Evaluation of Sample Preparation Strategies for Human Milk and Plasma Proteomics

Sample preparation is the most critical step in proteomics as it directly affects the subset of proteins and peptides that can be reliably identified and quantified. Although a variety of efficient and reproducible sample preparation strategies have been developed, their applicability and efficacy depends much on the biological sample. Here, three approaches were evaluated for the human milk and plasma proteomes. Protein extracts were digested either in an ultrafiltration unit (filter-aided sample preparation, FASP) or in-solution (ISD). ISD samples were desalted by solid-phase extraction prior to nRPC-ESI-MS/MS. Additionally, milk and plasma samples were directly digested by FASP without prior protein precipitation. Each strategy provided inherent advantages and disadvantages for milk and plasma. FASP appeared to be the most time efficient procedure with a low miscleavage rate when used for a biological sample aliquot, but quantitation was less reproducible. A prior protein precipitation step improved the quantitation by FASP due to significantly higher peak areas for plasma and a much better reproducibility for milk. Moreover, the miscleavage rate for milk, the identification rate for plasma, and the carbamidomethylation efficiency were improved. In contrast, ISD of both milk and plasma resulted in higher miscleavage rates and is therefore less suitable for targeted proteomics.     

高效液相色谱串联质谱法测定牛奶中四环素类抗生素及其代谢产物

建立了牛奶中四环素、金霉素、土霉素、强力霉素、去甲基金霉素、甲烯土霉素和二甲胺四环素等7种四环素类抗生素及差向四环素、差向金霉素、差向土霉素等3种代谢产物多残留的液相色谱串联质谱同时测定方法。方法采用Inertsil C8-3(5μm,150 mm×2.1 mm i.d)反相色谱柱,以pH 4.0的EDTA-M c llvaine缓冲溶液为提取溶液,以HLB固相萃取柱为净化柱,流动相为甲醇 0.01 mol/L三氟乙酸(梯度洗脱),流速0.3mL/m in,以正离子多反应监测模式测定,进样量30μL。方法的检出限为0.5~10μg/kg;测出限为50μg/kg;线性范围为50~1200μg/L,加标回收率为74.4%~101%,相对标准偏差为1.8%~8.3%。本方法具有灵敏、准确、简便、快速等优点,适用于牛奶中四环素类抗生素及其代谢产物多残留的同时确证检测。