Proteomics Dynamic Range Standard (UPS2) sample was acquired from Sigma-Aldrich (St. Louis, MO), the MassPREP E. coli Digest Standard was acquired from Waters (Milford, MA) and the MS compatible human protein extract digest was from Promega (Madison, WI). The UPS2 samples were reduced with 5 mM TCEP (tris(2-carboxyethyl)phosphine), alkylated with 50 mM iodoacetamide, and digested overnight with 1 μg trypsin (Promega, Madison, WI) in 100 mM Tris pH 8 at 37°C. UPS2, E. coli, and human peptides were acidified with formic acid and loaded at various concentrations, alone or in combination, onto an in-house made 75 μm x 12 cm analytical column emitter packed with 3 μm ReproSil-Pur C18-AQ (Dr. Maisch HPLC GmbH, Germany). A NanoLC-Ultra 1D plus (Eksigent, Dublin CA) nano-pump was used to deliver a 90 minute gradient from 2% to 35% acetonitrile with 0.1% formic acid, followed by a 30 minute wash with 80% acetonitrile prior to re-equilibration to 2% acetonitrile with 0.1% formic acid.
Nanolc ultra 1d plus
Manufactured by AB Sciex
Sourced in United States
The NanoLC Ultra 1D plus is a high-performance liquid chromatography system designed for nanoscale separations. It features a modular design and precise flow control for analytical and preparative applications.
Automatically generated - may contain errors
Lab products found in correlation
Tripletof 5600 mass spectrometer, by AB Sciex (8 mentions)
Tripletof 5600, by AB Sciex (3 mentions)
C18 nanoacquity beh analytical column, by Waters Corporation (3 mentions)
C18 pepmap trap column, by Thermo Fisher Scientific (3 mentions)
Acclaim pepmap100, by Thermo Fisher Scientific (2 mentions)
Acclaim pepmap 100 c18, by Thermo Fisher Scientific (2 mentions)
Acquity uplc m class peptide beh c18 column, by Waters Corporation (2 mentions)
Triple tof 5600 system, by AB Sciex (2 mentions)
Massprep e coli digest standard, by Waters Corporation (1 mentions)
Trypsin, by Promega (1 mentions)
Pierce magnetic rna protein pull down kit, by Thermo Fisher Scientific (1 mentions)
T7 or sp6 rna polymerase, by New England Biolabs (1 mentions)
Ltq orbitrap velos, by Thermo Fisher Scientific (1 mentions)
Mascot server, by Matrix Science (1 mentions)
High speed tripletof 5600 mass spectrometer, by AB Sciex (1 mentions)
Mascot software, by Matrix Science (1 mentions)
Peakview software, by AB Sciex (1 mentions)
Acclaim pepmap rslc, by Thermo Fisher Scientific (1 mentions)
Analyst tf 1, by AB Sciex (1 mentions)
Qubit fluorometric quantitation, by Thermo Fisher Scientific (1 mentions)
15 protocols using nanolc ultra 1d plus
1
Benchmarking Proteomics Standards for Mass Spectrometry
2
lnc-CTSLP4 Interactome Analysis
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In vitro transcription of lnc-CTSLP4 full-length sense, antisense, and serial truncated sequences were performed using T7 or SP6 RNA Polymerase (New England Biolabs, Ipswich, Suffolk, England) according to the manufacturer’s instructions (T7 RNA polymerase was used to perform in vitro transcription of lnc-CTSLP4 full-length sense and SP6 RNA polymerase was used for lnc-CTSLP4 full-length antisense). RNA pulldown assays were performed with the Pierce Magnetic RNA-Protein Pull-Down Kit according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA). lncRNA-interacting proteins were obtained and subjected to MS analysis. LC-MS/MS detection was carried out on a hybrid quadrupole-TOF mass spectrometer (TripleTOF 5600+, SCIEX) equipped with a nanoLC system (nanoLC-Ultra 1D Plus, Eksigent).
3
Phosphoproteomic Analysis of IP-Enriched Peptides
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The IP-enriched peptides were analyzed on an LTQ Orbitrap Velos (Thermo Fisher Scientific, San Jose, CA) coupled with a nanoLC system (Eksigent nanoLC-Ultra 1D plus, Dublin, CA). Peptides were separated on a C18 reversed phase column (100mm long, ID 75μm, 5μm 300Å BetaBasic, New Objective, Woburn, MA) using a 90-min linear gradient from 2% to 35% acetonitrile in 0.1% formic acid at a flow rate of 250nL/min. Mass analysis was conducted in data-dependent analysis (DDA) mode, where MS1 scanned mass range from 300 to 2000 m/z at 30,000 resolution in the Orbitrap, and 10 collision-induced dissociation (CID) MS2 scans were sequentially carried out in the ion trap.
The LC-MS data were searched against the SwissProt Human database using Mascot server (Matrix Science, London, UK; version 2.3). Searching parameters were set as: precursor mass tolerance at 20ppm, fragment ion mass tolerance at 0.8 Da, trypsin enzyme with 2 miscleavages, carbamidomethylation of cysteine as fixed modification, and deamidation of asparagine and glutamine, oxidation of methionine, and tyrosine/serine/threonine phosphorylation as variable modifications. Peptides identified from database search were filtered with a false discovery rate (FDR) of 0.05. Relative quantitation of phosphopeptides was calculated based on areas under the curve (AUC) of corresponding peptides from the control and treated samples.
