Temperature-dependent Proteome Study of Thermoanaerobacter tengcongensis

Comprehensive and quantitative information of the thermophile proteome is an important source for understanding of the survival mechanism under high growth temperature. Thermoanaerobacter tengcongensis (T. tengcongensis), a typical anaerobic thermophilic eubacterium, was selected to quantitatively evaluate its protein abundance changes in response to four different temperatures. Thermoanaerobacter tengcongensis proteins were trypsine digested, separated with high-pH RP, and identified with MS/MS analysis. The raw MS/MS data were converted into MGF format by Proteome Discoverer 1.2 (Thermo Fisher Scientific, Waltham, MA, USA). And the exported MGF files were searched by Mascot 2.3.02 (Matrix Science, Boston, MA, USA) against the database with all 2588 predicted proteins in T. tengcongensis downloaded from NCBI (NCBI reference sequence: NC_003869.1). An automatic decoy database search was performed. Several parameters in Mascot were set for peptide searching, tolerance of one missed cleavage of trypsin, Carbamidomethyl (C),iTRAQ8plex (K) and iTRAQ8plex (N-term) as fixed modification, iTRAQ8plex (Y),Oxidation (M) as variable modification. The precursor mass tolerance was 10 ppm, and the product ion tolerance was 0.02 Da.

嗜热菌蛋白质组的全面定量数据，是解析其在高温生长条件下生存机制的重要研究基础。选取典型的厌氧嗜热真细菌腾冲热厌氧杆菌（*Thermoanaerobacter tengcongensis*，简称T. tengcongensis），对其在四种不同温度条件下的蛋白质丰度变化开展定量分析。将该菌的蛋白质经胰蛋白酶酶解后，采用高pH反相液相色谱（high-pH RP）进行分离，并通过串联质谱（MS/MS）完成鉴定。原始串联质谱数据通过美国马萨诸塞州沃尔瑟姆市赛默飞世尔科技（Thermo Fisher Scientific）的Proteome Discoverer 1.2软件转换为MGF格式。随后使用美国马萨诸塞州波士顿市Matrix Science公司的Mascot 2.3.02软件，对导出的MGF文件进行数据库检索，检索数据库包含从NCBI下载的腾冲热厌氧杆菌全部2588个预测蛋白质（NCBI参考序列：NC_003869.1）。实验设置了自动反向数据库检索流程。Mascot软件的肽段检索参数配置如下：允许胰蛋白酶酶解出现1个漏切位点；固定修饰为羧酰胺甲基化（C）、iTRAQ8plex标记（K）以及iTRAQ8plex标记（N端）；可变修饰为iTRAQ8plex标记（Y）以及氧化（M）。前体离子质量容忍度设为10 ppm，产物离子质量容忍度设为0.02 Da。