Xcalibur 2

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany, Italy, France, United Kingdom

The Xcalibur 2.2 is a mass spectrometry data system that provides instrument control, data acquisition, and data analysis capabilities. It is a software package designed to support a variety of mass spectrometry instruments.

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Xcalibur 2.3 software program (2 citations) Xcalibur software 2.2 SP1.48 (3 citations) Xcalibur 2.2 SP1.48 software (26 citations) Thermo Scientific Xcalibur 2.1 (2 citations) Xcalibur 2.1 workstation (4 citations) XCalibur QuanBrowser 2.2 (9 citations) Xcalibur software ver. 2.0.7 (2 citations) Xcalibur 2.2 operation software (2 citations) XCalibur 2.1.0 software package (2 citations) Thermo Xcalibur v.2.2 (3 citations)

375 protocols using xcalibur 2

Peptide Separation and Data-Dependent MS/MS

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Protein expression was analysed by nano LC-MS/MS using a Surveyor HPLC system in line with a LTQ-Orbitrap XL controlled using XCalibur 2.0 software (Thermo Fisher Scientific, Waltham, MA, USA). Protocols were based on previously described conditions for peptide separation and data-dependent MS/MS [28 (link)]. The LTQ-Orbitrap XL was controlled using XCalibur 2.0 (Thermo Fisher Scientific, MA, USA) and operated in data-dependent acquisition mode where survey scans (m/z 460–2000) were acquired in the Orbitrap at a resolving power of 60,000. MS/MS spectra were concurrently acquired in the LTQ mass analyser on the eight most intense ions from the FT survey scan. Unassigned and singly charged precursor ions were not selected for fragmentation and 30-s dynamic exclusion (repeat count 1 exclusion list size 500) was used. Fragmentation conditions in the LTQ were: 35 % normalized collision energy, activation q of 0.25, 30 ms activation time and minimum ion selection intensity of 3000 counts.

Chen L., Wilson R., Bennett E, & Zosky G.R. (2016). Identification of vitamin D sensitive pathways during lung development. Respiratory Research, 17, 47.

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ESI-MS Analysis of ODN2-PtI2(DACH) and Protein Complexes

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ESI-MS spectrum of the ODN2-PtI2(DACH) mixture was recorded by direct injection at 5 μl/min flow rate in an Orbitrap high-resolution mass spectrometer (Thermo, San Jose, CA, USA), equipped with a conventional ESI source. The working conditions were as follows: negative polarity, spray voltage -2.7 kV, capillary voltage -20 V, capillary temperature 280° C, tube lens voltage -113 V. The sheath and the auxiliary gases were set at 23 and 4 (arbitrary units), respectively. For acquisition, Xcalibur 2.0. software (Thermo) was used and deconvoluted masses were obtained by using the ProMass 2.8 rev. 2 tool for Xcalibur (Novatia). ESI-MS spectra of the PtI2(DACH)-protein mixtures were recorded by direct injection at 3 μl/min flow rate in the same instrumentation. The working conditions were as follows: positive polarity, spray voltage 3.1 kV, capillary voltage 45 V, capillary temperature 220° C, tube lens voltage 230 V. The sheath and the auxiliary gases were set at 17 and 1 (arbitrary units), respectively. For acquisition, Xcalibur 2.0. software (Thermo) was used and deconvoluted masses were obtained by using the integrated Xtract tool. For spectra acquisition a nominal resolution (at m/z 400) of 100,000 was used.

Cirri D., Pillozzi S., Gabbiani C., Tricomi J., Bartoli G., Stefanini M., Michelucci E., Arcangeli A., Messori L, & Marzo T. (2017). PtI₂(DACH), the iodido analogue of oxaliplatin as a candidate for colorectal cancer treatment: chemical and biological features. Dalton transactions (Cambridge, England : 2003), 46(10).

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Quantitative Proteomics of Protein Modifications

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We used stable isotope labeling with amino acids in cell culture (SILAC; Ong et al, 2002), to measure changes in protein, lysine acetylation, and phosphorylation abundance. Peptide fractions were analyzed by online nanoflow LC‐MS/MS using a Proxeon easy nLC system (ThermoFisher Scientific) connected to an LTQ Orbitrap Velos (ThermoFisher Scientific) or Q‐Exactive (ThermoFisher Scientific) mass spectrometer. The LTQ Orbitrap Velos instrument was operated under Xcalibur 2.1 (ThermoFisher Scientific) with the LTQ Orbitrap Tune Plus Developers Kit version 2.6.0.1042 software in the data dependent mode to automatically switch between MS and MS/MS acquisition as described (Weinert et al, 2011). The Q‐Exactive was operated using Xcalibur 2.2 (ThermoFisher Scientific) in the data dependent mode to automatically switch between MS and MS/MS acquisition as described (Michalski et al, 2011; Kelstrup et al, 2012). All quantitative MS experiments performed in this study are summarized in Supplementary Table S1. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository (Vizcaino et al, 2013) with the dataset identifier PXD000507.

