Nanolc ultra 1d plus system

Manufactured by AB Sciex

Sourced in United States

The NanoLC Ultra 1D Plus system is a nano-flow liquid chromatography instrument designed for high-performance separations of complex samples. It features a compact design and precise flow control for efficient sample analysis.

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Lab products found in correlation

Sciex 5500 qtrap triple quadrupole mass spectrometer, by AB Sciex (2 mentions) Magic c18 aq 3 m resin, by Bruker (2 mentions) Picofrit analytical column, by New Objective (2 mentions) Betabasic 5 μm 300 particles, by New Objective (2 mentions) Ltq orbitrap velos, by Thermo Fisher Scientific (2 mentions) Ziptip, by Merck Group (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions) Zorbax 300sb c18 trap column, by Agilent Technologies (1 mentions) Ltq orbitrap elite mass spectrometer, by Thermo Fisher Scientific (1 mentions) Kinetex c18, by Phenomenex (1 mentions) Tripletof 5600 mass spectrometer, by AB Sciex (1 mentions) Xcalibur software v2, by Thermo Fisher Scientific (1 mentions) Ltq orbitrap xl mass spectrometer, by Thermo Fisher Scientific (1 mentions) Qtrap 5500, by AB Sciex (1 mentions) Q exactive hybrid quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

NanoLC Ultra 1D plus NanoLC-1D plus system NanoLC-Ultra 1D+, NanoLC 1D NanoLC Ultra system NanoLC Ultra NanoLC system

12 protocols using nanolc ultra 1d plus system

Pancreatic Protein Extraction and Trypsin Digestion

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Mechanical disruption of pancreatic tissue was performed using a Potter-Elvehjem homogenizer, in the presence of 7 M urea, 2 M thiourea, 4% CHAPS, 40 mM DTT and protease inhibitor cocktail (Roche Diagnostics). Individual sample homogenates were centrifuged for 5 min at 10000 g and cleared supernatans stored at −80 °C until further processing. Total protein was determined using a colorimetric method. After protein precipitation, 10 μg/sample was digested using a 1:25 trypsin:sample ratio, according to a method previously described [19 (link)]. After digestion, samples were desalted using ZipTip (Merck) according to manufacturer instructions.
Targeted nanoLC-MS/MS analyses were performed on a 1D Plus nanoLC Ultra system (Eksigent, Dublin, CA, USA) interfaced to a Sciex 5500 QTRAP triple quadrupole mass spectrometer (Sciex, Framingham, MA, USA) equipped with a nano-ESI source and controlled by Analyst v.1.5.2. software (ABSciex), according to Mora MI et al. [20 (link)].

Rius-Pérez S., Pérez S., Torres-Cuevas I., Martí-Andrés P., Taléns-Visconti R., Paradela A., Guerrero L., Franco L., López-Rodas G., Torres L., Corrales F, & Sastre J. (2019). Blockade of the trans-sulfuration pathway in acute pancreatitis due to nitration of cystathionine β-synthase. Redox Biology, 28, 101324.

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Quantitative Multiple Reaction Monitoring

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Samples were previously quantified by microBCA analysis (Pierce) and similar amounts (5 µg per sample) were individually dissolved in 8 M urea, 25 mM ammonium bicarbonate, reduced with DTT, and alkylated with iodoacetamide, according to a method described by López-Ferrer et al. (2004) (link). Digested samples were diluted with 0.2% trifluoroacetic acid in water and subjected to multiple reaction monitoring analysis using a 1D Plus nanoLC Ultra system (Eksigent, Dublin, CA, USA) interfaced to a Sciex 5500 QTRAP triple quadrupole mass spectrometer (Sciex, Framingham, MA, USA) equipped with a nano-electrospray ionization source and controlled by Analyst v.1.5.2. software (ABSciex). Trypsin-digested samples were loaded online on a C18 PepMap 300 µm internal diameter × 5 mm trapping column (5 µm, 100 Å, Thermo Scientific) and separated using a BioSphere C18 75 µm internal diameter × 150 mm capillary column (3 µm, 120 Å, Nanoseparations). A list of 84 transitions (usually 3–4 per peptide, with a preference toward higher-mass y series ions), corresponding to 21 unique peptides selected for 10 different proteins, was monitored. Skyline software determined automatically the collision energy values for the candidate peptides according to MacLean et al. (2010) (link).

