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Thermo easy nlc 1000

Manufactured by Thermo Fisher Scientific
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Sourced in Germany, United States

The Thermo Easy nLC 1000 is a liquid chromatography system designed for nanoflow applications. It is capable of delivering consistent and precise flow rates for high-performance liquid chromatography (HPLC) separations.

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Lab products found in correlation

Tmt 6plex, by Thermo Fisher Scientific (2 mentions) Mascot version 2, by Matrix Science (2 mentions) Proteome discoverer, by Thermo Fisher Scientific (2 mentions) Q exactive mass spectrometer, by Thermo Fisher Scientific (2 mentions) Trypsin, by Promega (2 mentions) Thermo quadrupole orbitrap q exactive mass spectrometer, by Thermo Fisher Scientific (1 mentions) Xcalibur version 2, by Thermo Fisher Scientific (1 mentions) Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions) Q exactive hf x mass spectrometer, by Thermo Fisher Scientific (1 mentions) Proteome discoverer 1, by Thermo Fisher Scientific (1 mentions) Orbitrap velos pro mass spectrometer, by Thermo Fisher Scientific (1 mentions) Proteome discoverer 2, by Thermo Fisher Scientific (1 mentions) Q exactive hf, by Thermo Fisher Scientific (1 mentions)

10 protocols using thermo easy nlc 1000

1

Proteomic Profiling of Amniotic Fluid

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Total protein concentration in AF samples was individually determined by a BCA assay (Micro BCA Protein Assay Kit, Thermo Fisher Scientific, Bremen, Germany). The pooled AF samples of SPTD case and TD control groups were generated by combining equal amounts of 10 individual AF samples from each group and further filtering them by centrifugation for 5 min at 16,000g and 4 °C. The 14 most abundant proteins were removed from 200 μg of pooled AF samples and subjected to tryptic digestion followed by high-pH reversed-phase fractionation as described in the Supplementary Material.
LC-MS/MS analyses of the fractionated peptide samples were performed using an online Thermo Easy nLC 1000 (Thermo Fisher Scientific, Bremen, Germany) system interfaced to a Thermo quadrupole-orbitrap Q-Exactive Mass Spectrometer (Thermo Fisher Scientific, Bremen, Germany), controlled by Xcalibur version 2.0.6 software (Thermo Fisher Scientific, San Jose, CA, USA) as described in the Supplementary Material.
Hong S., Lee J.E., Kim Y.M., Park Y., Choi J.W, & Park K.H. (2020). Identifying potential biomarkers related to pre-term delivery by proteomic analysis of amniotic fluid. Scientific Reports, 10, 19648.
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2

Peptide Separation and Identification via Orbitrap MS

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Peptides were loaded onto a 30-cm column (inner diameter: 75 microns; packed in-house with ReproSil-Pur C18-AQ 1.9-micron beads, Dr. Maisch GmbH) via the autosampler of the Thermo Easy-nLC 1000 (Thermo Fisher Scientific) at 60 °C. Using the nanoelectrospray interface, eluting peptides were directly sprayed onto the benchtop Orbitrap mass spectrometer Q Exactive HF (Thermo Fisher Scientific). Peptides were loaded in buffer A (0.1% (v/v) formic acid) at 250 nl/min and percentage of buffer B (80% acetonitrile, 0.1% formic acid) was ramped to 30% over 120 min followed by a ramp to 60% over 10 min then 95% over the next 5 min and maintained at 95% for another 5 min. The mass spectrometer was operated in a data-dependent mode with survey scans from 300 to 1750 m/z (resolution of 60000 at m/z = 200), and up to 12 of the top precursors were selected and fragmented using higher energy collisional dissociation (HCD with a normalized collision energy of value of 28). The MS2 spectra were recorded at a resolution of 15000 (at m/z = 200). AGC target for MS and MS2 scans were set to 3E6 and 1E5 respectively within a maximum injection time of 20 ms for MS1 and 50 ms for MS2 scans. Dynamic exclusion was set to 16 ms.
Galanti L., Peritore M., Gnügge R., Cannavo E., Heipke J., Palumbieri M.D., Steigenberger B., Symington L.S., Cejka P, & Pfander B. (2024). Dbf4-dependent kinase promotes cell cycle controlled resection of DNA double-strand breaks and repair by homologous recombination. Nature Communications, 15, 2890.
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3

