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

RATIONALE: Linear mode of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) has been routinely used for bacterial identification in the clinic, depending on the pattern analysis of spectral libraries rather than accurate mass measurement of ribosomal proteins (10-15 kDa). However, a demand for more accurate mass analysis of pathogens (e.g. KPC-2 carbapenemase) has been recently increasing for diagnostic purposes. METHODS: We introduced a 6xHIS-tagged KPC-2 (i.e. hKPC-2) and used it as an internal mass calibrator for the mass calibration of target proteins. After internal mass calibration (In-Cal), we evaluated the observed mass of KPC-2 against the theoretical mass of hKPC-2, which has 823 Da mass difference from the target protein. We further assessed the accuracy and precision of our calibration method regarding the identification of KPC-2 and other pathogens in clinical isolates (n = 42). RESULTS: Among several candidates for internal mass calibrators, the In-Cal using a 6xHIS-tagged protein on the target showed the highest mass accuracy and precision in the detection of target proteins (e.g. KPC-2). The application of hKPC-2 as an internal calibrator showed substantial improvement of mass accuracy, mass precision and also quantification of KPC in linearity and repeatability for KPC detection in the clinical isolates. CONCLUSIONS: Our In-Cal method using 6xHIS-tagged protein in MALDI-TOFMS allows successful mass calibration (<3.5 Da) of pathogenic proteins (>20 kDa) and provides high mass accuracy as much as that of medium- and high-resolution mass spectrometry.

Clinical Performance of the Osmotic Shock-MALDI MS Method to Detect Klebsiella pneumoniae Carbapenemase in Clinical Isolates.

The World Health Organization recently highlighted the serious worldwide problem of the emergence of antibiotic-resistant or antibiotic multidrug-resistant bacteria. Carbapenem-resistant Enterobacterales, including carbapenemase-producing Enterobacterales (CPE), are major antibiotic-resistant bacteria that can be identified by various methods, including antibiotic susceptibility testing, PCR, and immunologic assays. However, there is a need for a faster, more accurate, low-cost, and easy method to detect CPE strains. We previously developed an osmotic shock matrix-assisted laser desorption/ionization mass spectrometry (OS-MALDI MS) method for directly detecting intact Klebsiella pneumoniae carbapenemase (KPC) using osmotic shock cell lysis. In this study, we evaluated the OS-MALDI MS method and compared it with two other methods (octyl-glucoside-aided direct KPC detection method [OG-MALDI MS] and Bruker's MBT subtyping module indirect method [MBT-SM MALDI MS]). We first completed an analytical performance evaluation of the OS-MALDI MS method according to Clinical and Laboratory Standards Institute guidelines. Clinical testing was performed with 437 clinical isolates, including 292 KPC-producing bacteria and 145 non-KPC-producing bacteria. The OS-MALDI MS method exhibited 95.9% sensitivity, 100.0% specificity, and 100.0% precision for detecting KPC. Accuracy of the OS-MALDI MS, OG-MALDI MS, and MBT-SM MALDI MS methods was 97.3%, 55.9%, and 50.2%, respectively. In conclusion, the OS-MALDI MS method clearly outperformed the other methods, exhibiting the highest accuracy and sensitivity of the three methods. We propose the OS-MALDI MS method as a practical, useful method for clinic environments, which may help guide appropriate antibiotic treatment and contribute to the prevention of the spread of CPE.

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

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-ß-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.