Abstract
High-performance liquid tandem mass spectrometry (HPLC-MS) is widely employed for protein analysis in biological systems. However, conventional proteomic sample pretreatment methods suffer from multiple steps and poor reproducibility. In this study, we introduce EPURISp (Enzymatic Digestion with Ultrafiltration and Rapid In-situ Sample Purification), a novel proteomic pretreatment technique that combines enzymatic digestion, ultrafiltration, and one-step temperature-controlled vacuum drying for efficient desalting. The EPURISp method exhibits excellent protein recovery rates across a wide range of molecular weights and hydrophilicity, surpassing traditional C18 desalting approaches. Practical proteomic analysis (PXD044209) utilizing EPURISp demonstrates the highest protein identification yield with remarkable reproducibility, which is particularly advantageous in membrane protein identification. Notably, EPURISp exhibits superior performance in minimizing oxidation and deamidation modifications compared with conventional FASP methods. This innovative EPURISp method represents a significant advancement in proteomics analysis, providing reliable and efficient results for mass spectrometry.