TMT-Based Proteomic Analysis Workflow

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For this assay five samples were randomly selected from each device group. Equivalent amounts of protein (corresponding to similar surface areas from each sample) were reduced (10 mM dithiothreitol, 56 °C for 45 min) and alkylated (50 mM iodoacetamide, room temperature in the dark for 1 h). Proteins were subsequently digested with trypsin (sequencing grade; Promega, Madison, WI). trypsin activity was quenched by adding formic acid to a final concentration of 5%. Peptides were desalted using C18 SpinTips (Protea, Morgantown, WV) then lyophilized and stored at −80 °C. Peptides were labeled with TMT 6plex (Thermo Scientific) as per the manufacturer’s instructions. Peptides were then loaded on a precolumn and separated by reverse-phase HPLC (Thermo Easy nLC1000) over a 140-min gradient before nanoelectrospray using a QExactive mass spectrometer (Thermo Scientific). Raw mass spectral data files (.raw) were searched using Proteome Discoverer (Thermo Scientific) and Mascot version 2.4.1 (Matrix Science). TMT quantification was obtained using Proteome Discoverer and isotopically corrected as per the manufacturer’s instructions.
Bose S., Volpatti L.R., Thiono D., Yesilyurt V., McGladian C., Tang Y., Facklam A., Wang A., Jhunjhunwala S., Veiseh O., Hollister-Lock J., Bhattacharya C., Weir G.C., Greiner D.L., Langer R, & Anderson D.G. (2020). A retrievable implant for the long-term encapsulation and survival of therapeutic xenogeneic cells. Nature biomedical engineering, 4(8), 814-826.
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4

Purification and Identification of SBPs

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To purify SBPs, 2 mg samples of SIB were pre-equilibrated with PBS and incubated with the aforementioned sesame seedling extracts in 1 ml aliquots of PBS containing 0.1% NP-40; sesamin was used as a competitor at 2 mM. After washing with PBS, bound proteins were eluted with SDS containing PBS at 90 °C for 5 min, separated using SDS-PAGE and visualised by silver staining. The excised bands were then subjected to in-gel digestion with trypsin. Peptide mass fingerprint analyses were performed using nano liquid chromatography-mass spectrometry (nano LC-MS; Thermo Easy nLC1000 equipped Thermo Orbitrap Elite) and analysed by Mascot search software.
Tera M., Koyama T., Murata J., Furukawa A., Mori S., Azuma T., Watanabe T., Hori K., Okazawa A., Kabe Y., Suematsu M., Satake H., Ono E, & Horikawa M. (2019). Identification of a binding protein for sesamin and characterization of its roles in plant growth. Scientific Reports, 9, 8631.
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5

Peptide Separation by Reversed-phase HPLC

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Peptides were separated by reversed-phase HPLC (Thermo Easy nLC1000, Thermo Fisher Scientific, Waltham, MA) using a precolumn (made in house, 6 cm of 10-μm C18) and a self-pack 5-μm tip analytical column (12 cm of 5-μm C18; New Objective, Woburn, MA) over a 140-min gradient before nanoelectrospray using a Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific, Waltham, MA). Solvent A was 0.1% formic acid, and solvent B was 80% ACN/0.1% formic acid. The gradient conditions were 2 to 10% B (0 to 3 min), 10 to 30% B (3 to 107 min), 30 to 40% B (107 to 121 min), 40 to 60% B (121 to 126 min), 60 to 100% B (126 to 127 min), 100% B (127 to 137 min), 100 to 0% B (137 to 138 min), and 0% B (138 to 140 min), and the mass spectrometer was operated in a data-dependent mode. The parameters for the full-scan MS were resolution of 60,000 across 350 to 2000 m/z (mass/charge ratio), automatic gain control 3 x 106, and maximum ion injection time of 50 ms. The full-scan MS was followed by tandem mass spectrometry (MS/MS) for the top 15 precursor ions in each cycle with a normalized collision energy of 34 and dynamic exclusion of 30 s.
Wishart A.L., Conner S.J., Guarin J.R., Fatherree J.P., Peng Y., McGinn R.A., Crews R., Naber S.P., Hunter M., Greenberg A.S, & Oudin M.J. (2020). Decellularized extracellular matrix scaffolds identify full-length collagen VI as a driver of breast cancer cell invasion in obesity and metastasis. Science Advances, 6(43), eabc3175.
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6

