基于高分辨质谱的鸟分枝杆菌蛋白质组深度覆盖图谱构建与解析

Construction and analysis of a deep-coverage proteomic map of Mycobacterium avium based on high-resolution mass spectrometry

摘要:

鸟分枝杆菌(Mycobacterium avium)是一种重要的人畜共患非结核分枝杆菌(nontuberculous mycobacteria, NTM)，其感染率持续攀升，已构成严峻的公共卫生挑战。然而，受限于参考基因组蛋白注释的不完善及标准菌株蛋白质组数据的缺失，该菌编码蛋白的表达特征尚不明晰。为了弥补基因组预测注释与实际蛋白表达之间的信息缺口，亟需在标准菌株层面获得系统、可靠的蛋白质实验证据，本研究对M. avium标准菌株DSM 44156^T的全细胞蛋白与分泌蛋白，应用实验室自主研发的高活性乙酰化胰蛋白酶消化，结合高pH反相分级分离技术，进行质谱深度解析。研究共鉴定到3 655个非冗余蛋白质、32 917条肽段及91 744张谱图，覆盖了NCBI参考蛋白库中4 409个编码基因的82.90%，鉴定深度提升。全细胞与分泌蛋白组具有互补效应，共鉴定了2 445个蛋白质，且分别特异性鉴定了1 114个和96个独有蛋白。本研究构建了全面的、高精度的M. avium标准化蛋白质组图谱，不仅补充了该菌蛋白质的表达数据，更为深入揭示其致病机制、筛选新型诊断标志物与治疗靶点及制定NTM相关人兽共患病防控策略提供了关键的数据支撑。

Abstract:

Mycobacterium avium is an important zoonotic nontuberculous mycobacteria (NTM), and its continuously rising infection rate has posed a severe public health challenge. However, due to the incomplete annotation of protein-coding sequences in the reference genome and the absence of proteomic data for the type strain, the expression profiles of the proteins encoded by this bacterium remain poorly characterized. To address the gap between genome prediction annotation and protein expression, it is crucial to acquire systematic and reliable experimental evidence of proteins at the type strain level. This study conducted an in-depth mass spectrometry analysis of whole-cell and the secreted proteins from the type strain M. avium DSM 44156^T. We employed a highly active acetylated trypsin developed by our laboratory for digestion, combined with high-pH reversed-phase fractionation. A total of 3 655 non-redundant proteins, 32 917 peptides, and 91 744 spectra were identified, covering 82.90% of the 4 409 coding genes in the NCBI reference protein database. The whole-cell and secretory proteomes exhibited pronounced complementarity, with a total of 2 445 proteins in common, while 1 114 and 96 unique proteins were specifically detected in the whole-cell and secreted fractions, respectively. This study has constructed a comprehensive and high-precision standardized proteomic map of M. avium, which not only supplements the protein expression data for this species but also provides key data support for further elucidating its pathogenic mechanism, screening novel diagnostic markers and therapeutic targets, and formulating prevention and control strategies for NTM-related zoonoses.