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Optimization of a lysis method to isolate periplasmic proteins from Gram-negative bacteria for clinical mass spectrometry.
- Source :
-
Proteomics. Clinical applications [Proteomics Clin Appl] 2021 Nov; Vol. 15 (6), pp. e2100044. Date of Electronic Publication: 2021 Aug 18. - Publication Year :
- 2021
-
Abstract
- 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.<br />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.<br />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).<br />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.<br /> (© 2021 Wiley-VCH GmbH.)
- Subjects :
- Chromatography, High Pressure Liquid
Escherichia coli enzymology
Escherichia coli isolation & purification
Osmotic Pressure
Periplasmic Proteins isolation & purification
Periplasmic Proteins metabolism
Sodium Chloride chemistry
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
beta-Lactamases metabolism
Escherichia coli metabolism
Periplasmic Proteins analysis
Subjects
Details
- Language :
- English
- ISSN :
- 1862-8354
- Volume :
- 15
- Issue :
- 6
- Database :
- MEDLINE
- Journal :
- Proteomics. Clinical applications
- Publication Type :
- Academic Journal
- Accession number :
- 34370896
- Full Text :
- https://doi.org/10.1002/prca.202100044