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Cry1Ab is one of the most common Bacillus thuringiensis (Bt) proteins in genetically modified crops, which exhibits strong resistance against insect pests. In the present study, a sensitive and precise liquid chromatography stable isotope dilution multiple reaction monitoring tandem mass spectrometry (LC-SID-MRM-MS) assay was developed and validated to quantify the amount of Cry1Ab expression in transgenic maize leaves. The measurement of protein was converted to measurement of unique peptides to Cry1Ab protein. Two peptides unique to Cry1Ab were synthesized and labeled in H(2)(18)O to generate (18)O stable isotope peptides as internal standards. The validated method obtained superior specificity and good linearity. And the inter- and intra-day precision and accuracy for all samples were satisfactory. The results demonstrated Cry1Ab protein was 31.7 ± 4.1 μg g(-1) dry weight in Bt-176 transgenic maize leaves. It proved that the novel LC-SID-MRM-MS method was sensitive and selective to quantify Cry1Ab in the crude extract without time-consuming pre-separation or purification procedures.

Microgravity is one of the most important features in spaceflight. Previous evidence has shown that neurophysiological impairment signs occurred under microgravity. The present study was undertaken to explore the change in protein abundance in human SH-SY5Y neuroblastoma cells that were grown in a microgravity environment. The comparative proteomic method based on the (18)O labeling technique was applied to investigate the up-regulated proteins and down-regulated proteins in SH-SY5Y under simulated microgravity. Twenty-two differentially abundant proteins were quantified in human SH-SY5Y neuroblastoma cells. The cell microfilament network was disrupted under simulated microgravity, which was determined by the immunocytochemistry. The concentration of reactive oxygen species, malondialdehyde, and free Ca2+ ion significantly increased, and the level of ATP significantly decreased under simulated microgravity. However, there was no obvious cell apoptosis observed under simulated microgravity. These results provide new molecular evidence for the change in protein abundance in SH-SY5Y cells under simulated microgravity, which might unfold biological mechanisms and the development of effective countermeasures to deal with microgravity-related neurological problems. We believe that the state-of-the-art proteomic assay may be a means by which aerospace scientists will begin to understand the underlying mechanisms of space life activities at the protein level.

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Simulated microgravity has been reported to affect the gene, protein expression, and its function in the cells. Semicarbazide-sensitive amine oxidase (SSAO; E.C.1.4.3.6.) is widely distributed in vascular cells, smooth muscle cells, and adipocytes. It is noteworthy whether the expression of SSAO is affected under simulated microgravity or not. In this study, an SSAO-transformed Escherichia coli BL21 was constructed firstly. Then, a sensitive, selective, and accurate method based on high-performance liquid chromatography electrospray ionization triple quadrupole (HPLC-ESI-QQQ) was developed to determine the amount of SSAO in the E. coli BL21. The limit of detection and limit of quantification were 5.0 and 10 fmol, respectively. Finally, SSAO expression in the recombinant E. coli BL21 was evaluated with various gravity and temperature conditions by HPLC-ESI-QQQ analysis. It is interesting that the tendency in the alteration of SSAO under simulated microgravity showed temperature difference. At 18 °C, the amount of SSAO in the inclusion bodies and soluble fractions under the simulated microgravity increased by 83% and 116%, respectively, compared with normal gravity. However, the decrease by 38% and 49% in the inclusion bodies and soluble fractions under the simulated microgravity was observed at 37 °C. Results obtained here indicate that the SSAO expression under simulated microgravity is dramatically sensitive to the temperature. On the other hand, a novel bioreactor from this study may also be useful for the recombinant protein expression in the field of gene engineering.