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.

异常蛋白质合成与蛋白质表达是涵盖癌症至阿尔茨海默病等多种疾病状态的标志性特征。反映蛋白质组变化的血液生物标志物长期以来被认为在疾病预后与治疗方面具有潜在应用价值。然而，绝大多数生物标志物相关研究均聚焦于未标记的血浆蛋白质组学，这类研究包含非髓系来源蛋白质，但并未尝试动态标记蛋白质组的急性变化。本研究报道了一种可评估全血液体活检中从头蛋白质合成的方法。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，我们将啮齿类动物全血样本与L-叠氮高丙氨酸（L-azidohomoalanine, AHA）共同孵育，使该选择性反应性非天然氨基酸掺入新生多肽链中。值得注意的是，若在进行叠氮-炔烃"点击"反应前未使用乙二胺四乙酸（EDTA）孵育血液样本，则无法检测到探针掺入。该活细胞标记检测方法对茴香霉素抑制作用具有敏感性，且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。利用标记后的啮齿类动物血液样本，可通过标准液相色谱-串联质谱（LC/MS-MS）检测在多种实验条件下稳定识别已知血液蛋白质对应的标记多肽。此外，该检测方法可推广至人类血液样本的从头蛋白质合成检测。综上，本研究提出了一种快速便捷的从头蛋白质合成检测方法，可应用于全血活检，能够定量翻译水平变化并筛选出可能具有临床应用价值的差异表达蛋白质。