Chemical Engineering Department, Indian Institute of Technology Delhi, India.
Chemical Engineering Department, Indian Institute of Technology Delhi, India.
J Pharm Biomed Anal. 2023 Sep 20;234:115527. doi: 10.1016/j.jpba.2023.115527. Epub 2023 Jun 15.
Establishing analytical and functional comparability serves as the foundation of biosimilar development. A critical part of this exercise is sequence similarity search and categorization of post-translational modifications (PTMs), often by peptide mapping using liquid chromatography-mass spectrometry (LC-MS). When performing bottom-up proteomic sample preparation, efficient digestion of the protein and extraction of peptides for subsequent mass spectrometric analysis can be a challenge. Conventional sample preparation strategies face the risk of allowing interference of chemicals which are essential for extraction but are likely to interfere with digestion, resulting in complex chromatographic profiles due to semi-cleavages, insufficient peptide cleavages, and other unwanted reactions. Further, peptide cleanup through commonly used immobilized C-18 pipette tips can cause significant peptide loss as well as variability in individual peptide yields, thereby causing artifacts of various product-related modifications. In this study, we proposed a simple enzymatic digestion technique by incorporating different molecular weight filters and protein precipitation, with the objective to minimize interference of denaturing, reducing, and alkylating agents throughout overnight digestion. As a result, the need for peptide cleanup is significantly reduced and results in higher peptide yield. The proposed FAPP approach outperformed the conventional method across multiple metrics including, 30% more peptides, 8.19% more fully digested peptides, 14% higher sequence coverage rate, and 11.82% more site-specific alterations. Quantitative and qualitative repeatability of the proposed approach have been demonstrated. It can be concluded that the filter-assisted protein precipitation (FAPP) protocol proposed in this study offers an effective substitute for the traditional approach.
建立分析和功能可比性是生物类似药开发的基础。这项工作的一个关键部分是序列相似性搜索和翻译后修饰（PTM）的分类，通常通过液相色谱-质谱（LC-MS）进行肽图分析。在进行自上而下的蛋白质组学样品制备时，有效地消化蛋白质并提取肽以进行后续的质谱分析可能是一个挑战。传统的样品制备策略存在允许化学物质干扰的风险，这些化学物质对于提取是必不可少的，但很可能会干扰消化，从而导致由于半裂解、肽裂解不足和其他不需要的反应而导致复杂的色谱图谱。此外，通过常用的固定化 C-18 移液吸头进行肽净化会导致显著的肽损失以及个别肽产率的可变性，从而导致各种与产品相关的修饰的假象。在这项研究中，我们提出了一种简单的酶消化技术，通过结合不同分子量的过滤器和蛋白质沉淀，旨在最大限度地减少变性、还原和烷基化试剂在整个过夜消化过程中的干扰。结果，需要进行的肽净化显著减少，肽产量更高。与传统方法相比，所提出的 FAPP 方法在多个指标上都表现出色，包括增加 30%的肽、8.19%的完全消化肽、14%的更高序列覆盖率和 11.82%的更多特定位置改变。该方法的定量和定性重复性已得到证明。可以得出结论，本研究中提出的过滤器辅助蛋白质沉淀（FAPP）方案为传统方法提供了有效的替代方案。
