Microflex lt sh system

Following CFU enumeration, bacterial colonies from BHI agar plates were subcultured on 5% blood agar plates (Statens Serum Institute (SSI) Diagnostica) and incubated for 18–24 h at 35–37°C. After incubation, colonies from 5% blood agar plates were confirmed to be S. aureus with matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF) (Bruker Microflex, LT/SH system; Bruker Daltonik GMbH). To confirm rifampicin susceptibility, minimum inhibitory concentration (MIC) determination was performed by gradient test (Etest strips, bioMérieux). For the preparation of inoculum, inoculation, and incubation, we followed the guidelines recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (http://www.eucast.org). All MALDI-TOF and rifampicin susceptibility tests were performed at the reference laboratory at the Department of Clinical Microbiology at Aarhus University Hospital by trained staff.

l-Penetratin (l-PEN, RQIKIWFQNRRMKWKK) and d-Penetratin (d-PEN, rqikiwfqnrrmkwkk) as well as their carboxyfluorescein (CF)-labeled analogs (l-PEN_CF, d-PEN_CF, respectively) were synthesized by Fmoc-based solid-phase peptide synthesis on an automated CEM Liberty (Matthews, NC, USA) as previously described [38 (link)]. Briefly, N-terminal labeling was performed manually overnight in Teflon reactors. The labeling of insulin with TAMRA performed manually by dissolving insulin in water at an acidic pH (pH 1–2) and subsequently increasing the pH to >10 with 1 M Na₂CO₃. Hereafter, the solution was mixed with TAMRA (dissolved in DMSO) at a mass ratio of 2:1 insulin:TAMRA and was mixed overnight on a rotary mixer protected from light. The purification of the labeled CPPs, as well as TAMRA-insulin, were performed by RP-HPLC, and purities of >92% were obtained for all conjugates. Conjugate identity was confirmed by MALDI-ToF-MS (Bruker, Microflex LT/SH system). The compounds were freeze-dried, followed by storage at −18 °C.

Spectra were acquired using the Microflex LT/SH system (Bruker Daltonics Inc., Bremen). Each spot was analyzed three times, resulting in 24 single spectra per medium for each strain. A total of 72 spectra were generated for each strain.
As Bruker recommended, a Bacterial Test Standard (BTS 8255343) was used to calibrate the instrument, before each acquisition session. A BTS was also used on each acquisition plate, to check the quality of the acquisition. As the most distinct, clear, and significant spectra lie in a mass range of 5000 to 10,000 Da, the protein mass spectra of samples were acquired in a mass range of 2000–20,000 Da by the flexControl 3.4 program (Bruker Daltonics). The laser frequency for every run was 60 Hz with a linear positive mode. The default settings of the MALDI-TOF MS instrument were as follows: lens, 8.5 kV; ion source 1, 20 kV; and ion source 2, 18.1 kV. The flexControl program automatically acquired the spectrum of each spot. Each spectrum was generated by 240 laser shots (40 laser shot steps at six randomly selected positions of a single spot).