Tcherkez G., Ben Mariem S., Larraya L., García-Mina J.M., Zamarreño A.M., Paradela A., Cui J., Badeck F.W., Meza D., Rizza F., Bunce J., Han X., Tausz-Posch S., Cattivelli L., Fangmeier A, & Aranjuelo I. (2020). Elevated CO2 has concurrent effects on leaf and grain metabolism but minimal effects on yield in wheat. Journal of Experimental Botany, 71(19), 5990-6003.

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Quantitative Proteomics using DIA-SWATH

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1 µg of peptides was injected on a 6600 Sciex TripleTOF mass spectrometer interfaced with an Eksigent NanoLC Ultra 1D Plus system. The peptides were separated on a 75-µm-diameter, 20-cm-long new Objective emitter packed with Magic C18 AQ 3 µm resin (Michrom BioResources) and eluted at 300 nl/min with a linear gradient of 2–30% Buffer B for 60 min (Buffer B: 98% ACN, 0.1% FA). MS data acquisition for the individual meiotic time course samples was performed in data-independent acquisition (DIA) SWATH-MS mode using 64 variable precursor isolation windows with 1-Da overlaps acquired each for 50 ms plus one MS1 scan acquired for 250 ms, as described in Gillet et al. (2012 (link)). Library generation was performed in data-dependent acquisition mode (DDA, top20, with 20 s dynamic exclusion after 1 MS/MS). For either mode, the mass ranges recorded were 360–1,460 m/z for MS1 and 50–2,000 m/z for MS2, and the collision energy was set to 0.0625 × m/z—6.5 with a 15-eV collision energy spread regardless of the precursor charge state.

King G.A., Wettstein R., Varberg J.M., Chetlapalli K., Walsh M.E., Gillet L.C., Hernández-Armenta C., Beltrao P., Aebersold R., Jaspersen S.L., Matos J, & Ünal E. (2022). Meiotic nuclear pore complex remodeling provides key insights into nuclear basket organization. The Journal of Cell Biology, 222(2), e202204039.

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Mass Spectrometry Proteomics Workflow

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The samples were analyzed on an LTQ Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) coupled with an Eksigent NanoLC-Ultra 1D Plus system. Peptides were loaded onto a Zorbax 300SB-C18 trap column (Agilent) at a flow rate of 6 μL/min for 6 minutes and then separated on a reversed-phase PicoFrit analytical column (New Objective) using a short 15-minute linear gradient of 5% to 40% acetonitrile in 0.1% formic acid at a flow rate of 250 nL/min for 2D-DIGE protein spots and a 40-minute linear gradient for SNO-RAC samples. Mass analysis was carried out in data-dependent analysis mode, in which MS initially scanned the full MS mass range from m/z 300 to 2000 at 60 000 mass resolution, and then 6 collision-induced dissociation MS scans were sequentially carried out in the Orbitrap and the ion trap, respectively.

Menazza S., Aponte A., Sun J., Gucek M., Steenbergen C, & Murphy E. (2015). Molecular Signature of Nitroso–Redox Balance in Idiopathic Dilated Cardiomyopathies. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 4(9), e002251.

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Mass Spectrometry Proteomics Workflow

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The in-gel tryptic
digestion was
carried out by following the protocol described previously.^{44 (link)} Briefly, the gel bands were cut into 1 mm³ pieces, rinsed, and dehydrated, and the protein was reduced
with DTT and alkylated with iodoacetamide in the dark prior to overnight
digestion with trypsin at 37 °C in 50 mM ammonium bicarbonate.
The concentrated peptides were analyzed on an LTQ Orbitrap Velos (Thermo
Fisher Scientific, San Jose, CA) coupled with an Eksigent nanoLC-Ultra
1D plus system (Dublin, CA). Peptides were separated on a PicoFrit
analytical column (100 mm long, ID 75 μm, tip i.d. 10 μm,
packed with BetaBasic 5 μm 300 Å particles, New Objective,
Woburn, MA) using a 35 min linear gradient of 5–35% ACN in
0.1% FA at a flow rate of 250 nL/min. Mass analysis was carried out
in data-dependent analysis mode, where MS1 scanned the full MS mass
range from m/z 300 to 2000, at 30 000
mass resolution, and 10 CID MS2 scans were sequentially carried out
in the Orbitrap and the ion trap, respectively.

