Nano lc 1000

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Nano-LC 1000 is a high-performance liquid chromatography system designed for nanoscale separations. It features a compact design and precise flow control for sensitive analytical applications.

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Lab products found in correlation

Orbitrap elite mass spectrometer, by Thermo Fisher Scientific (1 mentions) Coomassie blue, by Thermo Fisher Scientific (1 mentions) Coomassie blue, by Merck Group (1 mentions) Thermo orbitrap velos, by Thermo Fisher Scientific (1 mentions) Q exactive, by Thermo Fisher Scientific (1 mentions) Nlc1000, by Thermo Fisher Scientific (1 mentions) Ltq orbitrap elite, by Thermo Fisher Scientific (1 mentions) Proteome discoverer 2.1 interface, by Thermo Fisher Scientific (1 mentions) Mascot 2, by Matrix Science (1 mentions)

Spelling variants of this product

Nano-LC 1000 system (4 citations) Easy-nLC 1000 nano-LC (3 citations) Easy-nLC 1000 nano-LC system (3 citations) Easy nano-LC Proxeon 1000 system (2 citations) Nano-LC Proxeon 1000 (2 citations) Thermo EASY nano-LC 1000 system (2 citations) Nano LC-1000 HPLC nanoflow system (2 citations)

9 protocols using nano lc 1000

Phosphopeptide Fractionation and Mass Spectrometry

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Phosphopeptide samples were fractionated followed a described protocol^{46 (link)}. Vacuum-dried peptides were dissolved in pH 10 buffer (10 mM ammonium bicarbonate, pH 10, adjusted by NH₄OH) and subjected to pH 10 C18 reverse-phase column chromatography. Peptides were eluted with a step gradient of 150 μL each of 6, 9, 12, 15, 18, 21, 25, 30, and 35% ACN (pH 10), pooled into six pools and vacuum-dried for nano-high-performance liquid chromatography-tandem MS.
Peptides were loaded onto a 10-cm column with a 150-μM inner diameter, packed in-house with 1.9 μM C18, and subjected to nano-high-performance liquid chromatography-tandem MS analysis by using a nano-LC 1000 coupled with an Orbitrap Elite mass spectrometer (ThermoFisher Scientific). The peptides were separated with a 75-min discontinuous gradient of 2–24%, 4–24%, or 8–26% ACN/0.1% formic acid at a flow rate of 800 nL/minute. The mass spectrometer was set to data-dependent mode, the precursor MS spectrum was scanned at 375–1300 m/z with 240k resolution at 400 m/z (2 × 10⁶ AGC target), and the 25 strongest ions were fragmented via collision-induced dissociation with 35 normalized collision energy and 1 m/z isolation width and detected by using an ion trap with 30 s of dynamic exclusion time, 1 × 10⁴ AGC target, and 100 ms of maximum injection time.

Chen Z., Lei C., Wang C., Li N., Srivastava M., Tang M., Zhang H., Choi J.M., Jung S.Y., Qin J, & Chen J. (2019). Global phosphoproteomic analysis reveals ARMC10 as an AMPK substrate that regulates mitochondrial dynamics. Nature Communications, 10, 104.

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Mouse Serum Proteome Profiling

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Mouse serum was pre-cleared by Proteome Purify 2 Mouse Serum Immunodepletion resin (MIDR002, RnD Systems) to remove albumin and immunoglobulins. Depleted serum was subjected to Mass Spectrometry-based whole proteome analysis [15 (link),16 (link)]. Briefly, trypsinized serum peptides were extracted by 50% acetonitrile/0.1% formic acid and 80% acetonitrile/0.1% formic acid solution. The proteome was profiled by nano-liquid chromatography-tandem mass spectrometry analysis with a nano-LC1000 coupled to a Thermo Q-Exactive (Thermo Fisher Scientific). The relative amount was normalized as the intensity-based absolute quantification algorithm and normalized to the intensity-based fraction of the total (iFOT).

Lee S.H., Choi J.M., Jung S.Y., Cox A.R., Hartig S.M., Moore D.D, & Kim K.H. (2020). The Bile Acid Induced Hepatokine Orosomucoid Suppresses Adipocyte Differentiation. Biochemical and biophysical research communications, 534, 864-870.

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Nano LC-MS Metabolite Profiling

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Twenty components were dissolved in 20 µl of nano LC A solution. A 2 µl sample was injected into the column gradient and eluted, analyzed and identified by Orbitrap Elite mass spectrometry. The sample was analyzed by a Thermo Nano LC 1000 high-performance liquid chromatography (HPLC) system, and an Acclaim PepMap^® 100 C18, 3 µm, 100 Å (75 µm × 2 cm) sample column and an Acclaim PepMap^® RSLC C18, 2 µm, 100 Å (50 µm × 15 cm) analysis column were applied in the mass spectrometric analysis.

