Proteolytic peptide analysis was performed using Orbitrap Elite Hybrid Mass Spectrometer (Thermo Electron, San Jose, CA) equipped with a Waters nanoAcquity UPLC system (Waters, Taunton, MA). A full scan at 120,000 resolution was obtained in the Oribtrap for eluted peptides in the range of 300–1800 Da followed by MS/MS scans on the twenty most abundant precursor ions by collision-induced dissociation at normalized collision energy of 35%. Raw LC-MS/MS data were searched through Mascot search engine (version 2.2.0, Matrix Science) against PEPT1 protein primary sequence with Met oxidation, Cys carbamidomethylation and NAP modification as variable modifications. The mass tolerance was set as 10 ppm for precursor ion and 0.8 Da for product ion. The significance threshold was P < 0.05.
Orbitrap elite hybrid mass spectrometer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Orbitrap Elite hybrid mass spectrometer is a high-performance analytical instrument designed for advanced mass spectrometry applications. It combines the benefits of a linear ion trap and an Orbitrap mass analyzer, providing high-resolution, accurate mass measurements, and excellent sensitivity. The core function of the Orbitrap Elite is to perform precise mass analysis and identification of complex molecular samples.
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Lab products found in correlation
Nanoacquity uplc system, by Waters Corporation (2 mentions)
Dionex lc, by Thermo Fisher Scientific (2 mentions)
Mascot search engine, by Matrix Science (1 mentions)
Nanoacquity system, by Waters Corporation (1 mentions)
Picoview nanospray interface, by New Objective (1 mentions)
Beh c18 column, by Waters Corporation (1 mentions)
Mascot demon, by Matrix Science (1 mentions)
Nanoelectrospray ion source, by Thermo Fisher Scientific (1 mentions)
Easy 2 nano uplc, by Thermo Fisher Scientific (1 mentions)
Xcalibur 2, by Thermo Fisher Scientific (1 mentions)
Reprosil pur c18 aq 3 μm resin, by Dr. Maisch (1 mentions)
M2 affinity gel, by Merck Group (1 mentions)
Ultraflextreme maldi tof mass spectrometer, by Bruker (1 mentions)
Silver staining kit, by Beyotime (1 mentions)
Easy nlc 1000 system, by Thermo Fisher Scientific (1 mentions)
Disuccinimidyl suberate, by Thermo Fisher Scientific (1 mentions)
Protein g mag sepharose, by GE Healthcare (1 mentions)
Sep pak c18, by Waters Corporation (1 mentions)
Ultimate 3000 rapid separation lc, by Thermo Fisher Scientific (1 mentions)
Reprosil pur beads, by New Objective (1 mentions)
15 protocols using orbitrap elite hybrid mass spectrometer
1
Proteolytic Peptide Analysis by Orbitrap LC-MS/MS
2
Nano LC-MS/MS Analysis of Tryptic Peptides
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Tryptic peptide extracts were subjected to nano LC-ESI-MS/MS analysis using a nanoAcquity system (Waters, Milford, MA, USA). The unit was connected to the orbitrap elite hybrid mass spectrometer (Thermo Electron, Bremen, Germany) equipped with a PicoView nanospray interface (New Objective, Woburn, MA, USA). The dried tryptic peptides were suspended in 40 µL mobile phase solution (water with 1% formic acid/acetonitrile, 98:2, v/v) and centrifuged with 15,000× g speed at 4 °C for 15 min (3500, Kubota, Japan). An aliquot of peptide mixtures (20 µL) was loaded into the sample vial. LC separation was done using a C18 BEH column (Waters, Milford, MA, USA) and a segment gradient mobile phase consisting of solvent A (0.1% formic acid in water; v/v) and solvent B (acetonitrile with 0.1% formic acid, v/v). Solvent B increased from 5% to 35% for 60 min at a flow rate of 300 nL/min and a column temperature of 35 °C. Mass spectrometry was operated in the dara-dependent mode and the MS spectra were acquired in the orbitrap (m/z 350–1600) with a resolution of 120 K at 400 m/z and automatic gain control (AGC) target at 108. The 20 most intense ions were sequentially isolated for CID MS/MS fragmentation and detection in the linear ion trap (AGC target at 10,000) with previously selected ions dynamically excluded for 60 s. Ions with single and unrecognized charge state were excluded.
