A novel filter-assisted protein precipitation (FAPP) based sample pre-treatment method for LC-MS peptide mapping for...

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.