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Published on: May 21, 2018
Optimization of a lysis method to isolate periplasmic proteins from Gram-negative bacteria for clinical mass
Dong Huey Cheon1, Saeyoung Lee1, Won Suk Yang1
1R&D Center for Clinical Mass Spectrometry, Seegene Medical Foundation, Seongdong-gu, Seoul, Korea.
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.
Insights
This study optimized osmotic shock lysis for isolating periplasmic proteins from Gram-negative bacteria, enhancing detection sensitivity for clinical mass spectrometry applications. The new method is rapid, affordable, and detergent-free.
Area of Science:
- Microbiology
- Analytical Chemistry
- Clinical Diagnostics
Background:
- Clinical mass spectrometry demands simplified sample preparation techniques.
- Efficient isolation of periplasmic proteins from Gram-negative bacteria is crucial for accurate diagnostics.
Purpose of the Study:
- To optimize a method for periplasmic protein isolation from Gram-negative bacteria.
- To apply the optimized method to clinical mass spectrometry.
Main Methods:
- Optimized osmotic shock (OS) lysis using Klebsiella pneumoniae carbapenemase (KPC)-producing E. coli standard cells.
- Analysis of supernatant by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS).
- Validation of the OS lysis method on KPC-2-producing Enterobacteriaceae clinical isolates using MALDI-TOF MS.
Main Results:
- Optimized OS lysis demonstrated high yield and enrichment of KPC-2 protein.
- Significantly increased detection sensitivity of KPC-2 protein in MALDI-TOF MS compared to other lysis methods.
- Validated on 19 clinical isolates, the OS method showed superior detection sensitivity (p < 0.001) versus methods like 1.5% n-octyl-β-D-glucopyranoside.
Conclusions:
- A straightforward, rapid, affordable, and detergent-free method for periplasmic protein analysis from Enterobacteriaceae was developed.
- This optimized osmotic shock lysis method enhances periplasmic protein detection sensitivity.
- The approach holds potential for advancing mass spectrometry-based clinical diagnostics.
