Polished steel target plate

The hydrolysis assay was developed using a Microflex™ (Bruker Daltonics, Billerica, MA, USA) mass spectrometer. The parameters settings were: ion source 1, 19.0 kV; ion source 2, 17.2 kV; lens, 6.0 kV; detector gain, 2.5 kV. Spectra were recorded in the mass range of 0–1000 Da with 60 Hz laser frequency. Each spectrum was obtained from 240 laser shots. The polished steel target plate (Bruker Daltonics, Bremen, Germany) and HCCA matrix (2.5 mg α-cyano-4-hydroxycinnamic acid dissolved in 50% acetonitril, 47.5% HPLC-pure H₂O and 2.5% trifluoroacetic acid, (Bruker Daltonics)) was used. For calibration the Peptide calibration standard II (Bruker Daltonics) was used. The peaks employed for calibration were CCA [M + H]⁺ at 190.05 Da, CCA [2 M + H]⁺ at 379.09 Da and Bradykinin (1–7) peak [M + H]⁺ at 757.40 Da.
The analysis of MALDI-TOF MS spectra was performed with the Flexanalysis 3.3 software (Bruker Daltonics). The spectra were smoothed and baseline subtracted and then manually examined for the specific ertapenem related peak patterns in the mass range of 4–600 Da previously described [4 (link)]. To approve a spectrum as reliable at least one sum buffer peak of hydrolysed or unhydrolysed ertapenem had to have a minimum intensity of 10⁴. The high intensity proves the specificity of the peaks and guarantees that no unspecific background noise is misinterpreted as a significant peak.

A saturated solution of α-cyano-4-hydroxycinnamic acid (αCHCA, 20 mg) in acetone (500 µL) was prepared as Mix 1. A precursor saturated solution of CHCA (20 mg) in 70% acetonitrile with 5% formic acid (500 µL) was prepared alongside a saturated solution of 2,5-dihydroxybenzoic acid (DHB, 20 mg, Sigma-Aldrich) in 70% acetonitrile with 0.1% trifluoroacetic acid (500 µL). Solutions were prepared at room temperature and vortexed thoroughly for 60 s before use. DHB (100 µL) and CHCA (100 µL) solutions were then combined to prepare Mix 2. Mix 1 (0.5 µL) was spotted onto a polished steel target plate (Bruker) and evaporated quickly to leave a thin layer of CHCA. A 0.5 µL aliquot of protein sample (typically 10 µM in PBS) was spotted directly onto the layer. Then, 0.5 µL of Mix 2 was added to the liquid droplet and allowed to dry.
A Bruker Microflex LRF was used to acquire the MALDI–TOF–MS data, in linear positive mode (laser 60 Hz, Ion Source 1: 19.5 kV, Ion Source 2: 18.15 kV, Lens: 7.00 kV, Pulsed Ion Extraction 240 ns, Detector Gain 2850 V). Data were processed using Bruker flexAnalysis v3.4.