Weinert B.T., Iesmantavicius V., Moustafa T., Schölz C., Wagner S.A., Magnes C., Zechner R, & Choudhary C. (2014). Acetylation dynamics and stoichiometry in Saccharomyces cerevisiae. Molecular Systems Biology, 10(1), 716.

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Mass Spectrometry-Based Compound Identification

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Identification was based on the following criteria: Retention time window (±0.2 min); exact mass of monoisotopic ion M+0 (mass accuracy _m, defined as (exact mass)–(accurate mass)/(accurate mass) × 106, less than 2 ppm); comparison of experimental and calculated isotopic patterns (Relative Isotopic Abundances RIA (M+1/M+0) and/or RIA (M+2/M+0) errors less than 15%).
Xcalibur 2.1 software (Thermo Fisher Scientific Inc., San Jose, CA, USA) was used to analyse and process all data for quantitative analysis [43 (link)].

Rubini S., Rossi S.S., Mestria S., Odoardi S., Chendi S., Poli A., Merialdi G., Andreoli G., Frisoni P., Gaudio R.M., Baldisserotto A., Buso P., Manfredini S., Govoni G., Barbieri S., Centelleghe C., Corazzola G., Mazzariol S, & Locatelli C.A. (2019). A Probable Fatal Case of Oleander (Nerium oleander) Poisoning on a Cattle Farm: A New Method of Detection and Quantification of the Oleandrin Toxin in Rumen. Toxins, 11(8), 442.

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Metabolite Identification Using Mass Spectrometry

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All MS data, including the retention times, m/z, and ion intensities, were extracted by SIEVE software (Thermo Fisher Scientific, Waltham, MA) and incorporated into the instrument, and the resulting MS data were assembled into a matrix. The SIEVE parameters were set as follows: m/z range 50–1,000; m/z width 0.02; retention time width 2.5; and m/z tolerance 0.005. Metabolites were searched within the following databases: ChemSpider (www.chemspider.com), Human Metabolome (www.hmdb.ca), Lipid MAPS (www.lipidmaps.org), KEGG (www.genome.jp/kegg), and MassBank (www.massbank.jp). To confirm the putative metabolites, MS/MS was performed. The MS/MS spectra were exported from the Xcalibur 2.1 software (Thermo Fisher Scientific, Waltham, MA) to the MS frontier software (Thermo Fisher Scientific, Waltham, MA) and then compared with references in the MS frontier software database or Human Metabolome, Lipid MAPS, MassBank MS/MS spectra databases.

Lee S.Y., Kim M., Jung S., Lee S.H, & Lee J.H. (2015). Altered Plasma Lysophosphatidylcholines and Amides in Non-Obese and Non-Diabetic Subjects with Borderline-To-Moderate Hypertriglyceridemia: A Case-Control Study. PLoS ONE, 10(4), e0123306.

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Proteomic Analysis of Pulled-down Proteins

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To prepare the eluted samples from the pull‐down assays for MS, the samples were subjected to SDS‐PAGE until the dye‐front had moved 1.5 cm into the gel. The gel was stained with Coomassie blue, destained, and a single gel slice encompassing proteins below 350 kDa was excised. The slice was subjected to in‐gel tryptic digestion with a ProGest automated digestion unit (Digilab UK). The resulting peptides were fractionated with a Dionex Ultimate 3000 nanoHPLC system in line with an LTQ‐Orbitrap Velos mass spectrometer (Thermo Scientific) controlled by Xcalibur 2.1 software (Thermo Scientific). The Orbitrap was set to analyse the survey scans at 60,000 resolution (at m/z 400) in the mass range m/z 300 to 2,000 and the top twenty multiply‐charged ions in each duty cycle were selected for MS/MS in the LTQ linear ion trap.

Haworth J.A., Jenkinson H.F., Petersen H.J., Back C.R., Brittan J.L., Kerrigan S.W, & Nobbs A.H. (2016). Concerted functions of Streptococcus gordonii surface proteins PadA and Hsa mediate activation of human platelets and interactions with extracellular matrix. Cellular Microbiology, 19(1), e12667.

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GA3 Extraction and Quantification

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GA₃ extraction was performed as described previously with minor modification^{31 (link)}. Initially, freeze-dried materials were extracted with 80% (v/v) methanol containing the internal standard of [²H₂]-GA₃. The aqueous phase was extracted and then purified on C₁₈ Sep-Pak and MCX SPE columns (Qasis; Waters, USA). The eluant was dried, re-dissolved in HPLC initial solution, and then detected with a liquid chromatography-mass spectrometry system (Thermo, USA). Tandem mass spectrometry data were analyzed using the Xcalibur 2.1 software (Thermo, USA).