Han M.H., Hu Z., Chen C.Y., Chen Y., Gucek M., Li Z, & Markey S.P. (2014). Dysbindin-Associated Proteome in the P2 Synaptosome Fraction of Mouse Brain. Journal of Proteome Research, 13(11), 4567-4580.

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Nanoscale LC-MS/MS Protein Quantification

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LC-MS/MS experiments were performed as above but using a nanoLC Ultra 1D plus system (Eksigent Corp., Dublin, CA) and an LTQ Orbitrap at a resolution of 60,000. Peptides were initially loaded onto an in-house pre-column (100 μm inner diameter × 40 mm, 3.6-μm particle size) from a nanoLC AS-2 autosampler with 0.5% formic acid at a flow rate of 1.5 μl/min, then further separated using an in-house reverse-phase nanocolumn (Phenomenex, kinetex C18, 75 μm inner diameter × 150 mm, 2.6-μm particle size) at a flow rate of 250 nl/min for 120 min. Peptide mass tolerance was ±20 ppm and fragment mass tolerance was 0.8 Da. Significance threshold was set to p > 0.05 and ion score cut off was set to 0. Protein abundance was estimated by calculating emPAI scores (21 (link)).

Li X.W., Rees J.S., Xue P., Zhang H., Hamaia S.W., Sanderson B., Funk P.E., Farndale R.W., Lilley K.S., Perrett S, & Jackson A.P. (2014). New Insights into the DT40 B Cell Receptor Cluster Using a Proteomic Proximity Labeling Assay. The Journal of Biological Chemistry, 289(21), 14434-14447.

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Peptide Analysis by Reversed-Phase Chromatography

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The peptides were diluted with 0.4% acetic acid to achieve concentrations of 1 μg μl⁻¹ and an aliquot containing approximately 1 μg was injected into a reversed-phase Magic C18aq column (15 cm × 75 μm) on an Eksigent nanoLC-ultra 1D plus system at a flow rate of 300 nl min⁻¹. Prior to use, the column was equilibrated with 95% buffer A (0.1% formic acid in H₂O) and 5% buffer B (0.1% formic acid in acetonitrile). The peptides were eluted with a linear gradient from 10% buffer B to 40% buffer B over 40 min. The HPLC system was coupled to a Q Exactive quadrupole mass spectrometer (Thermo Scientific) operated in the data dependent mode. Survey full-scan MS spectra (m/z 300–2000) were acquired in the Orbitrap with a resolution of 75,000. The MS/MS spectra of the 12 most intense ions from the MS1 scan with a charge state ≥2 were acquired with the following options: resolution, 17,500; isolation width, 2 m/z; normalized collision energy, 27%; dynamic exclusion duration, 30 s; and ion selection threshold, 4.00E+03 counts.

Hwang J., Suh H.W., Jeon Y.H., Hwang E., Nguyen L.T., Yeom J., Lee S.G., Lee C., Kim K.J., Kang B.S., Jeong J.O., Oh T.K., Choi I., Lee J.O, & Kim M.H. (2014). The structural basis for the negative regulation of thioredoxin by thioredoxin-interacting protein. Nature Communications, 5, 2958.

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Peptide Separation and Mass Analysis

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The samples were reconstituted with 0.1 % formic acid. Liquid chromatography was performed on an Eksigent nanoLC-Ultra 1D plus system (Dublin, CA). Peptide digest was first loaded on a Zorbax 300SB-C18 trap (Agilent, Palo Alto, CA) at 6 μL/min for 5 min, then separated on a PicoFrit analytical column (100 mm long, ID 75 μm, tip ID 10 μm, packed with BetaBasic 5 μm 300 Å particles, New Objective, Woburn, MA) using a 40-min linear gradient of 5-35 % ACN in 0.1 % FA at a flow rate of 250 nL/min. Mass analysis was carried out on an LTQ Orbitrap Velos (Thermo Fisher Scientific, San Jose, CA) with data-dependent analysis mode, where MS1 scanned full MS mass range from m/z 300 to 2000 at 30,000 mass resolution and six HCD MS2 scans were sequentially carried out at resolution of 7500 with 45 % collision energy, both in the Orbitrap.

