Optimization of Protocols for Detection of De Novo Protein Synthesis in Whole Blood Samples via Azide–Alkyne Cycloaddition

Aberrant
protein synthesis and protein expression are a hallmark
of many conditions ranging from cancer to Alzheimer’s. Blood-based
biomarkers indicative of changes in proteomes have long been held
to be potentially useful with respect to disease prognosis and treatment.
However, most biomarker efforts have focused on unlabeled plasma proteomics
that include nonmyeloid origin proteins with no attempt to dynamically
tag acute changes in proteomes. Herein we report a method for evaluating
de novo protein synthesis in whole blood liquid biopsies. Using a
modification of the “bioorthogonal noncanonical amino acid
tagging” (BONCAT) protocol, rodent whole blood samples were
incubated with l-azidohomoalanine (AHA) to allow incorporation
of this selectively reactive non-natural amino acid within nascent
polypeptides. Notably, failure to incubate the blood samples with
EDTA prior to implementation of azide–alkyne “click”
reactions resulted in the inability to detect probe incorporation.
This live-labeling assay was sensitive to inhibition with anisomycin
and nascent, tagged polypeptides were localized to a variety of blood
cells using FUNCAT. Using labeled rodent blood, these tagged peptides
could be consistently identified through standard LC/MS-MS detection
of known blood proteins across a variety of experimental conditions.
Furthermore, this assay could be expanded to measure de novo protein
synthesis in human blood samples. Overall, we present a rapid and
convenient de novo protein synthesis assay that can be used with whole
blood biopsies that can quantify translational change as well as identify
differentially expressed proteins that may be useful for clinical
applications.

蛋白质合成与表达异常是多种疾病的标志性特征，涵盖癌症至阿尔茨海默病等诸多病症。长期以来，能够反映蛋白质组（proteome）变化的血液生物标志物，被认为在疾病预后与治疗领域具有潜在应用价值。然而，绝大多数生物标志物相关研究均聚焦于含非髓系来源蛋白的未标记血浆蛋白质组学，并未尝试对蛋白质组的急性变化进行动态标记。本研究报道了一种可用于全血液体活检中蛋白质从头合成评估的方法。通过对"生物正交非经典氨基酸标记（bioorthogonal noncanonical amino acid tagging, BONCAT）"方案进行改良，研究人员将啮齿类动物全血样本与L-叠氮高丙氨酸（L-azidohomoalanine, AHA）共孵育，使该具有选择性反应性的非天然氨基酸能够掺入新生多肽链中。值得注意的是，若在进行叠氮-炔基"点击"反应前未使用乙二胺四乙酸（EDTA）对血液样本进行孵育处理，则无法检测到标记探针的掺入。该活细胞标记检测法对茴香霉素（anisomycin）抑制作用具有敏感性；通过荧光非经典氨基酸标记（FUNCAT）技术，可将带有标记的新生多肽定位于多种血细胞中。使用标记后的啮齿类动物血液样本，研究人员可在多种实验条件下，通过标准液相色谱-串联质谱（LC/MS-MS）检测已知血液蛋白的方式，稳定鉴定出这些带有标记的多肽。此外，该检测方法还可拓展应用于人类血液样本的蛋白质从头合成水平检测。综上，本研究开发了一种快速便捷的蛋白质从头合成检测方法，可应用于全血活检样本，能够定量分析翻译变化，并鉴定出可用于临床应用的差异表达蛋白。