Liquid Chromatography-Tandem Mass Spectrometry

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The dried peptides were reconstituted in 0.1%TFA and subjected to nanoflow liquid chromatography (Thermo Easy nLC 1000, Thermo Scientific) coupled to high resolution tandem MS (Q Exactive, HF, Thermo Scientific). The peptides were separated on an Acclaim PepMap HPLC analytical column (75 μM × 250 mm, 100 Å and 3 μM C18; Thermo Scientific) using a gradient of solvent B (80% ACN, 0.1% FA): 5-27% in 60 min, 27%-40% in 25 min, 40-98% in 10 min at a flow rate of 300 nl/min. MS scans were performed in the Orbitrap analyser at a resolution of 60,000 with an ion accumulation target set at 3e6 over a mass range of 380–1580 m/z, followed by MS/MS analysis at a resolution of 15,000 with an ion accumulation target set at 2e5. MS2 precursor isolation width was set at 1.4 m/z, HCD normalized collision energy at 27, and charge state one and unassigned charge states were excluded.
Laubscher D., Gryder B.E., Sunkel B.D., Andresson T., Wachtel M., Das S., Roschitzki B., Wolski W., Wu X.S., Chou H.C., Song Y.K., Wang C., Wei J.S., Wang M., Wen X., Ngo Q.A., Marques J.G., Vakoc C.R., Schäfer B.W., Stanton B.Z, & Khan J. (2021). BAF complexes drive proliferation and block myogenic differentiation in fusion-positive rhabdomyosarcoma. Nature Communications, 12, 6924.
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7

Protein Quantification by Dimethyl Labeling

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Protein samples were subjected to nanoflow liquid chromatography (Thermo Easy nLC 1000, Thermo Scientific) coupled to high resolution tandem MS (Fusion, Thermo Scientific). MS scans were performed in the Orbitrap analyzer at a resolution of 60,000 with an ion accumulation target set at 2e6 over a mass range of 350–1500 m/z, followed by MS/MS analysis in an iontrap with an ion accumulation target set at 1e5. MS2 precursor isolation width was setup at 1.6 m/z, normalized collision energy was 29, and charge state 1 and unassigned charge states were excluded. Acquired MS/MS spectra were searched against a human uniprot protein database, using a SEQUEST and the resulting peptides were filtered at a maximum of 1% FDR using the percolator validator algorithms in the Proteome Discoverer 1.4 software (Thermo Scientific, CA). The precursor ion tolerance was set at 10 ppm and the fragment ions tolerance was set at 0.6 Da along with methionine oxidation included as a dynamic modification. For dimethyl labeling on the N-terminus and lysine, 28.0313 and 32.0564 Da were filled in for light and medium labels respectively. More than 2 non-redundant peptides from each protein group was used for protein quantification.
Sebastian R., Hosogane E.K., Sun E.G., Tran A.D., Reinhold W.C., Burkett S., Sturgill D.M., Gudla P.R., Pommier Y., Aladjem M.I, & Oberdoerffer P. (2020). Epigenetic regulation of DNA repair pathway choice by macroH2A1 splice variants ensures genome stability. Molecular cell, 79(5), 836-845.e7.
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8

