Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, USA.
Mass Spectrometry and Proteomics Facility, Campus Chemistry Instrument Center, The Ohio State University, Columbus, OH, USA.
J Proteomics. 2021 Oct 30;249:104360. doi: 10.1016/j.jprot.2021.104360. Epub 2021 Sep 1.
We present an efficient protein extraction and in-solution enzymatic digestion protocol optimized for mass spectrometry-based proteomics studies of human skin samples. Human skin cells are a proteinaceous matrix that can enable forensic identification of individuals. We performed a systematic optimization of proteomic sample preparation for a protein-based human forensic identification application. Digestion parameters, including incubation duration, temperature, and the type and concentration of surfactant, were systematically varied to maximize digestion completeness. Through replicate digestions, parameter optimization was performed to maximize repeatability and increase the number of identified peptides and proteins. Final digestion conditions were selected based on the parameters that yielded the greatest percent of peptides with zero missed tryptic cleavages, which benefit the analysis of genetically variable peptides (GVPs). We evaluated the final digestion conditions for identification of GVPs by applying MS-based proteomics on a mixed-donor sample. The results were searched against a human proteome database appended with a database of GVPs constructed from known non-synonymous single nucleotide polymorphisms (SNPs) that occur at known population frequencies. The aim of this study was to demonstrate the potential of our proteomics sample preparation for future implementation of GVP analysis by forensic laboratories to facilitate human identification. SIGNIFICANCE: Genetically variable peptides (GVPs) can provide forensic evidence that is complementary to traditional DNA profiling and be potentially used for human identification. An efficient protein extraction and reproducible digestion method of skin proteins is a key contributor for downstream analysis of GVPs and further development of this technology in forensic application. In this study, we optimized the enzymatic digestion conditions, such as incubation time and temperature, for skin samples. Our study is among the first attempts towards optimization of proteomics sample preparation for protein-based skin identification in forensic applications such as touch samples. Our digestion method employs RapiGest (an acid-labile surfactant), trypsin enzymatic digestion, and an incubation time of 16 h at 37 °C.
我们提出了一种高效的蛋白质提取和溶液内酶解方案，针对的是基于质谱的人类皮肤样本蛋白质组学研究。人类皮肤细胞是一种蛋白质基质，可以实现个体的法医鉴定。我们对基于蛋白质的法医鉴定应用中的蛋白质组学样本制备进行了系统优化。通过系统地改变消化参数，包括孵育时间、温度以及表面活性剂的类型和浓度，来最大限度地提高消化的完全性。通过重复消化，优化参数以最大限度地提高重复性并增加鉴定的肽和蛋白质的数量。最终的消化条件是基于产生零漏切的肽百分比最高的参数来选择的，这有利于分析遗传变异肽（GVPs）。我们通过对混合供体样本进行基于 MS 的蛋白质组学分析，评估了最终消化条件对 GVP 的鉴定能力。结果是针对添加了由已知非同义单核苷酸多态性（SNP）构建的 GVP 数据库的人类蛋白质组数据库进行搜索的，这些 SNP 以已知的人群频率发生。本研究的目的是展示我们的蛋白质组学样本制备方法在未来通过法医实验室实施 GVP 分析的潜力，以促进人类识别。意义：遗传变异肽（GVPs）可以提供与传统 DNA 分析互补的法医证据，并可能用于人类识别。皮肤蛋白质的高效蛋白质提取和可重复消化方法是 GVP 分析的下游分析和该技术在法医应用中的进一步发展的关键贡献者。在这项研究中，我们优化了皮肤样本的酶解条件，如孵育时间和温度。我们的研究是首次尝试针对基于质谱的法医应用中基于蛋白质的皮肤识别的蛋白质组学样本制备进行优化，例如触摸样本。我们的消化方法采用了 Rapigest（一种酸不稳定的表面活性剂）、胰蛋白酶酶解以及在 37°C 下孵育 16 小时。
