R&D Center for Clinical Mass Spectrometry, Seegene Medical Foundation, Seongdong-gu, Seoul, Korea.
Department of Laboratory Medicine, Seegene Medical Foundation, Seongdong-gu, Seoul, Korea.
Proteomics Clin Appl. 2021 Nov;15(6):e2100044. doi: 10.1002/prca.202100044. Epub 2021 Aug 18.
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.
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.
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).
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.
临床质谱分析需要对样品制备进行简化。本研究旨在优化从革兰氏阴性菌中分离周质蛋白的方法，并将其应用于临床质谱分析。
使用产碳青霉烯酶肺炎克雷伯菌（KPC）的大肠埃希菌标准细胞来优化渗透休克（OS）裂解方法。通过 LC-MS/MS 和 MALDI-TOF MS 分析 OS 裂解后的上清液。然后通过 MALDI-TOF MS 确认 OS 裂解方法对产 KPC-2 的肠杆菌科临床分离株的有效性。
使用产 KPC-2 的大肠埃希菌标准细胞优化的 OS 裂解显示出高产量的 KPC-2 蛋白和丰富的周质蛋白。与其他裂解方法相比，MALDI-TOF MS 分析中 KPC-2 蛋白的检测灵敏度显著提高。19 株临床分离株通过 OS 法进行 MALDI-TOF MS 验证，与其他裂解方法（例如 1.5%正辛基-β-D-吡喃葡萄糖苷）相比，检测灵敏度也更高（p<0.001）。
本研究提供了一种直接、快速、经济且无去污剂的方法，用于分析肠杆菌科临床分离株的周质蛋白。这种方法可能有助于基于 MS 的临床诊断。