Liu G., Miao F., Wang Y., Kou J., Yang K., Li W., Xiong C., Zhang F., Wang X., Yan H., Wei C., Zhao C, & Yan G. (2022). Comparative proteomics analysis of Schistosoma japonicum developed in different Oncomelania snails as intermediate hosts. Frontiers in Cellular and Infection Microbiology, 12, 959766.

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Purification and Analysis of MYC Complexes

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MYC-binding protein complexes purified from GEN2.2 cells were lysed in lysis buffer (50 mM Tris-HCl pH 7.4, 250 mM NaCl, 1 mM EDTA, 1% TRITON-100, 10% glycerol supplemented with a complete protease inhibitor and phosphatase inhibitor cocktail (Sigma)) and subjected to ultracentrifugation. Cleared lysates were incubated overnight with protein G agarose beads (Pierce) with the indicated antibodies. Beads were washed extensively with lysis buffer, separated on a 4–20% gradient polyacrylamide gel, and stained with Coomassie blue (Sigma). Unstimulated GEN2.2 cells were lysed. Nuclear extracts were fractionated as shown in Figure 4A and then immunoprecipitated with MYC specific antibody and rabbit IgG control. Then, immune complexes from MYC-IP were separated on a 4–20% Gradient SDS-PAGE gel (Thermo Scientific) and stained with Coomassie blue for visualization. All bands were in–gel digested with trypsin, and the proteins were identified by nanoflow LC-MS/MS analysis with a nano-LC1000 (Thermo Scientific) coupled to a Thermo ORbitrap Velos™ (Thermo Scientific) mass spectrometer. Acquired MS spectra were processed by BioWorks software to convert data into peptide and protein composition information. All bands were analyzed by liquid chromatography-MS at the Proteomics Center, Baylor College of Medicine, Houston, TX.

Kim T.W., Hong S., Lin Y., Murat E., Joo H., Kim T., Pascual V, & Liu Y.J. (2016). Transcriptional repression of IRF7 by MYC is critical for type I interferon production in human pDC. Journal of immunology (Baltimore, Md. : 1950), 197(8), 3348-3359.

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Nano-LC-MS/MS Proteomic Workflow

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The experiment was carried out using Nano-LC 1000 LC-MS / MS with a proxeon easy NLC 1000 coupled with a mass spectrometer (Thermo Fisher Q exactive, United States) for fraction separation. Trypsinized fractions were loaded into buffer A (0.1% formic acid) connected to the reverse phase capture column of the C18 reverse phase analysis column, and separated using a linear gradient of buffer B (84% ACN and 0.1% formic acid). The eluent was analyzed by tandem mass spectrometry (MS/MS) in Q exactive after spraying via a nanoelectrospray source at 2.0 kV electrospray voltage. MS data were obtained using a data-dependent mode that dynamically selected the 20 most intense precursor ions present in the survey scan (350–18,000 M/Z) for higher-energy C-trap dissociation fragmentation. In the Orbitrap mass analyzer, survey scans were acquired at a resolution of 70,000 (full width at half maxima, FWHM) and ion fragments were detected at a resolution of 17,000 (FWHM). The collision energy was 28% in the MS survey scan with 10.0 s of dynamic exclusion (De Mandal et al., 2020 (link)).

Wang J., Fan P., Wei Y., Wang J., Zou W., Zhou G., Zhong D, & Zheng X. (2022). Isobaric tags for relative and absolute quantification-based proteomic analysis of host-pathogen protein interactions in the midgut of Aedes albopictus during dengue virus infection. Frontiers in Microbiology, 13, 990978.

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Nanoscale LC-MS/MS of Peptides

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Four injections were made into a NanoLC 1000 (Thermo Fisher Scientific, Inc., Waltham, MA, USA) interfaced to the LTQ OrbiTrap elite mass spectrometer (Thermo Fisher Scientific, Inc.) via a nanosource. The samples were loaded onto a 150 µm × 2 cm peptrap 300 A C18 pre-column (ReproSil-Pur, Dr. Maisch GmbH) in solvent A (99.9% water/0.1% formic acid) and desalted for 10 min. The peptides were eluted into a 75 µm × 25 cm 100 A C18 analytical column (self-packed; ReproSil-Pur, Dr. Maisch GmbH) and separated with a linear gradient of 5-30% solvent B (99.9% acetonitrile/0.1% formic acid) in 5 min, and then 69% solvent B for 115 min. The flow rate used was 500 nl/min. The survey scans were acquired in the OrbiTrap with a resolving power of 60,000 m/z 400 and an automated gain control target level of 1×10⁶. The 25 most abundant ions were selected for fragmentation using collision-induced dissociation in the linear ion trap. The precursor ions were fragmented with He gas for 30 ms with a normalized collision energy of 35. The dynamic exclusion parameters were set to exclude ions previously selected for fragmentation for 1 min. All data were acquired in reduced profile mode to accommodate further downstream processing.

