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.