Stable Isotope Labeling By Amino Acids In Cell Culture (Silac) Applied To Quantitative Proteomics Of Bacteria Under Cold Stress

The aim of this study was to conduct a comparative proteome quantitation of Vibrio parahaemolyticus and Edwardsiella tarda in response to 2-week prolonged cold stress in terms of demonstrating specifically characteristic changes at proteomic level,and to analyze the possibly potential risks based on cold-stressed strains.Comparative proteome quantitation analysis based on stable isotope labeling by amino acid in cell culture?SILAC?combined with high resolution mass spectrometry,which minimizes errors and contributes to the higher accuracy compared with chemical or label-free approaches.Quantitative analysis of mRNA-level and time course expression analysis were also conducted as auxiliary validations.Moreover,the research concerning reparation of cold-stress-damaged cell was conducted as well as exploring stable isotope labeling media and the optimal formula.A total of 1182 proteins were identified in Vibrio parahaemolyticus,among which601 were quantified?51 upregulated and 129 downregulated?.Catalase/peroxidase HPI,aromatic amino acid aminotransferase and 16S rRNA methyltransferase were significantly upregulated in spite of the inconsistency of some proteins expression and its corresponding mRNA expression.Glutamine synthetase,asparagine synthetase,polynucleotide phosphorylase and enzymes related to pentose phosphate pathway increased as well.Additionally,glucose phosphotransferase system?PTS?became active after cold stress.Some proteins associated with DNA synthesis,glycolysis,tricarboxylic acid cycle,transcription and translation were downregulated.A total of 1391 proteins were identified in Edwardsiella tarda,among which 898were quantified?72 upregulated and 164 downregulated?.Even if E.tarda is not psychrophile,some key proteins related to DNA synthesis?DNA Gyrase,ATP Synthase,DNA Topoisomerase and DNA Mismatch Repair Protein,MutS?,transcription?RNA Polymerase and Transcription-Repair-Coupling Factor?,Glycolysis?Fructose 1,6-Bisphosphatase and Glyceraldehyde-3-Phosphate Dehydrogenase?,glyoxylate cycle?Glyoxylate/Hydroxypyruvate Reductase A,G/HRA?,virulence?Hydrogenase,Lactate Permease and Out Membrane Protein A?and stress resistance?DNA starvation/stationary phase protection protein and universal stress proteins?were significantly upregulated.Furthermore,proteins related to hemolytic activities and gluconeogenesis were upregulated even if E.tarda ATCC 15497 is considered to be non-virulent or conditional pathogenic to aquaculture.Additionally,the amino acid composition and the expression of membrane proteins was also varied.The MS results demonstrated that most upregulated proteins in V.parahaemolyticus and E.tarda were related to enhance the stress resistance and self-adaptation after prolonged cold stress.We assumed that there might be correlation between upregulation and potential risks on seafood or human health.Most downregulated proteins contributed to energy conservation and self-protection in order to resist variously negative stresses.This study provides information on specific differences of proteome and offers some new clues to the differentiation and preliminary analysis of V.parahaemolyticus and E.tarda under prolonged cold stress.The addition of pyruvate in TCBS?selective medium?significantly enhanced the reparation of V.parahaemolyticus.When the concentration of pyruvate was 0.5%,the effect of reparation reached the maximum with efficiency of 52%;the addition of catalase in TSA?non-selective medium?significantly enhanced the reparation of E.tarda.When the concentration of catalase was 50 U/mL,the effect of reparation reached the maximum with efficiency of 26%.In the development of stable isotope labeling medium,?NH₄?₂SO₄ and NH₄Cl were selected as unique nitrogen sources,glucose and sodium acetate as carbon sources,and other components including KH₂PO₄,K₂HPO₄,MgSO₄ and NaCl were constant.Then orthogonal test including five-factor combined with four-level was designed and conducted.According to the visual analysis,variance analysis and effective curve of orthogonal test,the optimal formula of V.parahaemolyticus was as follows:3 g/L?NH₄?₂SO₄,2 g/L NH₄Cl,10 g/L glucose,the ¹⁵N labeled medium and non-labeled medium were as follows:2 g/L NH₄Cl,10 g/L glucose,0.6 g/L KH₂PO₄,0.9 g/L K₂HPO₄,0.2 g/L MgSO₄ and 20 g/L NaCl.The optimal formula of E.tarda was as follows:3 g/L?NH₄?₂SO₄,3 g/L NH₄Cl and 10 g/L glucose,the ¹⁵N labeled medium and non-labeled medium were as follows:3 g/L?NH₄?₂SO₄ or 3 g/L NH₄Cl,10 g/L glucose,0.6 g/L KH₂PO₄,0.9 g/L K₂HPO₄,0.2 g/L MgSO₄ and 20 g/L NaCl.