Proteomic analysis of turkey (Meleagris gallopavo) fresh and cryopreserved semen: raw data

The purpose of the study was to investigate the proteome alterations induced by equilibration and freezing/thawing process, both in spermatozoa and extracellular fluid (ECF). The differentially expressed proteins (DEPs) were analysed by 2-dimensional difference in-gel electrophoresis (2D-DIGE) and identified with matrix-assisted laser desorption/ionization mass spectrometry (MALDI TOF/TOF). The validation of the selected DEPs was performed by immunofluorescence and western blotting.This dataset contains 6 directories:1. The directory ‘DIGE CyDye labeled gels’ contains pictures of fresh, equilibrated and frozen/thawed spermatozoa described as SPERM and extracellular fluid described as ECF. The directory contains 54 pictures in .tif format which are described according to samples arrangement presented in Supplementary Tables 1 and 2.2.The directory ‘Immunofluorescence spermatozoa labeling validation’ contains six representative microphotographs in .tif format showing validation of semen smear fixation and antibody concentration. Specificity of the secondary antibody (omitting the primary antibody) is presented at Photo 1A- DAPI and Photo 1B- Cy3®-conjugated secondary antibody. Validation of semen smear fixation is presented at 2A. methanol_acetone fixation and 2B. 4% paraformaldehyde fixation. Secondary antibody dilution is presented at 3A. Cy3®-conjugated secondary antibody, dilution 1:200 and 3B. Cy3®-conjugated secondary antibody, dilution 1:300.3. The directory ‘Supplementary Figures’ contains 4 files in .tif format.- Supplementary Fig. 1A and 1B. 2D-DIGE gels showing the proteomic profiles of fresh (A) and frozen/thawed (B) turkey spermatozoa. Numbered protein spots in red correspond to the proteins that are more abundant, whereas numbered protein spots in green indicate proteins that are less abundant in frozen/thawed spermatozoa. Results of spot identification are presented in Supplementary Table 3.(.tif)- Supplementary Fig. 2A and 2B. 2D-DIGE gels showing the proteomic profiles of fresh (A) and frozen/thawed (B) turkey semen extracellular fluid. Numbered protein spots in red correspond to the proteins that are more abundant in frozen/thawed ECF. Results of spot identification are presented in Supplementary Table 4.(.tif)4. The directory ‘Supplemetary Tables’ contains 6 files, 2 in format .xlsx; 2 in format .ods and 2 in .csv format.- Supplementary Table 1. Fresh, equilibrated and frozen/thawed semen arrangement for a 2D-DIGE experiment of turkey spermatozoa in six replicates. The internal standard was generated when equal amount of protein from each of 12 samples was combined. (.xlsx)- Supplementary Table 2. Fresh, equilibrated and frozen/thawed semen arrangement for a 2D-DIGE experiment of turkey extracellular fluid in six replicates. The internal standard was generated when equal amount of protein from each of 12 samples was combined. (.xlsx)- Supplementary Table 3. List of differentially expressed proteins in fresh, equilibrated and frozen/thawed turkey spermatozoa identified by MALDI TOF/TOF analysis. (.ods)- Supplementary Table 4. List of differentially expressed proteins in fresh, equilibrated and frozen/thawed turkey extracellular fluid identified by MALDI TOF/TOF analysis. (.ods)- Supplementary Table 5. Gene ontology analysis of DEPs identified in spermatozoa between fresh and frozen/thawed semen. Gene ontology was performed using G:Profiler with the highlight driver terms GO at an adjusted p value < 0.05. (.csv)- Supplementary Table 6. Gene ontology analysis of DEPs identified in extracellular fluid between fresh and frozen/thawed semen. Gene ontology was performed using G:Profiler with the highlight driver terms GO at an adjusted p value < 0.05. (.csv)5. The directory ‘Western Blot pictures’ contains pictures of blots with target protein and stain free blots in .tif format (40 pictures). Pictures show blots for spermatozoa protein validation described as SPERM and for extracellular fluid proteins validation described as ECF. The following proteins were validated for spermatozoa: acrosin (ACR), aconitate hydratase (ACO2), alpha-enolase (ENO1), glycerol-3-phosphate dehydrogenase (GPD2), triosephosphate isomerase (TPI1); and for ECF: ACR (acrosin), alpha-enolase (ENO1), phosphoglycerate kinase (PGK1), pyruvate kinase PKM (PKM), and triosephosphate isomerase (TPI1).6. The directory 'Western Blot quantitative data' contains 14 files of raw data presenting relative intensity (arbitrary units) used for statistical analysis in .xlsx format for spermatozoa validation (SPERM ) and for extracellular fluid validation (ECF).The following proteins were validated for spermatozoa: acrosin (ACR), aconitate hydratase (ACO2), alpha-enolase (ENO1), glycerol-3-phosphate dehydrogenase (GPD2), triosephosphate isomerase (TPI1); and for ECF: ACR (acrosin), alpha-enolase (ENO1), phosphoglycerate kinase (PGK1), pyruvate kinase PKM (PKM), and triosephosphate isomerase (TPI1).

