Xcalibur v2

Manufactured by Thermo Fisher Scientific

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Xcalibur v2.2 is a software platform developed by Thermo Fisher Scientific for data acquisition, analysis, and management in mass spectrometry applications. It provides a comprehensive suite of tools for instrument control, data processing, and reporting.

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29 protocols using xcalibur v2

Metabolite Identification in Fungal Interactions

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The identification of metabolites was performed following a biologically-driven approach performing searches against the targeted in-house species-specific metabolic databases for Rhizoctonia and Stachybotrys. The libraries were constructed acquiring information from the literature and publicly available databases such as, KNApSAcK (http://kanaya.naist.jp/KNApSAcK/) and PubChem (http://pubchem.ncbi.nlm.nih.gov/). Identification of metabolites was based on mass accuracy (<2 ppm) and where available, on isotope and/or MS/MS fragmentation patterns (Supplementary Data Sets 1–4) using data from the databases of METLIN (http://metlin.scripps.edu/index.php) and mzCloud (https://www.mzcloud.org/) and the literature. In addition, the heuristic rules of Kind and Fiehn (2007 (link)), which are implemented in the MZmine 2 (Pluskal et al., 2010 (link)), were applied. These rules provide a valuable tool for reducing the number of candidate molecular formulae for a given ion. Detection of mass errors was confirmed by Xcalibur v.2.2 (Thermo Scientific).
Additionally, since the majority of the secondary metabolites have unique structures, the assignment of metabolites to the corresponding producing fungus during mycoparasitism was a feasible task at the applied mass resolution.

Chamoun R., Aliferis K.A, & Jabaji S. (2015). Identification of signatory secondary metabolites during mycoparasitism of Rhizoctonia solani by Stachybotrys elegans. Frontiers in Microbiology, 6, 353.

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UHPLC-MS/MS Untargeted Metabolomics

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Liquid chromatography was performed using a UHPLC system (Vanquish, Thermo Fisher Scientific) with a UPLC BEH Amide column (2.1 mm × 100 mm, 1.7 μm), coupled to a Q Exactive HFX mass spectrometer (Orbitrap, Thermo Fisher Scientific Inc., MA, USA). The mobile phase A was 25 mmol/L ammonium acetate and 25 ammonia hydroxide in water (pH 9.75), and the mobile phase B was acetonitrile. The auto-sampler temperature was 4 ^oC, and the sample injection volume was 4 µL. The QE HFX mass spectrometer was used to acquire MS/MS spectra with an information-dependent acquisition (IDA) mode using acquisition software (Xcalibur V2.2, Thermo Fisher Scientific Inc., MA, USA). In this mode, the acquisition software continuously evaluates the full scan MS spectrum. The ESI source conditions were set as follows: sheath gas flow rate as 25 Arb, Aux gas flow rate as 20 Arb, capillary temperature 350 ^oC, full MS resolution as 60,000, MS/MS resolution as 7,500, collision energy as 10/30/60 in NCE mode, spray Voltage as 3.6 kV (positive) or -3.2 kV (negative), respectively.

Yang Q., Luo J., Xu H., Huang L., Zhu X., Li H., Yang R., Peng B., Sun D., Zhu Q, & Liu F. (2023). Metabolomic investigation of urinary extracellular vesicles for early detection and screening of lung cancer. Journal of Nanobiotechnology, 21, 153.

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Peptide Separation and Identification

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Purified peptides were resuspend in buffer A* and separated on an EASY-nLC1000 system coupled to an LTQ-Orbitrap Velos (Thermo Scientific). Briefly, samples were loaded directly onto an in-house-packed 30-cm, 75-µm-inner-diameter, 360-µm-outer-diameter Reprosil-Pur C₁₈ AQ 3 µm column (Dr. Maisch, Ammerbuch-Entringen, Germany). Reverse-phase analytical separation was performed at 350 nl/min over a 180-min gradient by altering the buffer B concentration from 0 to 32% in 150 min, from 32 to 40% in the next 5 min, increasing it to 100% in 2.5 min, holding it at 100% for 2.5 min, and then dropping it to 0% for another 20 min. The LTQ-Orbitrap Velos was operated with Xcalibur v2.2 (Thermo Scientific) at a capillary temperature of 275°C with data-dependent acquisition and switching between collision-induced dissociation (CID) MS/MS (normalized collision energy [NCE], 35%; activation Q, 0.25; activation time, 10 ms; automated gain control [AGC] at 4 × 10⁴) and higher-energy collisional dissociation (HCD) MS/MS (resolution, 7,500; NCE, 45%; AGC at 2e5; maximum fill time, 200 ms).

Weber B.S., Hennon S.W., Wright M.S., Scott N.E., de Berardinis V., Foster L.J., Ayala J.A., Adams M.D, & Feldman M.F. (2016). Genetic Dissection of the Type VI Secretion System in Acinetobacter and Identification of a Novel Peptidoglycan Hydrolase, TagX, Required for Its Biogenesis. mBio, 7(5), e01253-16.

