Proteomic analysis of cold adaptation in a Siberian permafrost bacterium--Exiguobacterium sibiricum 255-15 by two-dimensional liquid separation coupled with mass spectrometry

Bacterial cold adaptation in Exiguobacterium sibiricum 255-15 was studied on a proteomic scale using a 2-D liquid phase separation coupled with MS technology. Whole-cell lysates of E. sibiricum 255-15 grown at 4 degrees C and 25 degrees C were first fractionated according to pI by chromatofocusing (CF), and further separated based on hydrophobicity by nonporous silica RP HPLC (NPS-RP-HPLC) which was on-line coupled with an ESI-TOF MS for intact protein M(r) measurement and quantitative interlysate comparison. Mass maps were created to visualize the differences in protein expression between different growth temperatures. The differentially expressed proteins were then identified by PMF using a MALDI-TOF MS and peptide sequencing by MS/MS with a MALDI quadrupole IT TOF mass spectrometer (MALDI-QIT-TOF MS). A total of over 500 proteins were detected in this study, of which 256 were identified. Among these proteins 39 were cold acclimation proteins (Caps) that were preferentially or uniquely expressed at 4 degrees C and three were homologous cold shock proteins (Csps). The homologous Csps were found to be similarly expressed at 4 degrees C and 25 degrees C, where these three homologous Csps represent about 10% of the total soluble proteins at both 4 degrees C and 25 degrees C.     

MALDI TOF MS在细菌检测和鉴定中的应用

近年来质谱技术有了快速的发展,新的软电离技术,基质辅助激光解析电离和电喷雾电离使得质谱能够对核酸或蛋白质,多肽等生物大分子进行微量分析,且具有高灵敏度和高质量检测范围,通过串联质谱的分析还可以得到结构信息。MALDI TOF MS能给出微生物多方面的信息,包括分子量和结构信息等,用于微生物的各种研究中,如根据细菌的组成成分获得指纹图谱检测和鉴定细菌,细菌体内含有大量的生物标志分子能用于细菌的化学分类和鉴定。     

Intermittent administration of morphine alters protein expression in rat nucleus accumbens

Repeated exposure to drugs of abuse causes time-dependent neuroadaptive changes in the mesocorticolimbic system of the brain that are considered to underlie the expression of major behavioral characteristics of drug addiction. We used a 2-D gel-based proteomics approach to examine morphine-induced temporal changes in protein expression and/or PTM in the nucleus accumbens (NAc) of morphine-sensitized rats. Rats were pretreated with saline [1 mL/kg subcutaneously (s.c.)] or morphine (10 mg/kg, s.c.) once daily for 14 days and the animals were decapitated 1 day later. The NAc was extracted and proteins resolved by 2-DE. Several protein functional groups were found to be regulated in the morphine-treated group, representing cytoskeletal proteins, proteins involved in neurotransmission, enzymes involved in energy metabolism and protein degradation, and a protein that regulates translation.     

N-Terminal protein modifications in an insect cell-free protein synthesis system and their identification by mass spectrometry

To evaluate the ability of an insect cell-free protein synthesis system to generate proper N-terminal cotranslational protein modifications such as removal of the initiating Met, N-acetylation, and N-myristoylation, several mutants were constructed using truncated human gelsolin (tGelsolin) as a model protein. Tryptic digests of these mutants were analyzed by MALDI-TOF MS and MALDI-quadrupole-IT-TOF MS. The wild-type tGelsolin, which is an N-myristoylated protein, was found to be N-myristoylated when myristoyl-CoA was added to the in vitro translation reaction mixture. N-myristoylation did not occur on the Gly-2 to Ala mutant, in which the N-myristoylation motif was disrupted, whereas this mutant was found to be N-acetylated after removal of the initiating Met. Analyses of Gly-2 to His and Leu-3 to Asp mutants revealed that the amino acids at positions 2 and 3 strongly affect the susceptibility of the nascent peptide chain to removal of the initiating Met and to N-acetylation, respectively. These results suggest that N-terminal modifications occurring in the insect cell-free protein synthesis system are quite similar to those observed in the mammalian protein synthesis system. Thus, a combination of the cell-free protein synthesis system with MS is an effective strategy to analyze protein modifications.