本研究旨在探究平衡与冻融过程在精子及细胞外液（extracellular fluid, ECF）中诱导的蛋白质组变化。采用双向荧光差异凝胶电泳（two-dimensional difference in-gel electrophoresis, 2D-DIGE）分析差异表达蛋白（differentially expressed proteins, DEPs），并通过基质辅助激光解吸电离飞行时间质谱（matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI TOF/TOF）对其进行鉴定。所选差异表达蛋白的验证采用免疫荧光法与蛋白质印迹法（western blotting）。
本数据集包含6个目录：
1. 目录“DIGE CyDye标记凝胶”：存储以.tif格式保存的54张图片，分别对应新鲜、经平衡处理及冻融处理的精子（标注为SPERM）与细胞外液（标注为ECF）样本，图片命名规则遵循补充表1与补充表2中呈现的样本排布方案。
2. 目录“精子免疫荧光标记验证”：包含6张.tif格式的代表性显微照片，用于验证精液涂片固定方法与抗体浓度。其中，照片1A（DAPI染色）与1B（Cy3®偶联二抗）展示了二抗的特异性对照（省略一抗的实验组）；照片2A（甲醇-丙酮固定法）与2B（4%多聚甲醛固定法）展示了精液涂片固定方法的验证结果；照片3A（Cy3®偶联二抗，稀释比例1:200）与3B（Cy3®偶联二抗，稀释比例1:300）展示了二抗稀释浓度的验证结果。
3. 目录“补充图”：包含4张.tif格式文件：
- 补充图1A与1B：展示新鲜（A）与冻融（B）火鸡精子蛋白质组图谱的2D-DIGE凝胶。红色编号蛋白点代表冻融精子中丰度更高的蛋白，绿色编号蛋白点代表冻融精子中丰度更低的蛋白，斑点鉴定结果详见补充表3。
- 补充图2A与2B：展示新鲜（A）与冻融（B）火精液细胞外液蛋白质组图谱的2D-DIGE凝胶。红色编号蛋白点代表冻融ECF中丰度更高的蛋白，斑点鉴定结果详见补充表4。
4. 目录“补充表”：包含6个格式各异的文件，其中2个为.xlsx格式、2个为.ods格式、2个为.csv格式：
- 补充表1：火鸡精子2D-DIGE实验中新鲜、经平衡处理及冻融处理精液的样本排布方案，设置6次生物学重复。内标样本由12份样本的等量蛋白混合制备，文件格式为.xlsx。
- 补充表2：火鸡细胞外液2D-DIGE实验中新鲜、经平衡处理及冻融处理精液的样本排布方案，设置6次生物学重复。内标样本由12份样本的等量蛋白混合制备，文件格式为.xlsx。
- 补充表3：经MALDI TOF/TOF分析鉴定的新鲜、经平衡处理及冻融处理火鸡精子的差异表达蛋白列表，文件格式为.ods。
- 补充表4：经MALDI TOF/TOF分析鉴定的新鲜、经平衡处理及冻融处理火鸡细胞外液的差异表达蛋白列表，文件格式为.ods。
- 补充表5：新鲜与冻融精液精子中差异表达蛋白的基因本体（Gene Ontology, GO）富集分析结果。分析采用G:Profiler工具，以校正后p值<0.05为阈值筛选核心驱动GO术语，文件格式为.csv。
- 补充表6：新鲜与冻融精液细胞外液中差异表达蛋白的基因本体富集分析结果。分析采用G:Profiler工具，以校正后p值<0.05为阈值筛选核心驱动GO术语，文件格式为.csv。
5. 目录“蛋白质印迹图片”：包含40张.tif格式的图片，涵盖靶蛋白免疫印迹图与总蛋白染色印迹图，用于精子（标注为SPERM）与细胞外液（标注为ECF）的蛋白验证实验。其中，精子验证的靶蛋白包括：顶体素（acrosin, ACR）、顺乌头酸水合酶（aconitate hydratase, ACO2）、α-烯醇化酶（alpha-enolase, ENO1）、甘油-3-磷酸脱氢酶（glycerol-3-phosphate dehydrogenase, GPD2）以及磷酸丙糖异构酶（triosephosphate isomerase, TPI1）；细胞外液验证的靶蛋白包括：顶体素（ACR）、α-烯醇化酶（ENO1）、磷酸甘油酸激酶（phosphoglycerate kinase, PGK1）、丙酮酸激酶PKM（pyruvate kinase PKM, PKM）以及磷酸丙糖异构酶（TPI1）。
6. 目录“蛋白质印迹定量数据”：包含14个.xlsx格式的原始数据文件，用于呈现精子（标注为SPERM）与细胞外液（标注为ECF）验证实验中用于统计分析的相对强度（任意单位）。其中，精子验证的靶蛋白包括：顶体素（ACR）、顺乌头酸水合酶（ACO2）、α-烯醇化酶（ENO1）、甘油-3-磷酸脱氢酶（GPD2）以及磷酸丙糖异构酶（TPI1）；细胞外液验证的靶蛋白包括：顶体素（ACR）、α-烯醇化酶（ENO1）、磷酸甘油酸激酶（PGK1）、丙酮酸激酶PKM（PKM）以及磷酸丙糖异构酶（TPI1）。