Maxis hd uhr tof

Digested proteins were analysed in technical triplicates by LC-MS/MS using a Proxeon EASY-nLCII (Thermo Fisher Scientific, Milan, Italy) chromatographic system coupled to a Maxis HD UHR-TOF (Bruker Daltonics GmbH, Bremen, Germany) mass spectrometer. Peptides were loaded on the EASY-Column C18 trapping column (2 cm L., 100 µm I.D, 5 µm ps, Thermo Fisher Scientific), and subsequently separated on an Acclaim PepMap100 C18 (75 µm I.D., 25 cm L, 5 µm ps, Thermo Fisher Scientific) nano scale chromatographic column. The flow rate was set to 300 nL/min and the gradient was from 3 to 35% of B in 80 min followed by 35 to 45% in 10 min and from 45 to 90% in 11 min. Mobile phase A was 0.1% formic acid in H₂O and mobile phase B was 0.1% formic acid in acetonitrile. The mass spectrometer was equipped with a nanoESI spray source and operated in positive ion polarity and Auto MS/MS mode (Data Dependent Acquisition - DDA). Precursors in the range 350 to 2,200 m/z (excluding 1,220.0–1,224.5 m/z) with a preferred charge state +2 to +5 (excluding singly charged ions). After acquiring one MS/MS spectrum, the precursors were actively excluded from selection for 30 seconds. In-source reference lock mass (1,221.9906 m/z) was acquired online throughout the runs.

A typical protocol of filter-aided sample preparation (FASP) for tryptic digestion was carried out overnight at 37 °C using trypsin (Promega, Madison, WI, USA) both for whole tears and FACS-sorted EVs. Digested proteins from each sample were analyzed in triplicate by LC-MS/MS using a Proxeon EASY-nLCII (Thermo Fisher Scientific, Milan, Italy) chromatographic system coupled to a Maxis HD UHR-TOF (Bruker Daltonics GmbH, Bremen, Germany) mass spectrometer. Digested peptides were analyzed on the EASY-Column C18 trapping column (2 cm L., 100 µm I.D, 5 µm ps, Thermo Fisher Scientific, Milan, Italy), and subsequently on an Acclaim PepMap100 C18 (75 µm I.D., 25 cm L, 5 µm ps, Thermo Fisher Scientific, Milan, Italy) The flow rate was set to 0.300 µL/min with a total run time of 90 min. Chromatographic parameters and mass spectrometer setting conditions have been reported in our previous work [14 (link)].

Each digested protein sample was analyzed in technical duplicate by LC-MS/MS using a Proxeon EASY-nLCII (Thermo Fisher Scientific, Milan, Italy) chromatographic system coupled to a Maxis HD UHR-TOF (Bruker Daltonics GmbH, Bremen, Germany) mass spectrometer as previously reported [20 (link)].

The number of purified EVs, established by FACS, was used to normalize proteomics analyses. In detail, 1 × 10⁶ EVs separated by FACS were used for each proteomic detection as previously reported [53 (link),63 (link)]. EVs digested proteins were acquired in triplicate by LC-MS/MS using a Proxeon EASY-nLCII (Thermo Fisher Scientific, Milan, Italy) chromatographic system coupled to a Maxis HD UHR-TOF (Bruker Daltonics GmbH, Bremen, Germany) mass spectrometer. In-source reference lock mass (1221.9906 m/z) was acquired online throughout runs. Protein identification was carried out by the MASCOT search engine, assuming the carbamidomethylation and the methionine oxidation as fixed and variable modification, respectively. The Exponentially Modified Protein Abundance Index (emPAI) [29 (link)] obtainable by MASCOT results from MS/MS data was used for approximate relative abundance of proteins in the mixture. Gene Ontology classification of identified proteins was carried out by PANTHER, which classified 58 identified EVs proteins. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [64 (link)] partner repository with the dataset identifier PXD022807.

To analyze the effects of ponatinib on the specific expressional signatures in endothelial cells, shotgun proteomics analyses were performed, accordingly with methods already in place in our laboratory [18 (link),19 (link)]. HUVECs were treated with 1.7 nM of ponatinib or DMSO or 1.7 nM of ponatinib + anti-Notch-1 antibody for 17 h. At the end of treatments, samples were prepared according to the Filter Aided Sample Preparation (FASP) method. Briefly, cellular pellets were lysed by sonication in a lysis buffer (urea 6 M in 100 mM Tris/HCl, pH 7.5) and after centrifugation of cell debris, the supernatants were assayed for protein concentration through Bradford assay (Bio-Rad, Hercules, CA, USA) using Bovine Serum Albumin (BSA, Sigma-Aldrich, St. Louis, MI, USA) standard for the calibration curve. Next, 50 µg of proteins was digested for each treatment by using trypsin (Promega, Madison, WI, USA). For protein label free identification and quantification, tryptic peptides from each sample were analyzed in triplicate with LC-MS/MS using a Proxeon EASY-nLCII (Thermo Fisher Scientific, Milan, Italy) chromatographic system coupled to a Maxis HD UHR-TOF (Bruker Daltonics GmbH, Bremen, Germany) mass spectrometer as already described [20 (link)].

The gel pieces were trypsin digested and analyzed by LC-MS as previously described [46 (link)]. Each digested spot sample was analyzed by LC-MS/MS using a Proxeon EASY-nLCII (Thermo Fisher Scientific, Milan, Italy) chromatographic system coupled to a Maxis HD UHR-TOF (Bruker Daltonics GmbH, Bremen, Germany) mass spectrometer. The raw data were processed using PEAKS Studio v7.5 software (Bioinformatic Solutions Inc, Waterloo, ON, Canada) using the function “correct precursor only”. The mass lists were searched against the nextprot database including isoforms (version as of June 2017; 42,151 entries) using 10 ppm and 0.05 Da as the highest error tolerances for parent and fragment ions, respectively. Carbamidomethylation of cysteines was selected as fixed modification and oxidation of methionines and deamidation of asparagine and glutamine as variable modifications allowing two missed cleavages.