RESEARCH ARTICLE Proteomic analysis on the temperature-dependent complexes in Thermoanaerobacter tengcongensis Bo Meng 1,2 , Zhong Qian 1,2 , Fan Wei 1,2 , Weiwei Wang 1 , Chuanqi Zhou 1,2 , Zhuowei Wang 1,2 , Quanhui Wang 3 , Wei Tong 1,2 , Qian Wang 1,2 , Yanhe Ma 3 , Ningzhi Xu 1,2 and Siqi Liu 1,2 1 Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, P. R. China 2 Beijing Proteomics Institute, Beijing, P. R. China 3 The Institute of Microbiology, Chinese Academy of Sciences, Beijing, P. R. China Received: August 6, 2008 Revised: February 24, 2009 Accepted: March 3, 2009 It is generally accepted that protein complexes play an active role in avoiding the protein degradation of the thermophiles. Thermoanaerobacter tengcongensis was cultured at three different temperatures (55, 75 and 801C) and the extracts of protein complexes were prepared. Through blue native PAGE, the changes of the relative band volumes in response to different temperatures were semi-quantitatively compared and six temperature- dependent bands were obtained. These bands were excised, digested with trypsin and then analyzed with MS for the identification of protein components. With the combination of the proteins identified by LC MS/MS and MALDI TOF/TOF MS, a total of 92 unique proteins were ascertained in these complexes. Besides, some protein components were examined with Western blot, which gave us insights into the survival mechanism of ther- mophiles. These included (i) the composition of complex at 801C was significantly different from that at the other two temperatures; (ii) HSPs presented in all temperature-dependent complexes; (iii) several proteins associated with the functional pathways existed in the same complexes, indicating that the complex structure provided facility for the functional efficiency. Keywords: Blue native PAGE / Chaperon / Protein complex / T. tengcongensis / Temperature 1 Introduction With the development of the techniques for investigation of protein interaction, people have realized that protein complexes play a critical role in biological processes. Moreover, some protein complexes are stress-responsive, whose compositions are highly sensitive to environmental changes. For instance, HSPs, a group of proteins with multiple functions, can avoid the aggregation of native proteins and refold the defective polypeptides, and they can be induced dramatically when cells are exposed to stress such as heat, UV, oxidizing or alkylating agents [1]. Exploring protein complexes responding to stress is believed to be significant for uncovering the molecular mechanisms of thermophilic adaptation. The proteins from thermophiles cultured at 45–751C or hyperthermophiles cultured at 801C or higher temperature usually show excellent thermal stability. In most cases, these thermostable proteins may not have special amino acid sequences because the proteins from thermophilic, hyper- thermophilic and mesophilic organisms share almost the same amino acid sequences and similar structures [2]. On the other hand, several physical factors, such as protein Abbreviations: BN, blue native; ENO, enolase; EF, elongation factor; FBP, fructose-1, 6-bisphosphatase; MBPP, maltose-bind- ing periplasmic protein; NUO, NADH: ubiquinone oxidoreduc- tase; PFOR, pyruvate:ferredoxin oxidoreductase; scFVs, single- chain antibodies; sHSP, small heat shock protein; TDPC, temperature-dependent proteins in a complex; TIPC, tempera- ture-independent proteins in a complex Correspondence: Dr. Siqi Liu, Center of Proteomic Analysis, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, P. R. China E-mail: siqiliu@genomics.org.cn Fax: 186-10-80485324 & 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com Proteomics 2009, 9, 3189–3200 3189 DOI 10.1002/pmic.200800650