Tryptic peptides derived from each gel slice were analyzed by an on-line C18 nano-flow reversed-phase liquid chromatography instrument (Easy nano-liquid chromatography) connected to an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific). Samples were concentrated onto an in-house packed 100-nm-inner diameter×1-cm C18 column (Magic C18, 5 µm, 300Å, Michrom Bioresources Inc.) then separated on 50-nm-inner diameter×15-cm C18 column at 300 nl/min with 75 min linear gradients from 0 to 35% acetonitrile in 0.1% formic acid. The LC eluent was directly nanosprayed into an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific) with an ionization voltage of 2.2 KV. During the chromatographic separation, the LTQ Orbitrap XL is operated in a data-dependent mode and under the direct control of Xcalibur (Thermo Fisher Scientific). The mass spectrometry (MS) data were acquired using the following parameters: 5 data-dependent collision-induced dissociation MS2 scan survey in the linear ion trap per every full scan in the Orbitrap with the resolution set to a value of 60,000; 35% normalized collision energy in CID; ±2 Da isolation window.
Ltq orbitrap xl
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, United Kingdom, France, Italy, Uruguay, Japan
The LTQ Orbitrap XL is a high-resolution mass spectrometer designed for advanced research applications. It combines a linear ion trap mass analyzer with a high-field Orbitrap mass analyzer, providing accurate mass measurements and high-resolution capabilities.
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Lab products found in correlation
Ultimate 3000, by Thermo Fisher Scientific (23 mentions)
Ultimate 3000 rslcnano system, by Thermo Fisher Scientific (20 mentions)
Xcalibur software, by Thermo Fisher Scientific (16 mentions)
Nanoacquity uplc system, by Waters Corporation (14 mentions)
Xcalibur 2, by Thermo Fisher Scientific (13 mentions)
Mascot, by Matrix Science (13 mentions)
Xcalibur, by Thermo Fisher Scientific (12 mentions)
Ultimate 3000 rslcnano, by Thermo Fisher Scientific (11 mentions)
Ultimate 3000 nano lc system, by Thermo Fisher Scientific (9 mentions)
Nanoacquity, by Waters Corporation (9 mentions)
Dionex ultimate 3000, by Thermo Fisher Scientific (9 mentions)
Proteome discoverer, by Thermo Fisher Scientific (9 mentions)
Trypsin, by Promega (9 mentions)
Picofrit column, by New Objective (8 mentions)
Easy nlc, by Thermo Fisher Scientific (7 mentions)
Acquity uplc beh c18 column, by Waters Corporation (6 mentions)
Q exactive, by Thermo Fisher Scientific (6 mentions)
Easy nlc 1000, by Thermo Fisher Scientific (6 mentions)
Sequencing grade modified trypsin, by Promega (6 mentions)
Triversa nanomate, by Advion (6 mentions)
774 protocols using ltq orbitrap xl
1
Quantitative Proteomic Analysis Pipeline
2
Xiaoyaosan Compound Analysis by HPLC-MS
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1 g Xiaoyaosan powder was put into 25 mL of 70% methanol-water solution, then the mix solution was ultrasonic extracted for 30 minutes at room temperature, filtered at 0.22 μm filter, stored at 4°C.
Accela High performance liquid chromatography and LTQ Orbitrap XL were purchased from Thermo Fisher Scientific Company (America); methanol (HPLC Grade) and formic acid (HPLC Grade) were purchased from Thermo Fisher Scientific Company (America); reference standards were purchased from the Chengmust Company, Sichuan Province, China.
Xiaoyaosan was performed on high performance liquid chromatography (HPLC) Accela 600 pump, LTQ Orbitrap XL (Thermo Fisher Scientific Company, America) using a SB-Aq column (4.6 × 250 mm, 5 micron, Agilent Technologies, USA), Capillary Voltage 2500 V–3000 V, Tubeleu 110 V, Scan range 100–1500, Sheath Gas 30 psi, and Aux Gas Flow 10 psi.
