Autoflex tof tof mass spectrometer

Preparations of bacterial isolates for MALDI-TOF MS measurement were done as previously described (Croxatto et al., 2012 (link)). The on-target extraction preparation method was used to identify all the isolates to establish a standardized rapid routine identification process. Briefly, one fresh colony material was picked and smeared onto a 384 polished steel MSP target (Bruker Daltonik GmbH, Germany) using a toothpick, then one μL of 70% formic acid was added onto the bacterial spot and allowed to air dry afterward add one μL of 10 mg/mL a-cyano-4-hydroxy-cinnamic acid (HCCA, Bruker Daltonik GmbH, Germany) matrix solution (50% acetonitrile; 2.5% trifluoroacetic acid; 47.5% distilled water) and allowed to air dry. The acquisition and analysis of mass spectra were then performed by an Autoflex TOF/TOF mass spectrometer (Bruker Daltonik GmbH, Germany) under the linear positive mode, MALDI Biotyper software package (version Compass) with the reference 7,854 database entries and default parameter settings was used. The Bruker Bacterial Test Standard (BTS) was used for calibration according to the manufacturer’s instructions. Two colony/sample material preparations were analyzed for each isolate, and the higher-scoring identification result was taken.

MALDI-TOF MS spectra were acquired on an Autoflex TOF/TOF mass spectrometer (Bruker Daltonics, Japan) equipped with 337 nm pulsed nitrogen laser under reflector mode. The accelerating voltage was 20 kV. Matrix, α-cyano-4-hydroxycinnamic acid (Aldrich), was prepared at a concentration of 10 mg/mL in 1:1 CH₃CN/ 0.1%TFA. External calibration was performed with [Ile⁷]-angiotensin III (m/z 897.51, monoisotopic, Sigma) and human ACTH fragment 18–39 (m/z 2465.19, monoisotopic, Sigma). The sample solution (0.5 μL) dropped onto the MALDI sample plate was added to the matrix solution (0.5 μL) and allowed to dry at room temperature. For TOF/TOF measurement, argon was used as a collision gas and ions were accelerated at 19 kV. The series of b and y ions were afforded, which enabled identification of whole amino acid sequence by manual analysis.

Before detection, serum and plasma were diluted with deionized water ten fold, and urine was diluted with deionized water five fold. After that, 1 µL of analyte solution (diluted serum, plasma, or urine) was spotted on the polish plate and dried in air at room temperature. Then 1 µL of matrix slurry (1 mg mL⁻¹ of SiO₂@Ag nanoshells, 10 mg mL⁻¹ of CHCA or DHB (dissolved in 0.1% TFA solution (water/acetonitrile = 7/3, v/v)) was added and dried for LDI MS analysis. Mass spectra were recorded on an AutoFlex TOF/TOF mass spectrometer (Bruker, Germany) equipped with an Nd:YAG laser (2 kHz, 355 nm).