Ultraflex tof tof

Thiocillin detection was analyzed using HPLC-MS-MS (Ultraflex TOF-TOF, Bruker) of cells and supernatants of 48 h cultures of B. cereus in TyJ medium incubated at 28 °C without shaking. The biofilm was collected and thoroughly suspended in PBS and then centrifuged at 12,000 × g to separate cells from the supernatant. Culture medium was centrifuged to separate floating cells from the supernatant. Previous to analysis, the samples were purified with C8 ZipTip® (Merck) to discard salts. To perform MS-MS, a low molecular weight matrix was used.

The molecular weights of CM-Ch samples were determined by MALDI-TOF MS using 2,5-dihydroxybenzoic acid (DHB) as a sample matrix. The experiment was performed in an ultraflex TOF/TOF (Bruker Daltonics, Germany) in the positive reflection mode (400–6000 m/z).

Molecular masses of compounds were measured on an Ultraflex TOF-TOF (Bruker Daltonik GmbH) spectrometer by the staff of laboratory of proteomics, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry.

Three mg of total protein from cell lysates were immunoprecipitated with an antibody for RPTPβ/ζ and subjected to reduction with dithiothreitol and alkylation with iodoacetamide. After SDS-PAGE and silver staining of the proteins, bands were excised and treated for in-gel digestion as described⁵³ . Briefly, the silver was destained and trypsin (porcine, modified, sequence grade, Promega Corporation, Madison, WI, USA) was introduced to the dried gel pieces. After overnight tryptic digestion, peptides were bound to a C18 µZipTip and after washing, they were eluted with acetonitrile containing the matrix (alfa-cyano 4-hydroxycinnamic acid) directly onto the target plate. The mass list was generated by MALDI-TOF mass spectrometry on an Ultraflex TOF/TOF from Bruker Daltonics, Bremen, Germany. The search for identity was performed using the search engine ProFound (http://prowl.rockefeller.edu/prowl-cgi/ProFound). The spectrum was internally calibrated using autolytic tryptic peptides, and the error was set at +/− 0.02 Da. One missed cleavage was allowed, and methionine could be oxidized. The significance of the identity was judged from the search engines scoring system. The occurrence of the few missed cuts was either on a terminal basic residue or surrounded by acidic amino acid residues.

Peptide profiles were analyzed with an ultraflex TOF/TOF (Bruker Daltonics, Massachusetts,USA). The samples were desalinated and concentrated using ZIP-TIPTM (Millipore, Perkin Elmer, UK) and then mixed with a-cyano-4-hydroxycinnamic acid matrix solution according to the proportion of 1:1. The mixture was deposited in the AnchorChip (Bruker Daltonics, Massachusetts,USA) for future use. The reflex pattern was automatically controlled by the FlexControl software. The Mascot software (Matrix Science Ltd, London, UK) was used to analyze the PMF and LIFT MS peak. The BioTools software (Bruker Daltonics, Massachusetts,USA) was used to search and identify PMF and MS/MS data with a peptide mass tolerance of 100 ppm. Protein identifications were accepted when the observed and predicted isoelectric points and relative molecular weights were consistent, and scores indicated nonrandom identifications at a significance level of P < 0.05.

The mass spectra were acquired using a MALDI‐TOF mass spectrometer (Ultraflex TOF/TOF; Bruker Daltonik, Bremen, Germany). Ions were generated using a pulsed 337‐nm nitrogen laser and were accelerated to 23.5 kV. All spectra were obtained in the linear mode with a delayed extraction of 60 ns. For sample preparation, 0.5 μL of a matrix solution prepared by dissolving sodium 2,5‐dihydroxybenzoate (1 mg·mL⁻¹) and 2,5‐dihydroxybenzoic acid (19 mg·mL⁻¹) in 30% ethanol was spotted onto a target plate (MTP 384 target plate ground steel; Bruker Daltonik) and dried. Subsequently, an aliquot (0.5 μL) of the glycan solution was spotted onto the matrix crystal and dried.