Nano lc 1d plus

Manufactured by AB Sciex

Sourced in United States

The Nano-LC 1D plus is a high-performance liquid chromatography system designed for separation and analysis of complex samples. It features a nanoflow-optimized design and advanced control software for precise and reproducible separations. The core function of the Nano-LC 1D plus is to provide efficient and sensitive separation of analytes prior to mass spectrometric analysis.

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Lab products found in correlation

5800 maldi tof tof analyzer, by AB Sciex (1 mentions) Qtrap 5500 mass spectrometer, by AB Sciex (1 mentions) Proteomics grade trypsin, by Merck Group (1 mentions) Sep pak c18 cartridge, by Waters Corporation (1 mentions) Ltq orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions) As 1 autosampler, by AB Sciex (1 mentions) C18 particles, by Dr. Maisch (1 mentions) Ltq orbitrap xl etd, by Thermo Fisher Scientific (1 mentions)

4 protocols using nano lc 1d plus

iTRAQ-based Proteomic Analysis Workflow

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The iTRAQ-labeled mixture was first separated by an ICAT^TM strong cation-exchange cartridge (AB SCIEX, FosterCity, CA, USA) according to the manufacturer’s instructions. After being desalted by Ultramicrospin^TM columns (The Nest Group, Southborough, MA, USA), the eluted fractions were spotted and mixed with a MALDI matrix solution (5 mg/mL α-Cyano-4-hydroxycinnamic acid (CHCA) in 70% acetonitrile and 0.1% v/v TFA) with the nano-LC 1D plus (Eksigent, Redwood, CA, USA).
The sample spots were analyzed using a 5800 MALDI TOF/TOF Analyzer (AB SCIEX, FosterCity, CA, USA). Seven hundred and fifty laser shots were accumulated from each sample well, and the precursor ions were selected from MS spectra ranged from 800 to 4000 Da. MS/MS analysis was achieved with air used as the collision gas with a collision energy of 2 kV.
All MS data were analyzed using ProteinPilot software v4.0.8085 (AB SCIEX, FosterCity, CA, USA) using the Paragon algorithm 4.0.0.0 searched against the Uniprot human protein database.

Guo H., Dong J., Hu S., Cai X., Tang G., Dou J., Tian M., He F., Nie Y, & Fan D. (2015). Biased random walk model for the prioritization of drug resistance associated proteins. Scientific Reports, 5, 10857.

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Nano-LC-MS/MS Analysis of Digested Peptides

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Digested samples with synthetic peptides included were injected into an nHPLC (Eksigent nano LC 1D plus) where peptides were concentrated in a trap column (Eksigent nanoLC trap) for 5 min at a flow of 2 μl/min of 2% ACN and 0.1% FA before their separation in a 15-cm analytical column (Eksigent nanoLC column 2C18-CL). Peptide elution was achieved by a 5 to 35% ACN gradient for 30 min at 300 nl/min. Both trap and analytical columns were heated at 50°C to get more constant retention times between samples. At least three biological replicates of each condition were analyzed, with at least 2 technical replicates of each one. All analyses were performed in an AB Sciex QTRAP 5500 mass spectrometer.

Amador-García A., Zapico I., Borrajo A., Malmström J., Monteoliva L, & Gil C. (2021). Extending the Proteomic Characterization of Candida albicans Exposed to Stress and Apoptotic Inducers through Data-Independent Acquisition Mass Spectrometry. mSystems, 6(5), e00946-21.

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Deacetylation Assay for Histone Acetyltransferases

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HATs (p300/CBP, PCAF, GCN5, Tip60) plasmids were co-transfected into HeLa cells, TSA (1 μM) of nicotinamide (20 mM) for 12 h, lysed in IP buffer, and followed by an in vitro deacetylation assay. The samples were digested by 12.5 ng/μl proteomics-grade trypsin (Sigma; T6567) at a ratio of 1:40 enzyme to protein, re-IPed with acetyl-lysine-conjugated beads (Immunchem; ICP0388), and peptides were then desalted by solid-phase extraction (Sep-pak C18 cartridges, Waters Corporation, Milford, MA) (37 (link)). LC-MS-MS analysis of the peptides was performed using a LTQ-Orbitrap mass spectrometer (Thermo Scientific) with a nanospray source and an Eksigent NanoLC 1D Plus and AS1 Autosampler, as previously described (11 ).

Park S.H., Ozden O., Liu G., Song H.Y., Zhu Y., Yan Y., Zou X., Kang H.J., Jiang H., Principe D.R., Cha Y.I., Roh M., Vassilopoulos A, & Gius D. (2016). SIRT2-mediated deacetylation and tetramerization of pyruvate kinase directs glycolysis and tumor growth. Cancer research, 76(13), 3802-3812.

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Peptide Separation and Mass Spectrometry Analysis

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Peptides of each sample were separated by online reversed-phase nanoscale capillary liquid chromatography and analyzed by electrospray mass spectrometry in tandem (ESI-MS/MS). The experiments were performed in the mass spectrometry facility RPT02H of Carlos Chagas Institute (Fiocruz, Parana) with a nanoLC-1D plus (Eksigent) coupled to LTQ Orbitrap XL ETD (Thermo Scientific) mass spectrometer. Chromatographic separation of the peptide mixtures was carried out on an analytical silica column of 15 cm, 75-µm ID and with a 3 µM diameter C18 particles (Dr. Maisch), flow rate of 250 nL/min of mobile phase (ACN, 0.1% formic acid, 5% DMSO) with a linear gradient from 5 to 40% ACN in 180 min. Peptides were ionized by nano-electrospray (voltage 2.7 kV) and injected into the MS. Full-scan MS spectra (at 300.0–1800.0 m/z range) were acquired on an Orbitrap analyzer with a resolution of R = 60,000. The 10 most intense peaks were fragmented by CID and analyzed in the Ion trap. A dynamic exclusion list of 90 sec was applied and the “lock mass” option was enabled (m/z = 401.922718). The MS data was deposited into the Mendeley Data repository with the dataset identifier http://dx.https://doi.org/10.17632/4pbttmb3kx.1.

Zamith-Miranda D., Amatuzzi R.F., Munhoz da Rocha I.F., Martins S.T., Lucena A.C., Vieira A.Z., Trentin G., Almeida F., Rodrigues M.L., Nakayasu E.S., Nosanchuk J.D, & Alves L.R. (2021). Transcriptional and translational landscape of Candida auris in response to caspofungin. Computational and Structural Biotechnology Journal, 19, 5264-5277.

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