α cyano 4 hydroxycinnamic acid matrix

Bacterial recovery from whole intestines, tissues and fecal contents was performed as described by Del Chierico et al. (2014) (link). Briefly, intestines were mechanically homogenized, washed and resuspended in Hanks’ balanced salt solution (HBSS) to obtain the enriched bacterial suspensions (EBSs). Ten microliters of 500 μL of an EBS were cultured for 48–72 h under aerobic, microaerophilic, and anaerobe growth conditions. Bacterial cell density was estimated by serial dilutions and plate counting. Based on morphology and growth conditions, colonies were characterized and re-isolated in order to proceed with MALDI–TOF MS-based IDs performed with a Microflex LT mass spectrometer (Bruker Daltonics, GmbH, Bremen, Germany) using Flex Control (version 3.0) and MALDI Biotyper automation control (version 2.0) software. Specifically, bacterial cells were directly picked from isolated colonies, smeared in triplicate onto an MSP 96 polished steel target (Bruker Daltonics) and overlaid with α-cyano-4-hydroxycinnamic acid matrix (Bruker Daltonics). Spectral analyses and bacterial IDs were automatically performed by matching against a reference library (version 2.0 SR 1; Bruker Daltonics; Del Chierico et al., 2014 (link)).

The C. auris isolates used in this study were clinical clade I isolates that are part of a collection at the Centre of Expertise in Mycology, Nijmegen, The Netherlands. Additionally, a single C. auris isolate that was derived from the first diagnosed case of candidemia in Central Italy was also studied. In total, eight isolates that were confirmed to be C. auris by PCR and sequencing of the ribosomal DNA internal transcribed spacer (ITS) region were submitted to protein extraction according to a previously developed MALDI-TOF MS protocol (De Carolis et al., 2014 (link)). Briefly, yeast cells were suspended in 10% formic acid and then vortexed; one µL of lysate was placed on the MALDI target plate to obtain 12 technical replicates, which were overlaid each with one µL of absolute ethanol before allowing co-crystallization with the α-cyano-4-hydroxycinnamic acid matrix (Bruker Daltonics). A total of ≥5000 laser shots were used to generate a main spectrum profile (MSP) for each isolate, which was then added to the Bruker MALDI Biotyper^® database. Isolates were also submitted to antifungal susceptibility testing (AFST), which was performed using a MALDI-TOF MS based assay (see below).

PCR was performed in volumes of 50 μl as described above. Primer pairs PNOC-F/PNOC-R and PCR cycles were performed as described by Haouas et al. [25 (link)] and sequencing was performed as described above. Obtained sequences were submitted to GenBank. A male Ph. mascittii specimen and filtered H₂O were used as negative controls.
MALDI-TOF peptide mass mapping analysis of host-specific hemoglobin peptides was performed according to a protocol by Hlavackova et al. [26 (link)]. Blood from engorged abdomens was digested using trypsin (Promega) and the resulting peptides were mixed with an α-cyano-4-hydroxycinnamic acid matrix (Bruker Daltonics). Peptide mass maps were acquired with an Ultraflex III MALDI-TOF instrument (Bruker Daltonics) and at least two peptides per female were selected for MS/MS sequencing. MS/MS spectra were searched against the SwissProt 2019_05 database subset of vertebrate proteins using an in-house MASCOT search engine (Matrix Science).

Samples were spotted on the MALDI plate following the dried-droplet method. Briefly, 1 μl of the reconstituted in-gel digest sample was spotted on a BigAnchorChip target plate (Bruker Daltonics), followed by 1 μl of matrix (10 mg/ml α-cyano-4-hydroxycinnamic acid matrix (Bruker Daltonics) in 50% ACN, 0.1% TFA). Sample and matrix mixture was dried at room temperature. Mass spectra were obtained on an UltrafleXtreme (Bruker Daltonics, Bremen, Germany) matrix-assisted laser desorption ionization–tandem time of flight (MALDI-TOF/TOF) mass spectrometer. Mass spectra were recorded in positive ionization reflectron mode in the mass range of 700–3500 Da. Operating conditions were as follows: ion source 1 = 25.00 kV, ion source 2 = 24.40 kV, lens voltage = 8.50 kV, reflector voltage = 26.45 kV, optimized pulsed ion extraction time = 130 ns, matrix suppression = 500 Da. 1500 single-shot spectra were accumulated by recording 50-shot spectra at 10 random positions using fixed laser attenuation. Mass spectra were externally calibrated using a standard peptide mixture (Bruker); calibration was considered good when a value below 1 ppm was obtained.

Protein masses were determined on purified solution samples. An amount of 1 μL of protein at ~20 μM was mixed with 1 μL of α-Cyano-4-hydroxycinnamic acid matrix (Bruker Daltonics, Wissembourg, France) solution in 0.3% TFA/CH3CN (50:50 v/v). One μL of the mix was spotted on the target and analysed by MALDI-TOF on an Ultraflex III spectrometer (Bruker Daltonics, Wissembourg, France) controlled by the Flexcontrol 3.0 package (Build 51) and operated in the linear mode, using a maximum accelerating potential of 25 kV and a 5000–15,000 m/z range (LP_Protmix_Method). The laser frequency was fixed to 100 Hz and ~1000 shots per sample were cumulated. Four external standards (Protein Calibration Standard I, Bruker Daltonics, Wissembourg, France) were used to calibrate each spectrum to a mass accuracy within 100 ppm. Peak picking was performed using the FlexAnalysis 3.0 software with an adapted analysis method. Parameters used were: centroid peak detection algorithm, S/N threshold fixed to 5 and a quality factor threshold of 30.