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.

本数据集呈现了对三种沙门氏菌（Salmonella enterica）菌株的全面蛋白质组学分析，包括两种污水分离株——沙门氏菌（Salmonella enterica）血清型Diarizonae（61:c:1,5,7）和沙门氏菌（Salmonella enterica）血清型Enteritidis（9:g,m:-），以及已充分表征的沙门氏菌（Salmonella Typhimurium）UK-1实验室菌株。为了在各个不同菌株之间提供高质量的蛋白质鉴定和定量，本数据集的制备和分析方法均经过精心优化。该分析允许进行蛋白质组学的比较研究。

为了生成本数据集，对每个菌株的稳态培养进行了细致的处理。将培养物在4℃下以高速（13,000 x g）离心，以获得细胞沉淀物，随后进行两次洗涤——初次以含有5 mM EDTA的磷酸盐缓冲盐溶液（PBS）去除金属离子污染物，随后以纯化的PBS进行洗涤。使用超声波细胞破碎仪对细胞进行裂解，这是一种基于超声的技术，可保持蛋白质的完整性，随后再次离心以获得富含蛋白质的上清液，该上清液随后使用0.22-µm PES滤膜进行过滤灭菌。

为了进行蛋白质分离，每个样本加载了25 µg蛋白质到SDS-PAGE凝胶中，每个菌株有三个生物学重复，以确保测量的可重复性和稳健性。每个凝胶通道被分割，并在凝胶内进行胰蛋白酶消化，以产生适合质谱分析的肽段。所得肽段样本使用250-mm超高效液相色谱（UHPLC）系统，连接到Orbitrap Fusion质谱仪进行分析。这种配置实现了高分辨率检测和精确的质谱数据收集。

使用Sequest和X! Tandem对质谱数据进行分析，这些分析将光谱数据与Salmonella Uniprot数据库进行比较，假设胰蛋白酶消化的特异性。Scaffold软件通过Scaffold Local FDR算法使用严格的概率阈值（>95.0%）验证肽段和蛋白质的鉴定。此外，使用蛋白质先知算法进一步细化蛋白质概率，蛋白质鉴定至少需要两个肽段。具有重叠肽段序列的蛋白质被分组，以简化解释，并报告得分最高的匹配项。

本数据集达到了肽段级别的假发现率（FDR）为0.35%，远低于通常接受的阈值1%，蛋白质FDR为3.3%，在标准范围1-5%之内，确保蛋白质鉴定的高度可靠性。在菌株间进行定量分析时使用了归一化光谱计数，并使用t检验评估统计显著性。

本数据集为理解两个环境来源的沙门氏菌污水分离株和实验室菌株之间的蛋白质组学景观提供了一个宝贵资源。