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Shotgun Proteomic Analysis by LC-MS/MS

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Samples were separated on Dionex Acclaim PepMap 100 75 uM × 2 cm nanoViper (C18, 3 µm, 100 Å) trap and Dionex Acclaim PepMap RSLC 50 uM × 15 cm nanoViper (C18, 2 µm, 100 Å) separating columns (Sunnyvale, CA). An EASY n-LC (Thermo Fisher Scientific, Waltham, MA) ultra-high-performance liquid chromatography system was used with buffer A = 2% (v/v) acetonitrile/0.1% (v/v) formic acid and buffer B = 80% (v/v) acetonitrile/0.1% (v/v) formic acid as mobile phases. A Nanospray Flex source (Thermo Fisher Scientific) was used to position the end of the emitter near the ion transfer capillary of the mass spectrometer. An Orbitrap Elite–ETD mass spectrometer (Thermo Fisher Scientific) was used to collect data from the LC eluate. An Nth Order Double Play with ETD Decision Tree method was created in Xcalibur v2.2 (Thermo Fisher Scientific; see the Supporting Materials for more details).

Massey V.L., Dolin C.E., Poole L.G., Hudson S.V., Siow D.L., Brock G.N., Merchant M.L., Wilkey D.W, & Arteel G.E. (2016). The Hepatic “Matrisome” Responds Dynamically to Injury: Characterization of Transitional Changes to the Extracellular Matrix in Mice. Hepatology (Baltimore, Md.), 65(3), 969-982.

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Top-Down Mass Spectrometry of Neutrophil Myeloperoxidase

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Top-down/native MS was performed of (i) the intact α-chain of nMPO and (ii) intact nMPO. For (i) nMPO was reduced, desalted and injected on a C4 LC column connected to an Agilent 6538 quadrupole-time-of-flight mass spectrometer operating in high-resolution positive polarity mode. Mass spectra were deconvoluted using MassHunter vB.06 (Agilent Technologies). Assignments were guided by the LC-MS/MS peptide data, see Table S3A and ii) intact nMPO was infused into a modified Q-Exactive (Thermo Scientific) operating in positive ion polarity via nano-ESI using custom-made gold-coated capillaries (75 (link)). Data were processed with Xcalibur v2.2 (Thermo Scientific), spectra deconvoluted with UniDec (76 (link)) and annotated using in-house software.
Intact nMPO was analysed using single-molecule mass photometry as described (44 (link)). Coverslips were assembled for sample delivery using silicone CultureWell gaskets (Grace Bio-Labs). Data were acquired, processed and analysed using in-house software (43 (link)), see Extended experimental procedures for details.

Tjondro H.C., Ugonotti J., Kawahara R., Chatterjee S., Loke I., Chen S., Soltermann F., Hinneburg H., Parker B.L., Venkatakrishnan V., Dieckmann R., Grant O.C., Bylund J., Rodger A., Woods R.J., Karlsson-Bengtsson A., Struwe W.B, & Thaysen-Andersen M. (2020). Hyper-truncated Asn355- and Asn391-glycans modulate the activity of neutrophil granule myeloperoxidase. The Journal of Biological Chemistry, 296, 100144.

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MALDI-TOF and FT-MS Analysis of Peptides

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For MALDI-MS, peptide samples were desalted by C18 ZipTip according to manufacturer instructions and analyzed using a Bruker Daltonics UltrafleXtreme MALDI-TOF/TOF instrument operating in reflector/positive mode with α-cyano-4-hydroxycinnamic acid as the matrix.
For high-resolution mass spectrometry and MS/MS, desalted peptides were resuspended in 80% (v/v) MeCN, 1% AcOH and centrifuged at 11000 x g for 5 min prior to analysis by direct infusion Fourier transform mass spectrometry (FT-MS). An Advion NanoMate 100 was used to directly infuse samples to an LTQ-FTMS/MS (ThermoFisher) operating at 11 T. The MS was calibrated weekly, following the manufacturer’s instructions, and tuned daily using Pierce LTQ Velos ESI Positive Ion Calibration Solution (ThermoFisher). Spectra were collected with a resolution of 100,000. Ions were selected for ion trap fragmentation or FT-MS/MS fragmentation based on signal intensity, and spectra were collected using the following parameters: isolation width of 5 m/z, normalized collision energy of 35, activation q value 0.4, activation time 30 ms. Data analysis was performed using the Qualbrowser application within ThermoFisher Xcalibur v 2.2.

Deane C.D., Burkhart B.J., Blair P.M., Tietz J.I., Lin A, & Mitchell D.A. (2016). In vitro biosynthesis and substrate tolerance of the plantazolicin family of natural products. ACS chemical biology, 11(8), 2232-2243.