Method. The mobile phases comprised eluent A (0.1% formic acid) and eluent B (methanol). The gradient flow was as follows: 0~5 minutes, 30% B; 5~40 minutes, 30–90% B; 40~45 minutes, 90% to 100% B; 45~50 minutes, 100% B. The analysis was performed at a flow rate of 1.0 mL/min. The injection volume was 10 μL.
Accela High performance liquid chromatography and LTQ Orbitrap XL were purchased from Thermo Fisher Scientific Company (America); methanol (HPLC Grade) and formic acid (HPLC Grade) were purchased from Thermo Fisher Scientific Company (America); reference standards were purchased from the Chengmust Company, Sichuan Province, China.
Xiaoyaosan was performed on high performance liquid chromatography (HPLC) Accela 600 pump, LTQ Orbitrap XL (Thermo Fisher Scientific Company, America) using a SB-Aq column (4.6 × 250 mm, 5 micron, Agilent Technologies, USA), Capillary Voltage 2500 V–3000 V, Tubeleu 110 V, Scan range 100–1500, Sheath Gas 30 psi, and Aux Gas Flow 10 psi.
Method. The mobile phases comprised eluent A (0.1% formic acid) and eluent B (methanol). The gradient flow was as follows: 0~5 minutes, 30% B; 5~40 minutes, 30–90% B; 40~45 minutes, 90% to 100% B; 45~50 minutes, 100% B. The analysis was performed at a flow rate of 1.0 mL/min. The injection volume was 10 μL.
3
Pesticide Analysis in Microcosms
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Two hours and 18 days after application of the treatments, 4 mL-water subsamples of three microcosms per ARO treatment were taken and frozen at −20°C. Later, the samples were filtered (0.2 µm) and analyzed for their concentration of the three pesticides used in the ARO mixture. Measurement of the pesticides was conducted using a UltiMate3000 HPLC System combined with an LTQ-OrbiTrap XL (Thermo Scientific, USA). These samples were then analyzed with an UltiMate3000 HPLC System (column: Phenomenex, Art.-No. 00B-4462-Y0) and an attached LTQ Orbitrap XL (Thermo Scientific) operated in positive ionization mode.
4
High-Resolution LC-MS for Metabolite Profiling
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LC-MS was performed using a system of Liquid Chromatography coupled to Electrospray Ionization and High-Resolution Mass Spectrometry (LC-ESI/HRMS) characterized by a quaternary Accela 600 pump and an Accela autosampler coupled to an LTQOrbitrap XL mass spectrometer (LTQ-Orbitrap XL, Thermo Fisher Scientific, Bremen, Germany) working in negative ion mode. A Phenomenex C18 Kinetex Evo-RP (150 mm × 2.1 mm, 5 µm) column at a flow rate of 0.2 mL/min was selected; water plus 0.1% formic acid and acetonitrile plus 0.1% formic acid were used as mobile phase A and B, respectively. A linear gradient method from 5 to 95% of B in 35 min was performed. The autosampler was set to inject 4 μL of methanol extract (1 mg/mL). The mass range was from m/z 120 to 1600 with a resolution of 30,000. A data-dependent scan experiment was performed for fragmentation, selecting precursor ions corresponding to the first and the second most intense ions from the LC-HRMS spectrum and using collision energy at 30%, a minimum signal threshold of 300, and an isolation width of 2.0.
5
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.
6
Quantitative proteomic analysis of NDH-PSI supercomplex
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In vivo chloroplast protein labeling was performed as described previously [45 (link), 65 (link)]. For comparative analysis, the leaves of wild type and pbr1-1 or overexpression line of PBR1 (OE-5) were pulse-labeled with 200 mg l−113C615N4 L-Arginine (‘heavy’, H) and 15N4 L-Arginine (‘medium heavy’, M), respectively. Thylakoid membranes were extracted from the labeled leaves as described [66 (link)]. The extracted thylakoid membranes were separated by BN-PAGE as described [66 (link), 67 (link)]. Gel slices corresponding to NDH–PSI supercomplex [30 (link)] were excised, and the extracted peptides were analyzed by LC-MS/MS on a high-performance mass spectrometer (LTQ-Orbitrap XL,Thermo Finnigan, San Jose, CA, USA) as described previously [65 (link)]. Raw data were processed using the MaxQuant 1.1.36 software package (http://www.maxquant.org/ ) for protein identification and quantitation.
