Purpose
Clinical mass spectrometry requires a simple step process for sample preparation. This study aims to optimize the method for isolating periplasmic protein from Gram‐negative bacteria and apply to clinical mass spectrometry.
Experimental Design
The Klebsiella pneumoniae carbapenemase (KPC)‐producing E. coli standard cells were used for optimizing the osmotic shock (OS) lysis method. The supernatant from OS lysis was analysed by LC‐MS/MS and MALDI‐TOF MS. The effectiveness of the OS lysis method for KPC‐2‐producing Enterobacteriaceae clinical isolates were then confirmed by MALDI‐TOF MS.
Results
The optimized OS lysis using KPC‐2 producing E. coli standard cells showed a high yield of KPC‐2 protein and enriches periplasmic proteins. Compared with other lysis methods, the detection sensitivity of KPC‐2 protein significantly increased in MALDI‐TOF MS analysis. Nineteen clinical isolates were validated by MALDI‐TOF MS using the OS method, which also showed higher detection sensitivity compared to other lysis method (e.g., 1.5% n‐octyl‐β‐D‐glucopyranoside) (p < 0.001).
Conclusions and Clinical Relevance
This study provides a straightforward, rapid, affordable, and detergent‐free method for the analysis of periplasmic proteins from Enterobacteriaceae clinical isolates. This approach may contribute to MS‐based clinical diagnostics.