceutical R&D, AstraZeneca, Boston, MA 02451, USA.
Molecular, Cellular and Biomedical Sciences, University of New Hampshire, Durham, NH 03824, USA.
Int J Mol Sci. 2020 Mar 16;21(6):2016. doi: 10.3390/ijms21062016.
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, HO, EPO, SU11274, Staurosporine and Vorinostat.
定量探测多种蛋白质及其翻译后修饰（PTMs）的能力将有助于广泛的生物学、生物化学和药理学研究。我们报告了一种新颖的、基于微阵列的分析技术，该技术将免疫亲和捕获与基质辅助激光解吸电离质谱（MALDI MS）相结合，能够支持高度多重、靶向蛋白质组学分析。该技术称为“亲和珠辅助质谱（Affi-BAMS）”，它是一种无 LC 的技术，能够为同时分析多种蛋白质和 PTM 而开发高度特异性和可定制的分析面板。虽然亲和珠以前曾与 MS 结合使用，但 Affi-BAMS 工作流程在单个珠子上进行富集，该珠子包含一种类型的抗体，通常捕获单个分析物（蛋白质或 PTM），同时具有足够的结合能力，能够在大约 3 个数量级内进行定量。通过将 Affi-BAMS 珠子与不同的蛋白质特异性相结合，实现了多重化能力。为了使 MS 能够筛选珠捕获的分析物，我们进一步开发了一种从排列在显微镜载玻片上的单个珠子上空间定位洗脱目标的新方法。由此产生的微斑点阵列包含高度浓缩的分析物，定位在直径为 0.5 毫米的斑点内，可直接使用 MALDI MS 进行测量。虽然 Affi-BAMS 可以监测完整蛋白质和蛋白质片段，但我们最初专注于将该技术应用于基于自上而下的蛋白质组学，通过将强大的基于磁珠的工作流程与 MALDI MS 采集的高通量性质相结合，每天能够筛选数百个样本。为了展示 Affi-BAMS 的各种应用和稳健性，我们展示了几项研究，重点关注 rapamycin、HO、EPO、SU11274、Staurosporine 和 Vorinostat 等刺激物对 4EBP1、RPS6、ERK1/ERK2、mTOR、Histone H3 和 C-MET 的响应。
