Ultraflex 3 maldi tof mass spectrometer

All mass spectrometry measurements were performed by using Ultraflex III MALDI-TOF mass spectrometer (Bruker, Bremen, Germany) equipped with a UV Nd:YAG laser (355 nm) and LIFT MS/MS unit. The molecular weights of the recombinant Lc-def and digestion products were determined in a linear and reflector mode.

Protein membrane fractions were extracted by trifluoroethanol/chloroform mixture as described previously [38 (link)]. Resulting aqueous and insoluble fractions were solubilized in lysis buffer (7 M urea, 2 M thiourea, 2 % Triton X-100, 0,5 % amido sulfobetaine-14 (ASB14), 1 % Ampholytes 3–10, 50 mM DTT) and purified with 2-D Clean-Up Kit (GE Healthcare). The protein concentration was determined using 2-D Quant kit (GE Healthcare). 2D SDS-PAGE was performed on IPG strips pH 4–7 NL (Amersham Biosciences) as described previously [23 (link)]. Gels were either stained with colloidal Coomassie Brilliant Blue G-250 (CBB G-250) or electroblotted. The protein spots selected for mass spectrometric analysis were destained using 50 mM 4-ethylmorpholine acetate (pH 8.1) in 50 % acetonitrile (MeCN) and in-gel digested overnight with trypsin (100 ng; Promega) in a cleavage buffer containing 25 mM 4-ethylmorpholine acetate. The resulting peptides were extracted to 40 % MeCN/0.1 % TFA and measured on an Ultraflex III MALDI-TOF mass spectrometer (BrukerDaltonics) in a mass range of 700–4000 Da. For protein identification the peptide mass spectra were searched against SwissProt or NCBI (National Center for Biotechnology Information) bacterial database using an in-house Mascot search engine. The identity of protein candidates was confirmed using MS/MS analysis.

Formation of disulphide bond between cysteine residues 22/44 within HMGB1 domain A was determined by MALDI-TOF mass-spectrometry. Briefly, proteins were subjected to trypsin digestion. The digests (1:l) were mixed with CHCA (α-cyano-4-hydroxycinnamic acid) matrix solution on the Anchor Chip target in a 2:1 ratio. The digests were analyzed by using an Ultraflex III MALDI-TOF mass spectrometer (Bruker Daltonik, Bremen, Germany). An external calibration procedure was employed, using a mixture of seven peptide standards (Bruker Daltonik). Peptide maps were acquired in reflectron positive mode (25 kV acceleration voltage) with 800 laser shots and peaks with 0.70–4.1 kDa mass range and minimum S/N 10 were picked out for MS/MS analysis employing LID-LIFT arrangement with 600 laser shots for each peptide. The Flex Analysis 3.4 and MS Biotools 3.2 (Bruker Daltonik) software were used for data processing. MASCOT 2.4 (MatrixScience, London, UK) search engine was used for evaluation of the MS and MS/MS data. Database searches were done against the NCBI protein database. A mass tolerance of 100 ppm was allowed during processing MALDI MS data for PMF and 0.6 Da during processing LID-LIFT data for MS/MS ion searches. The MS/MS data of peptides containing disulphide bridges were interpreted manually.

All reagents were purchased from Sigma Aldrich, Thermo Fisher, Alfa Aesar, Apollo Scientific, or Fluorochem and used without further purification. Restriction enzymes, polymerases, and ligases were purchased from New England Biolabs. MALDI-TOF MS was carried out using ground steel target plates on a Bruker ultraFlex III MALDI-TOF mass spectrometer. NMR spectra were obtained using a Bruker 400 MHz NMR spectrometer (Bruker AV3400HD). ESI-MS data were obtained on a Bruker MicroTOF spectrometer.

All of the protein spots generated by 2-DE were analyzed by MALDI-TOF/TOF MS. The protein spots were carefully excised from the gel, destained using destaining solution (50% acetonitrile, 25 mM acid ammonium carbonate), and then digested for 13 h using sequencing-grade modified trypsin (Roche). Peptides from the digested proteins were used for MALDI-TOF/TOF analysis. MALDI-TOF MS was performed using an Ultraflex^III MALDI-TOF mass spectrometer (Bruker Daltonics) operating in reflectron mode with 20 kV accelerating voltage and 23 kV reflecting voltage. A saturated solution of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 0.1% trifluoroacetic acid was used as the matrix. A 1-μL volume of a mixture of the matrix and sample solutions at a 1:1 ratio was applied to the Score384 target well. The SNAP algorithm (S/N threshold: 5; Quality Factor Threshold: 30) in FlexAnalysis v.2.4 was used to select the 150 most prominent peaks in the mass range m/z 700–4,000. The subsequent MS/MS analysis was performed in a data-dependent manner, and the 10 most abundant ions were subjected to high energy collision-induced dissociation analysis. The collision energy was set to 1 keV, and nitrogen was used as the collision gas.

Twenty picomoles of native CP (nCP), wtCP and mutCP were dissolved in 50% v/v acetonitrile containing 0.05% v/v trifluoroacetic acid, diluted 1:1 in a saturated sinapinic acid matrix, applied on the anchor-chip target plate and allowed to dry. Samples were analyzed with a Ultraflex III MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) by using the Flex Control 3.0 data acquisition software. Spectra were obtained in LINEAR mode over the m/z range 1500–20000 for a total of 500 shots. The instrumental parameters were chosen by setting the ion source 1 at 25 kV, ion source 2 at 21.5 kV, the pulsed ion extraction at 20 ns. The instrument was externally calibrated prior to analysis using the Bruker Peptide Calibration standard kit.