Proteomic Profiling of Salmonella enterica Strains from Environmental and Laboratory Sources: Comparative Analysis of Wastewater Isolates and Lab Strain UK-1

This dataset represents a comprehensive proteomic analysis of three Salmonella enterica strains, specifically two wastewater isolates—Salmonella enterica serovar Diarizonae (61:c:1,5,7) and S. enterica serovar Enteritidis (9:g,m:-)—alongside the well-characterized S. Typhimurium UK-1 lab strain. The preparation and analysis methods were carefully optimized to provide high-quality protein identifications and quantifications across these distinct strains, allowing for comparative proteomic analysis. To generate the dataset, stationary-phase cultures of each strain were processed meticulously. Cultures were centrifuged at high speed (13,000 x g) at 4ºC to obtain cell pellets, which were washed twice—initially with PBS containing 5 mM EDTA to eliminate metal ion contaminants and then with PBS alone to enhance sample purity. The cells were lysed using a Sonifier Cell Disruptor, a sonication-based technique that preserves protein integrity, and then centrifuged again to yield a protein-rich supernatant, which was subsequently filter-sterilized using a 0.22-µm PES filter. For protein separation, 25 µg of protein from each sample was loaded onto SDS-PAGE gels, with three biological replicates for each strain, ensuring reproducibility and robustness in measurements. Each gel lane was sectioned, and in-gel digestion with trypsin was performed to produce peptides suitable for mass spectrometry. The resulting peptide samples were analyzed using a 250-mm Ultrahigh-Performance Liquid Chromatography (UHPLC) system coupled to an Orbitrap Fusion mass spectrometer. This setup enabled high-resolution detection and precise mass spectrometry data collection. Mass spectrometry data were analyzed using Sequest and X! Tandem, which compared spectral data against a Salmonella Uniprot database, assuming trypsin digestion specificity. Scaffold software validated peptide and protein identifications using a stringent probability threshold (>95.0%) through the Scaffold Local FDR algorithm. Additionally, protein probabilities were refined with the Protein Prophet algorithm, requiring at least two peptides for protein identification. Proteins with overlapping peptide sequences were grouped to streamline interpretation, with the top-scoring hit reported. The dataset achieved a peptide-level false discovery rate (FDR) of 0.35%, well below the commonly accepted threshold of 1%, and a protein FDR of 3.3%, within the standard 1-5% range, ensuring high reliability in protein identifications. Quantification across strains was performed using normalized spectral counts, and statistical significance was evaluated using t-test. This dataset provides a resource for understanding the proteomic landscape across two environmentally derived Salmonella wastewater isolates and compared to a lab strain.本数据集针对3株肠炎沙门氏菌（Salmonella enterica）开展了全面蛋白质组学分析，具体涵盖两株污水分离株——肠炎沙门氏菌迪阿里佐纳血清型（Salmonella enterica serovar Diarizonae，61:c:1,5,7）与肠炎沙门氏菌肠炎血清型（S. enterica serovar Enteritidis，9:g,m:-），以及一株已被充分表征的肠炎沙门氏菌鼠伤寒亚种UK-1实验室菌株。研究人员对实验流程进行了精心优化，以实现上述不同菌株的高质量蛋白质鉴定与定量，为比较蛋白质组学分析提供支撑。 为构建该数据集，我们对每株菌株的稳定期培养物开展了严谨处理：将培养物于4℃下以13000×g高速离心获取细胞沉淀，随后进行两次洗涤——首次使用含5mM乙二胺四乙酸（Ethylenediaminetetraacetic acid, EDTA）的磷酸盐缓冲液（Phosphate Buffered Saline, PBS）以去除金属离子污染物，随后仅用PBS洗涤以提升样品纯度。采用超声细胞破碎仪（Sonifier Cell Disruptor）进行细胞裂解，该超声裂解技术可维持蛋白质结构完整性；裂解后再次离心得到富含蛋白质的上清液，随后使用0.22μm聚醚砜（Polyethersulfone, PES）滤器完成过滤除菌。 蛋白质分离环节中，取每份样品25μg蛋白质上样至十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, SDS-PAGE）凝胶，每株菌株设置3次生物学重复，以保障实验的可重复性与结果的稳健性。将每条凝胶泳道切割分块，采用胰蛋白酶进行胶内酶解，得到适用于质谱分析的肽段。 所得肽段样品通过250mm超高效液相色谱（Ultrahigh-Performance Liquid Chromatography, UHPLC）系统与Orbitrap Fusion质谱仪联用进行分析，该配置可实现高分辨率检测与精准的质谱数据采集。 质谱数据采用Sequest与X! Tandem软件进行分析，将谱图数据与肠炎沙门氏菌UniProt数据库进行比对，设定胰蛋白酶酶切特异性。通过Scaffold软件结合Scaffold Local FDR算法，以严格的概率阈值（>95.0%）验证肽段与蛋白质鉴定结果；同时借助Protein Prophet算法优化蛋白质概率，要求蛋白质鉴定至少需匹配2条肽段。对于存在重叠肽段序列的蛋白质，将其进行分组以简化结果解读，并报告得分最高的匹配结果。 本数据集的肽段水平错误发现率（false discovery rate, FDR）为0.35%，远低于普遍接受的1%阈值；蛋白质水平FDR为3.3%，处于1%~5%的标准范围内，确保了蛋白质鉴定结果的高可靠性。菌株间的定量分析采用归一化光谱计数法完成，统计学显著性通过t检验进行评估。 本数据集为解析两株环境来源的污水分离沙门氏菌的蛋白质组特征提供了宝贵资源，并可与实验室菌株开展对比分析。