Ultraflextreme mass spectrometer

Digestion of Cry6Aa with trypsin results in two fragment fractions, a large fragment and a small fraction containing two peptides. The small fragment sample was analyzed by matrix-assisted laser desorption/ionization - time of flight mass spectrometry (MALDI-TOF/TOF MS) to determine the peptide masses and was subsequently fragmented in the LIFT mode to confirm the sequence of the peptides. The MALDI-TOF MS and tandem mass spectrometry (MS/MS) were conducted with a Bruker UltraFlextreme mass spectrometer. The C-terminal peptide (CTP) sample was diluted with 0.2 % trifluoroacetic acid (TFA) at 1:1 (v/v) and desalted using a C-18 Ziptip (Millipore). The peptides were eluted with 60 % acetonitrile (ACN) in 0.1 % TFA and mixed with a 2,5-dihydroxybenzoic acid (DHB) matrix [15 mg/ml in ACN:H₂O (50:50)]. After spotting 1 μl of the mix on a MALDI sample plate, the peptide mass was analyzed using reflection-positive mode and the peptide was fragmented using the LIFT mode. The instrument was calibrated with CM 2 (calibration mixture 2, Peptide Mass Standards Kit, Applied Biosystems Sciex, P/N P2-3143-00). The mass spectrum was collected and analyzed using flex analysis software. The sequence was verified using BioTools software (Bruker) and the MASCOT search engine (Matrix Science).

The probiotic strain was isolated from sweet whey at pH > 6 (Dairy Cooperative in Drzycim, Poland) according to the previous protocol [26 (link)]. The molecular identification of bacterial strain was performed by MALDI-TOF-MS identification by the ultrafleXtreme mass spectrometer (Bruker Daltonics, Hamburg, Germany) using formic acid-acetonitrile extraction and BioTyper identification [26 (link)]. Finally, the isolated strains of L. paracasei LPC20 were deposited in the Polish Collection of Microorganisms (PCM) under deposit no. B/00287.

Matrix assisted laser desorption ionisation time of flight (MALDI-TOF) mass spectrometry was performed on a UltrafleXtreme mass spectrometer (Bruker (UK) Ltd, Coventry, UK) using a matrix of containing a saturated solution of α-Cyano-4-hydroxycinnamic acid in acetonitrile with 0.1% trifluoroacetic acid in a 1:1 ratio of sample in H₂O mixed and spotted onto a 384-well plate with a ground steel surface prior to ionization and detection (100% laser power/+ve ion mode).

Excised SDS-PAGE gel-bands of rLifA^C1480A were incubated in 50 mM ammonium bicarbonate containing porcine trypsin (Promega) in a trypsin:lymphostatin ratio of ~1:30, overnight at 32 °C. Peptides were identified by MALDI mass spectroscopy on an ultrafleXtreme™ mass spectrometer (Bruker) using an α-cyano-4-hydroxycinnamic acid matrix. A peptide mass map was generated from spectral data using Compass DataAnalysis 4.4 software (Bruker). Peptide masses were searched against the National Center for Biotechnology Information database of non-identical protein sequences (NCBInr) with the MASCOT search engine,⁵⁰ (link) mass tolerance of 10 ppm (Matrix Science). Peptide masses were also compared to the sequence of full-length LifA from EPEC E2348/69 (Protein Prospector software).12 (link), 13 (link), 51 The instrument was calibrated prior to each data acquisition and an internal calibration performed on the digest products of porcine trypsin.

Samples were analyzed with a MALDI-TOF mass spectrometer in positive ion mode. For MS analysis, the dried sample was resuspended in 10 μL of methanol/trichloromethane (1:1, v/v). A total of 0.5 μL of matrix solution (10 mg of 2,5-DHB dissolved in 1 mL of 30% ethanol) and 0.5 μL of the diluted analyte solution were spotted on the MALDI target plate (Bruker Daltonics). Then, the plate was analyzed by an ultrafleXtreme mass spectrometer (Bruker Corporation, Germany), which was controlled by flexControl 3.4 software (build 119) (Bruker Daltonics).