3
Quantitative Analysis of Hsp20 Peptide in Rat Lenses
4
Chromatographic Separation and Orbitrap Mass Spectrometry
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Chromatographic separation was performed using an Ultimate 3000 nanoLC system coupled with an Orbitrap Elite Hybrid Mass Spectrometer (Thermo Scientific, USA), and capillary column IF100-100H035 (NewObjective, USA). Mobile phases for analysis were (i) Mobile phase A: 450 µl H2O, 180 µl Tributylamine, 50 µl acidic acid buffered at pH 9.2 with ammonium hydroxide (ii) Mobile phase B: methanol. Separation was performed using a gradient from 90 % A to 10 % A in 15 min after injection and maintained with 10 % A for 10 min before re-equilibration with a constant flow of 0.5 µl/min. Injection was performed in low dispersion mode using 25 % of a 1 µl loop (250 nl). Mass acquisition was performed in negative mode with a resolution of 60000 and the inspected mass range was between 80 and 1100 m/z.
5
Proteomic Analysis of Immunoprecipitated Peptides
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The dried immunoprecipitated peptides were resuspend in Buffer A (0.2% Formic Acid, 2% ACN, nanoLC grade 97.8% H2O) and subjected to proteomic analysis using an EASY II nano-UPLC (Thermo Fisher Scientific) connected on-line to an Orbitrap Elite hybrid mass spectrometer with a nanoelectrospray ion source (Thermo Scientific) using settings similar to those previously described (Porras-Yakushi et al., 2015 (link)). Peptides were separated using a 15 cm silica analytical column with a 75 μm inner diameter packed in-house with reversed phase ReproSil-Pur C18AQ 3 μm resin (Dr Maisch GmbH, Amerbuch-Entringen, Germany). The flow rate was set to 350 nl/min, using a linear gradient of 2%-32% B (0.2% Formic Acid, 80% ACN, 19.8% nanoLC grade H2O). Mass spectrometry detectable samples were analyzed on a 159 min gradient, while basic reversed phase immunoprecipitated samples were analyzed on a 90 min gradient. The mass spectrometer was set to collect data in a data-dependent mode, switching automatically between full-scan MS and tandem MS acquisition. All samples were analyzed by ETD and decision tree fragmentation. For ETD fragmentation, the fifteen most intense precursor ions were selected, while the 20 most intense ions were selected for fragmentation using the decision tree method. Data acquisition was managed using Xcalibur 2.0.7 and Tune 2.4 software (Thermo Fisher Scientific).
6
Mass Spectrometry Analysis of LATS1 and CDK8 Interactions
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HEK293T cells were transfected with either Flag-LATS1/HA-Tyk2 or Flag-CDK8/HA-LATS1. Forty-eight hours after transfection, cells were harvested by the NP-40 lysis buffer [150 mM NaCl, 1% NP-40, tris-HCl (20 mM, pH 7.4), phenylmethylsulfonyl fluoride (PMSF; 50 mg/ml), 0.5 mM EDTA, and protease inhibitors mixtures]. M2 affinity gel (Sigma-Aldrich, A2220) was used to pull down Flag-LATS1 or Flag-CDK8. Following procedures previously described (21 (link)), we performed the mass spectrometry analysis as follows: SDS–polyacrylamide gel electrophoresis (SDS-PAGE) gels were minimally stained with Coomassie brilliant blue and then cut into 1 × 1–mm gel block, followed by digestion with trypsin. Next, the resulting tryptic peptides were purified using the C18 Zip Tip. Then, the peptides were analyzed by an Orbitrap Elite hybrid mass spectrometer (Thermo Fisher Scientific) coupled with a Dionex LC, and tandem mass spectra were collected for the selected precursor ion within a 0.02-Da mass isolation window. Then, spectral data were searched using the Proteome Discoverer 1.4 against a UniProt protein database. The peptide spectrum matches for LATS1 or CDK8 were obtained after database search.
