Optimization of a lysis method to isolate periplasmic proteins from Gram‐negative bacteria for clinical mass spectrometry

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. 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.

A Graphene-Coated Silicon Wafer Plate Improves the Sensitivity and Reproducibility of MALDI-TOF MS Analysis of Proteins

“…KPC-2 is a major virulence factor that confers resistance to carbapenem antibiotics in Gram-negative bacteria. 32 Our results show that the G/SiO 2 plate can be very useful for general protein analysis and identification of disease-related target proteins.…”

“…We compared the performance of the G/SiO 2 plate with that of a conventional SUS plate for protein detection by MALDI-TOF MS. To evaluate the performance of the G/SiO 2 plate and assess its clinical applicability, we used the clinically important Klebsiella pneumoniae carbapenemase (KPC-2) protein as a main target protein. KPC-2 is a major virulence factor that confers resistance to carbapenem antibiotics in Gram-negative bacteria . Our results show that the G/SiO 2 plate can be very useful for general protein analysis and identification of disease-related target proteins.…”

“…Extraction of KPC-2 Protein from Standard Cells and Clinical Isolates. As previously reported, 32 we prepared a KPC-2 protein standard by transforming a recombinant KPC-2 gene into recombinant Escherichia coli TOP10 cells. The transformed cells were cultured at 37 °C for 16 h in Luria− Bertani liquid medium (Becton, Dickinson and Company, USA) containing 50 mg/L ampicillin.…”

Improving mass accuracy in matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry analysis of pathogenic proteins using 6xHIS‐tagged internal calibration

“…Next, the harvested cells were lysed via an osmotic lysis method. 18 The hKPC-2 protein (6xHIS-tagged KPC-2 protein) was then isolated from the lysate using Ni 2+ -affinity chromatography with Ni-NTA agarose resin (QIAGEN Inc., MD, USA). After washing the resin with 25 mM Tris-HCl buffer (pH 8.0) five times, the hKPC protein was eluted with an elution buffer containing 300 mM imidazole in 25 mM Tris-HCl (pH 8.0).…”

“…13,15 In this context, we have recently reported on clinical studies for the identification of KPC-2 from clinical isolates 16,17 and the technical development of a MALDI-TOFMS-compatible assay using osmotic lysis. 18 In fact, intact protein analysis using MALDI-TOFMS has advantages in time-saving, being less laborious and simple application without proteolytic digestion process. [19][20][21] However, due to analytes with heterogeneous crystallinity comprising matrix and sample mix, there are challenging issues in terms of reproducibility, mass accuracy and sensitivity in MALDI-TOFMS analysis.…”