Guo Y., Cui L., Jiang S., Wang D., Jiang S., Xie C, & Jia Y. (2016). S100A1 transgenic treatment of acute heart failure causes proteomic changes in rats. Molecular Medicine Reports, 14(2), 1538-1552.

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Mass Spectrometry Proteomic Analysis

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Conditioned medium was subjected to unbiased mass-spec profiling as previously described (71 (link)). Briefly, samples were denatured and digested, followed by extraction with 50% acetonitrile and 2% formic acid. Vacuum-dried peptides were subjected to reverse phase column chromatography with a micro-pipette tip C18 column and fractionated with stepwise ACN gradient into different elution groups. Eluent was pooled and vacuum-dried for analysis with a nano-LC 1000 coupled with an Orbitrap Fusion^™ mass spectrometer (ThermoFisher Scientific). Data were analyzed with Proteome Discoverer 2.1 interface (ThermoFisher Scientific) and detected peptides were assigned into gene products by Py Grouper and Tackle analysis flatform from iSpec(75 (link)).

Ye Q., Jo J., Wang C.Y., Oh H., Choy T.J., Kim K., D’Alessandro A., Reshetnyak Y.K., Jung S.Y., Chen Z., Marrelli S.P, & Lee H.K. (2023). Astrocytic Slc4a4 regulates blood-brain barrier integrity in healthy and stroke brains via a NO-CCL2-CCR2 pathway. bioRxiv.

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Proteomic Analysis of Mouse Liver Tissue

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The detailed procedure has been described.20 Briefly, liver tissue was homogenized in a lysis buffer (50 mM NH₅CO₃ and 1 mM CaCl₂). After denaturation and subsequent trypsinization, peptides were extracted by 50% acetonitrile/0.1% formic acid and 80% acetonitrile/0.1% formic acid solution. The hepatic proteome was profiled by nano‐liquid chromatography–tandem mass spectrometry (LC/MS‐MS) analysis with a nano‐LC1000 coupled to a Thermo Q‐Exactive (Thermo Fisher Scientific). Obtained MS‐MS spectra were searched against the target‐decoy mouse Reference Sequence database in the Proteome Discoverer 1.4 interface (PD1.4; Thermo Fisher Scientific) with the Mascot algorithm (Mascot 2.4; Matrix Science, Boston, MA). The relative amount was calculated by the intensity‐based absolute quantification algorithm and normalized to the intensity‐based fraction of the total.

Kim K.H., Choi J.M., Li F., Dong B., Wooton‐Kee C.R., Arizpe A., Anakk S., Jung S.Y., Hartig S.M, & Moore D.D. (2018). Constitutive Androstane Receptor Differentially Regulates Bile Acid Homeostasis in Mouse Models of Intrahepatic Cholestasis. Hepatology Communications, 3(1), 147-159.

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Proteome Analysis by Q Exactive Mass Spectrometry

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Mass spectrometry was performed using a Thermo Nano LC 1000 high-performance liquid chromatography (HPLC) system with Proxeon EASY-nLC 1000 coupled to a Q Exactive. An Acclaim PepMap® 100 C18 (3 µm, 100 Å, 75 µm × 2 cm) trap column and an Acclaim PepMap® RSLC C18 (2 µm, 100 Å, 50 µm × 15 cm) analysis column were used in the mass spectrometric analysis. Trypsin digested fractions were reconstituted and eluted from the trap column and then loaded onto a reversed-phase analytical column. The eluent was sprayed via an NSI source at a 1.8 kV electrospray voltage and then analyzed by MS/MS in Q Exactive. The mass spectrometer was operated in data-dependent mode, automatically switching between MS and MS/MS.

Miao F., Wang Y., Yang K., Li W., Xiong C., Zhang J., Wang X., Yan H., ChangYin W., Yan G., Chang-Lei Z., Liu G., & Liu X. (2021). Comparative Proteomics of Schistosoma Japonicum Developed From Different Oncomelania Snails Permissive Areas.

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