Gimenez M., Marie S.K., Oba-Shinjo S., Uno M., Izumi C., Oliveira J.B, & Rosa J.C. (2015). Quantitative proteomic analysis shows differentially expressed HSPB1 in glioblastoma as a discriminating short from long survival factor and NOVA1 as a differentiation factor between low-grade astrocytoma and oligodendroglioma. BMC Cancer, 15, 481.

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Shotgun Proteomics Using SWATH-MS

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1 µg of peptides were injected on a 5600 TripleTof mass spectrometer (ABSciex, Concord, Ontario, Canada) interfaced with an Eksigent NanoLC Ultra 1D Plus system (Eksigent, Dublin, CA, USA). The peptides were separated on a 75-µm-diameter, 20-cm long New Objective emitter packed with Magic C18 AQ 3 µm resin (Michrom BioResources) and eluted at 300 nl/min with a linear gradient of 5–35% Buffer A for 120 min (Buffer A: 2% acetonitrile, 0.1% FA; Buffer B: 98% acetonitrile, 0.1% FA). MS data acquisition was performed in either data-dependent acquisition (DDA, top20, with 20 s dynamic exclusion) or data-independent acquisition (DIA) SWATH-MS mode (32 fixed precursor isolation windows of 25 Da width [+1 Da overlap] each acquired for 100 ms plus one MS1 scan acquired for 250 ms) as described in Gillet et al. (2012) (link). The mass ranges recorded were 360–1460 m/z for MS1 and 50–2000 m/z for MS2. For either mode, the collision energy was set to 0.0625 × m/z – 6.5 with a 15 eV collision energy spread regardless of the precursor charge state.

Klingauf-Nerurkar P., Gillet L.C., Portugal-Calisto D., Oborská-Oplová M., Jäger M., Schubert O.T., Pisano A., Peña C., Rao S., Altvater M., Chang Y., Aebersold R, & Panse V.G. (2020). The GTPase Nog1 co-ordinates the assembly, maturation and quality control of distant ribosomal functional centers. eLife, 9, e52474.

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Peptide Sequencing by Orbitrap Mass Spectrometry

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The peptide samples were reconstituted in 0.4% acetic acid and sonicated in a sonication bath at 35 °C for 10 min. An LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific) was used, and 0.5 µg of the sample was injected into a reversed-phase C18 column (20 cm × 75 μm i.d., 3 μm, 120 Å, packed in-house; Dr. Maisch, Beim Brückle, Ammerbuch-Entringen, Germany) on an Eksigent NanoLC-ultra 1D plus system at a flow rate of 400 μL/min. The column was pre-equilibrated with 95% solvent A (0.1% formic acid in water) and 5% solvent B (0.1% formic acid in acetonitrile) for 16 h. The peptides were eluted at a flow rate of 400 μL/min with a linear gradient of 5–40% B for 240 min, followed by 80% B wash at 300 nL/min for 35 min and 5% B re-equilibration at a flow rate of 300 nL/min for 10 min. ESI spray voltage was set to 2.1 kV, capillary voltage to 21 V and the temperature of the heated capillary to 250 °C. MS survey was scanned from 350 to 1800 m/z, and the top 10 ions were selected for data-dependent MS/MS scans with the following parameters: charge state, ≥2; isolation width, 2 m/z; normalized collision energy, 35%; dynamic exclusion duration, 60 s. All data were acquired using Xcalibur software v2.2 (Thermo Fisher Scientific).

Shin S., Lee J., Kwon Y., Park K.S., Jeong J.H., Choi S.J., Bang S.I., Chang J.W, & Lee C. (2021). Comparative Proteomic Analysis of the Mesenchymal Stem Cells Secretome from Adipose, Bone Marrow, Placenta and Wharton’s Jelly. International Journal of Molecular Sciences, 22(2), 845.

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