7
Identification of DNA2 Ubiquitination Sites
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To detect the DNA2 ubiquitination sites, 3xFlag-hDNA2, myc-hTRAF6 and 6His-tagged ubiquitin were co-transfected in 293T cells. Total 3xFlag-hDNA2 proteins were purified using anti-Flag M2 beads. Purified 3xFlag-hDNA2 proteins were resolved in an 8% SDS-PAGE gel. The gel was stained with Coomassie, and the DNA2 bands were cut and subjected to mass spectrometry (Shanghai Institute of Material Medica, Chinese Academy of Sciences). Briefly, the excised gel containing 3xFlag-hDNA2 was cut into pieces and washed with water. It was then destained and dehydrated with acetonitrile. Dithiothreitol (DTT) and NH4HCO3 were added to final concentrations of 20 mM and 50 mM, respectively. The mixture was incubated at 56°C for 30 min. The gel was destained with acetonitrile again. Iodoacetamide (IAA) and NH4HCO3 were added to final concentrations of 100 mM and 50 mM, respectively. After incubation for 20 min in the dark, in-gel tryptic digestion was carried out at 37°C overnight. The digested sample was dissolved in 0.1% formic acid and cleared by centrifugation (room temperature, 20,000 × g, 15 min). The supernatant was analyzed using an Orbitrap Elite Hybrid Mass Spectrometer (Thermo Fisher). The data was analyzed using MaxQuant software (43 (link)).
8
Determination of Alginate Oligosaccharides
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The reactions were conducted at 37 °C for 24 h with a 100 μl solution of 50 mM Tris buffer pH 7.5, 0.4% (w/v) alginate (500 cps), and 0.01 mg/ml enzyme. After incubation, two volumes of ethanol were added to the solution. Then, the mixture was centrifuged at 15,000 g for 10 min and the supernatant was transferred to a tube. The concentration of the unsaturated alginate oligosaccharides was determined using the extinction coefficient of 6150 1/M cm44 (link). The conditions of the samples and mass analyser (Orbitrap Elite hybrid mass spectrometer; Thermo Scientific, Waltham, MA) are described in the Supplementary Information.
9
MALDI-TOF and Orbitrap HRMS Analysis
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MALDI-TOF mass spectra were recorded with an UltrafleXtreme MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a UV laser (Nd) in the reflectron positive-ion mode.
High-resolution mass spectra (HRMS) were recorded with an Orbitrap Elite Hybrid mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an electrospray ionization (ESI) source in positive-ion mode. The detailed HRMS data for compounds1 –5 are presented in the Supplementary Materials, Section S3 .
High-resolution mass spectra (HRMS) were recorded with an Orbitrap Elite Hybrid mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an electrospray ionization (ESI) source in positive-ion mode. The detailed HRMS data for compounds
10
Peptide Identification by Mass Spectrometry
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SDS-PAGE gels were minimally stained with the Silver Staining kit (Beyotime, P0017S). Then the gels were cut into 1 × 1 mm gel block and digested with trypsin. The resulting tryptic peptides were purified using a C18 Zip Tip. Next, the peptides were analyzed by an Orbitrap Elite hybrid mass spectrometer (Thermo Fisher) coupled with the Dionex LC. MS/MS spectra were collected for the selected precursor ion within a 0.02 Da mass isolation window. All spectral data were searched using the Proteome Discoverer 1.4 against a UniProt protein database (https://www.uniprot.org ). The peptide spectrum matches for OTUD1 were obtained after database search.
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