β-Lactamase ragged ends detected by electrospray mass spectrometry correlates poorly with multiple banding on isoelectric focusing

Abstract

Purified preparations of TEM-2, P99, Bacillus cereus I and B. cereus II beta-lactamases were examined by electrospray (ES) mass spectrometry. The ES mass spectra of the B. cereus enzymes revealed the presence of four to five components of different mass, corresponding to the loss of different numbers of N-terminal amino acids (ragged ends). The ES mass spectra of both TEM-2 and P99 consisted of a single component with no evidence of ragged ends. All four beta-lactamase preparations were visualized on isoelectric focusing (IEF) gels stained with nitrocefin to investigate a possible correlation between IEF patterns and ragged ends. Multiple banding patterns were seen with each beta-lactamase preparation. Although these may correlate with the presence of ragged ends in the two B. cereus preparations, the satellite bands seen with P99 and TEM-2 were not associated with differences detected by ES mass spectrometry. In this study we have shown for the first time that beta-lactamase satellite bands seen on IEF are not always associated with ragged ends. Furthermore, we have illustrated the use of ES mass spectrometry to characterize the extent of ragged end formation in protein samples. This is of particular significance if the sample is required for detailed biochemical or crystallography experiments.

Broad antibiotic resistance profile of the subclass B3 metallo‐β‐lactamase GOB‐1, a di‐zinc enzyme

“…To verify the hypothesis that GOB-1 has ragged ends (not a unique phenomenon with respect to MBLs [32]), the N-terminus of the enzyme was sequenced. The presence of two N-terminal sequences QVVKE and LNAQV confirmed that the signal peptide was cleaved at two positions.…”

Inhibition of metallo-beta-lactamases by a series of mercaptoacetic acid thiol ester derivatives

“…has been utilized previously to characterize inhibitors covalently bound to serine ␤-lactamases (2,9,19). Although the substrate hydrolysis of metallo-␤-lactamases is not expressed by means of covalent intermediates, the presence of a cysteine residue close to the active site of metallo-␤-lactamases affords the prospect of covalent intervention at this residue in achieving inhibition.…”

“…ESMS of the B. cereus II enzyme gave 6 peaks, A through F (Fig. 2), each of which corresponded to ragged ends comprising the loss of N-terminal amino acid residues (19). Following a 1:10,000 dilution, B. cereus II enzyme was inhibited 95% by each of the inhibitors, SB214751, SB214752, or SB213079.…”

Substrate Selectivity and a Novel Role in Inhibitor Discrimination by Residue 237 in the KPC-2 β-Lactamase

“…ESI-MS of intact KPC-2 ␤-lactamase revealed that multiple forms of the KPC-2 ␤-lactamase are produced when the full-length protein is expressed in E. coli (data not shown). Thus, the N terminus of KPC-2 appears ragged when expressed in E. coli DH10B or Origami2 DE3 pLys (32,38). All purified forms of KPC-2 behaved identically in kinetic assays (data not shown).…”