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Proteomic Analysis of Maize Roots

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Initially, the RAW files were viewed in Xcalibur v.2.2 (Thermo Scientific) and a subsequent data search was conducted of the Zea mays database downloaded from Uniprot in September 2015, using PatternLab (v.4.0.0.5) for the proteomics computational environment^{62 (link)} and the Comet algorithm (v.20144.011)⁶³. Parameters used were as follows: fully tryptic peptides; up to two missed cleavages; carbamidomethylation as fixed modification; oxidation of methionine as a variable modification; and a peptide precursor tolerance of 40 ppm. The Search Engine Processor^{64 (link)} was used for post-processing of the peptide spectrum matches to achieve a protein list with less than 1% false discovery range (FDR). Proteins identified in at least two replicate samples were considered for further analysis. The PatternLab TFold module was used to identify differentially expressed proteins in the two treatments⁶³. The TFold module uses a p-value-dependent fold-change cutoff to maximize the number of identifications that satisfy a theoretical FDR estimator (in this case, the Benjamini-Hochberg). Fold-changes were calculated as the average spectral counts obtained in the reference control roots (CR) divided by those obtained in humic acid roots (HAR).

Nunes R.O., Domiciano G.A., Alves W.S., Melo A.C., Nogueira F.C., Canellas L.P., Olivares F.L., Zingali R.B, & Soares M.R. (2019). Evaluation of the effects of humic acids on maize root architecture by label-free proteomics analysis. Scientific Reports, 9, 12019.

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Orbitrap Elite ETD Mass Spectrometry Acquisition

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An Orbitrap Elite ETD mass spectrometer (Thermo Fisher Scientific) was used to collect data. An Nth Order Double Play method was created in Xcalibur v2.2 (Thermo Fisher Scientific). Scan event one of the method obtained an FTMS MS1 scan (normal mass range; 60,000 resolution, full scan type, positive polarity, centroid data type) for the range 300–2000 m/z. Scan event two obtained FTMS HCD MS2 scans (normal mass range; 60,000 resolution; centroid data type) on up to 10 peaks that had a minimum signal threshold of 5000. The lock mass option was enabled (0% lock mass abundance) using the 371.101236 m/z polysiloxane peak as an internal calibrant.

Schneider G., Kaliappan A., Nguyen T.Q., Buscaglia R., Brock G.N., Hall M.B., DeSpirito C., Wilkey D.W., Merchant M.L., Klein J.B., Wiese T.A., Rivas-Perez H.L., Kloecker G.H, & Garbett N.C. (2021). The Utility of Differential Scanning Calorimetry Curves of Blood Plasma for Diagnosis, Subtype Differentiation and Predicted Survival in Lung Cancer. Cancers, 13(21), 5326.

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Negative Ion Mass Spectrometry Protocol

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Mass spectrometry was performed with a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, IQLAAEGAAPFALGMAZR). Samples dissolved in 0.1% formic acid were infused with a syringe at a flow rate of 5 µl/min. A mass spectrometer equipped with a microspray ESI source was operated in full mass-spectral-acquisition mode for MS scan data in a mass range of m/z 100–500. A resolving power setting of 70,000 was used in negative ion mode. MS/MS scan data were also acquired in negative ion scan mode; a normalized collision energy (NCE) of 35% was applied to a precursor ion selected, m/z 321.05. The MS and MS/MS scan AGC target value was set at 1e⁶. All data acquisition and analysis were carried out with XCalibur v.2.2 software (Thermo Fisher Scientific).

Park S.H., Park J., Lee S.J., Yang W.S., Park S., Kim K., Park Z.Y, & Song H.K. (2019). A host dTMP-bound structure of T4 phage dCMP hydroxymethylase mutant using an X-ray free electron laser. Scientific Reports, 9, 16316.

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UbK1 Phosphorylation Analysis by MS

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Phosphorylation of UbK1 was determined by mass spectrometry as previously described (Liu et al., 2017 (link)). In brief, samples were reduced, alkylated, and digested with endoproteinase AspN (New England Biolabs). Free carboxyl were methyl-esterified prior to phosphopeptide enrichment using a High-Select TiO₂ Phosphopeptide Enrichment Kit (ThermoFisher). Peptides were separated by reversed phase chromatography and analyzed by mass spectrometry using an Orbitrap Elite – ETD mass spectrometer (ThermoFisher) with an Nth Order Double Play created in Xcalibur v2.2 (ThermoFisher). RAW data files were searched in Peaks Studio X (Bioinformatics Solutions Inc., Waterloo, ON) using the Denovo, PeaksDB, PeaksPTM, and Label Free Q algorithms, and a protein database containing only the UbK protein. A peptide.csv file was exported from the Label Free Q result for curation in Microsoft Excel. Peptides were accepted if the Best Peptide Score ≥ 20, all PTM Ascores ≥ 50, and the Avg. ppm < 5.

Perpich J.D., Yakoumatos L., Johns P., Stocke K.S., Fitzsimonds Z.R., Wilkey D.W., Merchant M.L., Miller D.P, & Lamont R.J. (2021). Identification and Characterization of a UbK Family Kinase in Porphyromonas gingivalis which Phosphorylates the RprY Response Regulator. Molecular oral microbiology, 36(5), 258-266.

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