Wang H., Jin Y., Wang C., Li B., Jiang C., Sun Z., Zhang Z., Kong F, & Zhang H. (2017). Fasciclin-like arabinogalactan proteins, PtFLAs, play important roles in GA-mediated tension wood formation in Populus. Scientific Reports, 7, 6182.

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Peptide Separation and Analysis

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The peptide samples were chromatographically separated on an EASY-nLC II system (Thermo Fisher Scientific Inc., Bremen, Germany) with two columns set up: a trap column C18-A1, 2 cm (SC001, Thermo Fisher Scientific, Waltham, MA, USA) and an analytical column PepMap C18, 15 cm × 75 µm, 3 µm particles, and 100 Å pore size (ES800, Thermo Fisher Scientific, Waltham, MA, USA). Samples (4–8 μL) were analyzed with an LTQ Orbitrap XL hybrid mass spectrometer (Thermo Fisher Scientific Inc., Bremen, Germany). MS data were acquired in the data-dependent MS² mode. Data acquisition was controlled by XCalibur 2.1 software (Thermo Fisher Scientific Inc., Bremen, Germany).

Prodić I., Smiljanić K., Simović A., Radosavljević J, & Ćirković Veličković T. (2019). Thermal Processing of Peanut Grains Impairs Their Mimicked Gastrointestinal Digestion While Downstream Defatting Treatments Affect Digestomic Profiles. Foods, 8(10), 463.

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Kinetic Study of B-CeP1 Reactive Species

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The formation of B-CeP 1 reactive and hydrolyzed species upon incubation at 37°C in 1× BPE buffer (pH 7.4) was followed in time by ESI-MS. A fresh solution of test compound was prepared at the concentration of 80 μM and incubated at 37°C. Samples were taken after 5 min, 15 min, 30 min, 1 h, 2 h and 24 h, diluted 1:10 with methanol and analyzed on a Thermo Fisher Scientific (West Palm Beach, CA, USA) LTQ-Orbitrap Velos mass spectrometer. The analyses were performed in nanoflow mode by using quartz emitters produced in house by using a Sutter Instruments Co. (Novato, CA, USA) P2000 laser pipette puller. Up to 5 μl samples were typically loaded onto each emitter by using a gel-loader pipette tip. A stainless steel wire was inserted in the back-end of the emitter to supply an ionizing voltage around 1 kV. Typical source temperature was 200°C, whereas desolvation voltage was in the range of 40–50 V. Data were processed by using Xcalibur 2.1 software (Thermo Scientific). We calculated the percentage of each species over total compound in each sample to obtain relative percentages of B-CeP 1 reacted species, whose chemical structures are schematized in panel B of Supplementary Figure S1.

Sosic A., Göttlich R., Fabris D, & Gatto B. (2021). B-CePs as cross-linking probes for the investigation of RNA higher-order structure. Nucleic Acids Research, 49(12), 6660-6672.

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Quantification of Kidney Fatty Acid Metabolites

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Fatty acid metabolites in kidneys were measured, as described previously, with a slight modification [22 (link)–24 (link)]. High-performance liquid chromatography (HPLC) was combined with ESI–MS using a TSQ quantum mass spectrometer (Thermo Fisher Scientific K.K., Tokyo, Japan). HPLC was performed using a Luna 3u C18(2) 100Å LC column (100 × 2.0 mm, Phenomenex, Torrance, CA, USA) at 30°C. Samples were eluted in a mobile phase comprising acetonitrile–methanol (4:1, v/v) and water–acetic acid (100:0.1, v/v) in a 27:73 ratio for 5 min, ramped up to a 70:30 ratio after 15 min, to a 80:20 ratio after 25 min, held for 8 min, ramped up to 100:0 ratio after 35 min, and held for 10 min with flow rate of 0.1 mL/min. MS–MS analyses were conducted in negative ion mode, and fatty acid metabolites were detected and quantified by selected reaction monitoring (SRM). Conditions for the detection of each compound by SRM are listed (S1 Table). Peaks were selected and their areas were calculated using the Xcalibur 2.1 software (Thermo Fisher Scientific K.K.).

Katakura M., Hashimoto M., Inoue T., Mamun A.A., Tanabe Y., Arita M, & Shido O. (2015). Chronic Arachidonic Acid Administration Decreases Docosahexaenoic Acid- and Eicosapentaenoic Acid-Derived Metabolites in Kidneys of Aged Rats. PLoS ONE, 10(10), e0140884.

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