Kune Christopher, Tielens Sylvia, Baiwir Dominique, Fléron Maximilien, Vandormael Denis, Eppe Gauthier, Nguyen Laurent, Mazzucchelli Gabriel
Mass Spectrometry Laboratory, MolSys Research Unit, University of Liege, Liege B-4000, Belgium.
Laboratory of Molecular Regulation of Neurogenesis, GIGA-Stem Cells, University of Liège, Liège B-4000, Belgium.
Anal Chem. 2025 Feb 25;97(7):3836-3845. doi: 10.1021/acs.analchem.4c03709. Epub 2025 Feb 11.
Proteomics, essential for understanding gene and cell functions, faces challenges with peptide loss due to adsorption onto vial surfaces, especially in samples with low peptide quantities. Using HeLa tryptic digested standard solutions, we demonstrate preferential adsorption of peptides, particularly hydrophobic ones, onto polypropylene (PP) vials, leading to nonuniform signal loss. This phenomenon can alter protein quantification (e.g., Label-Free Quantification, LFQ) if no appropriate data processing is applied. Our study is based on understanding this adsorption phenomenon to establish recommendations for minimizing peptide loss. To address this issue, we evaluated the nature of surface material and buffer additives to reduce peptide-surface noncovalent binding. Here, we report that using vials made from polymer containing polar monomeric units such as poly(methyl methacrylate) (PMMA) or polyethylene terephthalate (PET) drastically reduces the hydrophobic peptide loss, increasing the global proteomics performance (4-fold increase in identified peptides for the single-cell equivalent peptide content range). Additionally, the incorporation of nonionic detergents like poly(ethylene oxide) (PEO) and n-Dodecyl-Beta-Maltoside (DDM) at optimized concentrations (0.0001% and 0.0075%, respectively) improves the overall proteomic performance and consistency, even across different vial materials. Implementing these recommendations on 0.2 ng/μL HeLa tryptic digest results in a 10-fold increase in terms of peptide signal. Application to True Single-Cell sample preparation without specialized instrumentation dramatically improves the performance, allowing for the identification of approximately 650 proteins, a stark contrast to none detected with classical protocols.
蛋白质组学对于理解基因和细胞功能至关重要，但由于肽段吸附在小瓶表面而面临肽段损失的挑战，尤其是在肽段量较低的样品中。使用HeLa胰蛋白酶消化的标准溶液，我们证明了肽段，特别是疏水性肽段，优先吸附在聚丙烯（PP）小瓶上，导致信号损失不均匀。如果不进行适当的数据处理，这种现象会改变蛋白质定量（例如，无标记定量，LFQ）。我们的研究基于对这种吸附现象的理解，以建立减少肽段损失的建议。为了解决这个问题，我们评估了表面材料的性质和缓冲添加剂，以减少肽段与表面的非共价结合。在此，我们报告使用由含有极性单体单元的聚合物制成的小瓶，如聚甲基丙烯酸甲酯（PMMA）或聚对苯二甲酸乙二酯（PET），可大幅减少疏水性肽段的损失，提高整体蛋白质组学性能（在单细胞等效肽段含量范围内，鉴定出的肽段增加了4倍）。此外，以优化浓度（分别为0.0001%和0.0075%）加入聚环氧乙烷（PEO）和正十二烷基-β-麦芽糖苷（DDM）等非离子去污剂，可提高整体蛋白质组学性能和一致性，即使在不同的小瓶材料中也是如此。在0.2 ng/μL HeLa胰蛋白酶消化物上实施这些建议，肽段信号增加了10倍。将其应用于无需专门仪器的真正单细胞样品制备，可显著提高性能，能够鉴定出约650种蛋白质，这与传统方法未检测到任何蛋白质形成鲜明对比。
