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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.
摘要：
在多个样品中定量探测不同组的蛋白质及其翻译后修饰（PTM）的能力将有助于广泛的生物学，生化和药理研究。我们报道了一部小说,结合免疫亲和捕获与基质辅助激光解吸电离质谱（MALDIMS）的微阵列分析技术，能够支持高度多路复用，靶向蛋白质组学测定。称为“亲和微珠辅助质谱”(Affi-BAMS)，ThisLC-freetechnologyenablesdevelopmentofhighlyspecificandcustomizableassaypanelsforsimultaneouslyprofilingofmultipleproteinsandPTM.WhileailredbeadshavebeenusedpreviouslyincombinationwithMS,Affi-BAMS工作流程在包含一种抗体的单个珠子上使用富集，通常捕获单一分析物(蛋白质或PTM),同时具有足够的结合能力以使得能够在大约3个数量级内进行定量。通过组合具有不同蛋白质特异性的Affi-BAMS珠实现多路复用能力。为了能够通过MS筛选珠子捕获的分析物，我们进一步开发了一种新的方法，从排列在显微镜载玻片上的单个珠子中进行目标的空间局部洗脱。所得的微点阵列包含位于0.5mm直径点内的高度浓缩的分析物，可以使用MALDIMS直接测量。虽然完整的蛋白质和蛋白质片段都可以通过Affi-BAMS进行监测，我们最初专注于将这项技术应用于自下而上的蛋白质组学,通过将稳健的基于磁珠的工作流程与MALDIMS采集的高通量性质相结合,每天筛选数百个样品.为了展示Affi-BAMS的各种应用和鲁棒性，一些研究集中在4EBP1，RPS6，ERK1/ERK2，mTOR，组蛋白H3和C-MET刺激包括雷帕霉素，H2O2,EPO,SU11274，星形孢菌素和伏立诺他。
