Development of a mass spectrometric method for the analysis of periplasmic proteins from Gram-negative bacteria

Mass spectrometry-based proteomics has emerged as a powerful technique in the field of clinical diagnostics but requires a multistep process for sample preparation. The purpose of clinical mass spectrometry is to detect and quantify protein targets using well-established methods. Here, we found that the osmotic shock (OS) process allowed us to reduce the salt concentration and simultaneously enrich periplasmic proteins, specifically target proteins associated with antibiotic resistance in Gram-negative bacteria. Furthermore, we demonstrated that an optimized condition of the OS lysis allows the highest sensitivity in the MS analysis, and the OS lysate containing the proteins of interest (e.g., KPC-2) can be directly injected into multiple mass spectrometers without desalting process. Our study provides a detergent-free, straightforward method for periplasmic protein analysis using mass spectrometry.