Microflex lrf maldi tof ms

Microbial colony smears or liquid samples were applied to a target location on a stainless steel MALDI plate. The target plate was incubated in a humidified chamber for 30 min at 110 °C. The MALDI plate was washed with deionized water from a squeeze bottle, allowed to air dry, then 1 μL of norharmane matrix solution was applied (10 mg/mL in 12:6:1, v/v/v chloroform/methanol/water) to each target location. Following the method of Leung et al.^{16 (link)}, spectra were acquired from target locations in negative ion mode using a Microflex LRF MALDI-TOF MS (Bruker, Billerica MA) in reflectron mode with a limited mass range of 1000–2400 m/z. Typically, 300 laser shots were summed to acquire each mass spectrum.

Isolates were cultured onto lysogeny broth (LB) plates and grown for 16 h at 37°C. To express resistance, samples were also cultured on LB with 2 μg/mL colistin sulfate plates. Isolates with MICs less than 2 μg/mL had reduced growth. Lipid A analysis was conducted using FLAT per the methods in Sorensen et al. (31 (link)). Briefly, a single colony was spotted on a MALDI plate, overlaid with 1 μL of buffer of 0.2 M anhydrous citric acid and 0.1 M trisodium citrate dihydrate, incubated at 110°C for 30 min, and rinsed with endotoxin-free water (31 (link)). Each isolate was spotted in triplicate. Subsequently, 1 μL of 10 mg/mL norharmane matrix suspended in 2:1 chloroform-methanol was spotted onto the extracted lipid sample on a MALDI plate. A Bruker Microflex LRF MALDI-TOF MS in the negative ion and reflectron mode was used to collect mass spectra. The MALDI-TOF MS comprises a 337 nm nitrogen laser. Analyses were conducted at 43% global intensity with approximately 300 laser shots for each spectrum acquisition. Spectra were recorded in triplicate. Electrospray tuning mix (Agilent, Palo Alto, CA) was used for mass calibration. Flex analysis (v3.4) software processed the mass spectra with smoothed and baseline corrections. Signal-to-noise ratios above three were considered optimal for inclusion of signature ions (52 (link)).