TMT-Based Proteomic Analysis Workflow

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For this assay five samples were randomly selected from each device group. Equivalent amounts of protein (corresponding to similar surface areas from each sample) were reduced (10 mM dithiothreitol, 56 °C for 45 min) and alkylated (50 mM iodoacetamide, room temperature in the dark for 1 h). Proteins were subsequently digested with trypsin (sequencing grade; Promega, Madison, WI). trypsin activity was quenched by adding formic acid to a final concentration of 5%. Peptides were desalted using C18 SpinTips (Protea, Morgantown, WV) then lyophilized and stored at −80 °C. Peptides were labeled with TMT 6plex (Thermo Scientific) as per the manufacturer’s instructions. Peptides were then loaded on a precolumn and separated by reverse-phase HPLC (Thermo Easy nLC1000) over a 140-min gradient before nanoelectrospray using a QExactive mass spectrometer (Thermo Scientific). Raw mass spectral data files (.raw) were searched using Proteome Discoverer (Thermo Scientific) and Mascot version 2.4.1 (Matrix Science). TMT quantification was obtained using Proteome Discoverer and isotopically corrected as per the manufacturer’s instructions.
Bose S., Volpatti L.R., Thiono D., Yesilyurt V., McGladian C., Tang Y., Facklam A., Wang A., Jhunjhunwala S., Veiseh O., Hollister-Lock J., Bhattacharya C., Weir G.C., Greiner D.L., Langer R, & Anderson D.G. (2020). A retrievable implant for the long-term encapsulation and survival of therapeutic xenogeneic cells. Nature biomedical engineering, 4(8), 814-826.
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9

Nano-LC-MS/MS Proteomic Analysis

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Purified trypsin-digested peptides were analyzed by nano-reverse-phase liquid chromatography (LC)-tandem mass spectrometry (MS/MS) using a Thermo Easy-nLC 1000 (Thermo Fisher Scientific, Waltham, MA, USA) coupled with Orbitrap Velos Pro mass spectrometer (Thermo Fisher Scientific). The experimental conditions are presented in Table 1. The linear trap quadrupole was calibrated using the positive ion calibrant solution (Cat no., 88323, Pierce) and tuned to optimize the response of the ion at m/z 524 of the tetra peptide Met-Arg-Phe-Ala MS/MS.
Rajasekaran S., Soundararajan D.C., Tangavel C., K. S. S.V., Nayagam S.M., Matchado M.S., Muthurajan R., Shetty A.P., Kanna R.M, & Dharmalingam K. (2020). Proteomic Signature of Nucleus Pulposus in Fetal Intervertebral Disc. Asian Spine Journal, 14(4), 409-420.
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10

High-Resolution Tandem Mass Spectrometry for Proteomics

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The dried peptides were reconstituted in 0.1%TFA and subjected to nanoflow liquid chromatography (Thermo Easy nLC 1000, Thermo Scientific) coupled to high resolution tandem MS (Q Exactive, HF, Thermo Scientific). MS scans were performed in the Orbitrap analyser at a resolution of 60,000 with an ion accumulation target set at 3e 6 over a mass range of 380-1580 m/z, followed by MS/MS analysis at a resolution of 15,000 with an ion accumulation target set at 2e 5 . MS2 precursor isolation width was set at 1.4 m/z, normalized collision energy at 27, and charge state one and unassigned charge states were excluded.
Proteomics Data Processing: The raw data was searched against the full human uniprot protein database using the SEQUEST algorithm in the Proteome Discoverer 2.2 software (Thermo Scientific, CA). The precursor ion tolerance was set at 10 ppm and the fragment ions tolerance was set at 0.02 Da along with methionine oxidation included as dynamic modification. Only fully tryptic peptides with up to two mis-cleavages and FDR of 1% using the percolator validator algorithms were accepted.
Laubscher D., Gryder B.E., Sunkel B.D., Andrésson Þ., Das S., Roschitzki B., Wolski W., Wu X., Chou H., Song Y., Wang C., Wei J.S., Wang M., Wen X., Marques J.G., Wachtel M., Vakoc C.R., Schäfer B.W., Stanton B.Z., & Khan J. (2021). BAF complexes drive proliferation and block myogenic differentiation in fusion-positive rhabdomyosarcoma.
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