7
Affinity Purification of OSKM Interactors
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MBP-CCP6-wt and MBP-CCP6-mut23 (link),29 (link) proteins were expressed in E. coli and purified using the amylose resin (New England BioLabs, Ipswich, USA) according to the manufacturer’s instruction. Proteins were immobilized with Affi-gel10 resin to go through OSKM-transduced MEF lysates for affinity chromatography. Eluted fractions were visualized by SDS-PAGE followed by silver staining. Differential bands in SDS-PAGE gels were trypsinized for mass spectrometry with LTQ Orbitrap XL (Thermo Finnigan)29 (link).
8
Comprehensive Spectroscopic Profiling of Natural Products
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NMR spectra were recorded on DRX-400 and DRX-600 MHz NMR spectrometer (Bruker, Billerica, MA, USA) with tetramethylsilane (TMS) as an internal standard. HRESIMS were carried out on a LTQ-Orbitrap XL (Thermo Finnigan, San Jose, CA, USA). Specific rotation was obtained on an IP-digi300 polarimeter (InsMark, Shanghai, China) at 20 °C. SC-XRD data were collected on a Bruker D8 venture diffractometer (Bruker, Billerica, MA, USA). Silica gel (200–300 mesh; Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), LiChroprep RP-18 (40–63 mm; Darmstadt, Germany) and Sephadex LH-20 (25–100 μm; Pharmacia Biotek, Copenhagen, Denmark) were used for column chromatography. Thin-layer chromatography (TLC) was carried out with glass precoated Silica gel GF254 plates (Qingdao Haiyang Chemical Co., Ltd.). Compounds were visualized under UV light and by spraying with phosphomolybdic acid followed by heating. All solvents were analytical grade.
9
Determination of Ribonucleoside m/z Values
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For determination of m/z precursor and product values 5 mL cultures of each growth medium was inoculated with a colony of S. cerevisiae BY4741. Total RNA was isolated at OD 6 and digested as described for NAIL-MS experiments. The ribonucleosides were separated using a Dionex Ultimate 3000 HPLC system on an Interchim Uptisphere120–3HDO C18. Mobile phase A was 2 mM ammonium acetate and mobile phase B was 80% acetonitrile containing 2 mM ammonium acetate. Gradient elution started with 0% B and increased to 12% B after 10 minutes and to 80% after 12 minutes. After 4 minutes elution at 80% B and subsequently regeneration of starting conditions to 100% A after 5 minutes, the column was equilibrated at 100% A for 8 minutes. The flow rate was 0.2 mL/minute and the column temperature 30°C. High-resolution mass spectra of precursor and product ions were recorded by a ThermoFinnigan LTQ Orbitrap XL.
10
HPLC Separation of Ribonucleosides
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The ribonucleosides were separated using a Dionex Ultimate 3000 HPLC system on an Interchim Uptisphere120-3HDO C18 or a Synergi, 2.5 μm Fusion-RP C18, 100 Å, 100 × 2 mm (Phenomenex®, Torrance, California, USA). Mobile phase A was 2 mM ammonium acetate and mobile phase B was 80% acetonitrile containing 2 mM ammonium acetate. Gradient elution started with 0% B and increased to 12% B after 10 min and to 80% after 12 min. After 4 min elution at 80% B and subsequently regeneration of starting conditions to 100% A after 5 min, the column was equilibrated at 100% A for 8 min. The flow rate was 0.2 mL/min and the column temperature 30 °C. High-resolution mass spectra of precursor and product ions were recorded by a ThermoFinnigan LTQ Orbitrap XL. The parameters of the mass spectrometer were tuned with a freshly mixed solution of adenosine (5 μM). The parameters were sheath gas flow rate, 16 arb; auxiliary gas flow rate, 11 arb; sweep gas flow rate, 4 arb; spray voltage, 5.0 kV; capillary temperature, 200 °C; capillary voltage, 20 V, tube lens 65 V.
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