The LC-MS data were searched against the SwissProt Human database using Mascot server (Matrix Science, London, UK; version 2.3). Searching parameters were set as: precursor mass tolerance at 20ppm, fragment ion mass tolerance at 0.8 Da, trypsin enzyme with 2 miscleavages, carbamidomethylation of cysteine as fixed modification, and deamidation of asparagine and glutamine, oxidation of methionine, and tyrosine/serine/threonine phosphorylation as variable modifications. Peptides identified from database search were filtered with a false discovery rate (FDR) of 0.05. Relative quantitation of phosphopeptides was calculated based on areas under the curve (AUC) of corresponding peptides from the control and treated samples.
4
Nano-LC-MS/MS for Peptide Profiling
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One-microgram aliquots of tryptic peptides per sample (injection volume 5 μL) were analyzed on a nano liquid chromatography system (Eksigent Technologies nanoLC Ultra 1D plus, AB SCIEX, Foster City, CA) coupled to a 5600 Triple TOF mass spectrometer (AB SCIEX) with a nanoelectrospray ion source. Samples were injected into a C18 PepMap trap column (5 µm, 100 µm I.D. × 2 cm, Thermo Scientific) at 2 µL/min, in 0.1% formic acid in water, and the trap column was connected on-line to a C18 nanoAcquity BEH analytical column (1.7 µm, 100 Å, 75 µm I.D. × 15 cm, Waters). The nanopump provided a flow-rate of 250nL/min and the gradient elution conditions were as follows: 0.1% formic acid in water as mobile phase A, and 0.1% formic acid in acetonitrile as mobile phase B, from 5 to 40% B in 120 min. The mass spectrometer operated in data-dependent acquisition mode. For MS1 scans, the accumulation time was set to 250 ms and up to 10 precursor ions were acquired per spectrum (100 ms for each MS2), which represents a total cycle time of 1.3 s. This shorter duty cycle allows for the acquisition of a greater number of points per MS1 ion precursor, which is essential to improve quantitation in label-free based quantitative approaches.
5
2D nanoLC-MS/MS Proteomics Workflow
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Five microliters of sample (0.4 µg/µL) was injected into a 2D nano liquid chromatography (nanoLC) ESI-MS/MS system (Eksigent Technologies nanoLC Ultra 1D Plus, AB SCIEX, Foster City, CA, USA) coupled to a High Speed TripleTOF 5600 Mass Spectrometer (SCIEX, Foster City, CA, USA) with a Nanospray III source. The trap column was an Acclaim PepMap 100 (ThermoFisher Scientific, Rockford, IL, USA) with a 5 µm particle diameter and 100 Å pore size, switched online with the analytical column. The loading pump delivered a solution of 0.1% formic acid in water at 2 µL/min. The nanopump provided a flow rate of 300 nL/min and was operated under gradient elution conditions, using 0.1% formic acid in water as mobile phase A and 0.1% formic acid in acetonitrile as mobile phase B. These assays were performed in the Proteomic Laboratory at the National Center for Biotechnology (CNB), Madrid, Spain.
6
LC-MS/MS Proteomics Workflow for Wheat and Barley
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For the LC–MS/MS (nanoLC-Ultra 1D Plus and TripleTOF 5600, both are produced by AB SCIEX, Waltham, MA, America) sample processing steps of the target protein, refer to Kong et al. [28 (link)]. Protein identification was performed with MASCOT software (2.5.1 Version, Matrix Science, Columbia, MO, USA) (http://www.matrixscience.com/ ) by searching the uniprot database. The database used for the search was a self-built database including “xylan”, “xylanase” and “GH10 domain containing protein” from wheat and barley. The searching parameters were as Table 6 :
7
Nanoliquid Chromatography-Mass Spectrometry Analysis of Muropeptides
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Samples of digested PG (muropeptides) were analysed on a nano liquid chromatography system (Eksigent Technologies nanoLC Ultra 1D plus, AB SCIEX, Foster City, CA) coupled to a 5600 Triple TOF mass spectrometer (AB SCIEX, Foster City, CA) with a nano-electrospray ion source. Samples were injected on a C18 PepMap trap column (5 μm, 100 μm I.D. x 2 cm, Thermo Scientific) at 2 μL/min, in 0.1% formic acid in water, and the trap column was switched on-line to a C18 nanoAcquity BEH analytical column (1.7 μm, 100 Å, 75 μm I.D. x15 cm, Waters). Equilibration was done in mobile phase A (0.1% formic acid in water), and peptide elution was achieved in a 40 min linear gradient from 5% - 40% B (0.1% formic acid in acetonitrile) at 250 nL/min. Instrument calibration was continuously updated through automatic injection and calibration of a commercial peptide mixture (Beta-gal tryptic digest, AB SCIEX). Mass accuracy ranged between 5–10 ppm. The mass spectrometer operated in data-dependent acquisition mode. For TOF scans, the accumulation time was set to 250 ms, and per cycle, up to 15 precursor ions were monitored. MS1 and MS2 spectra were analysed manually using PeakView software (SCIEX).
8
Peptide Sample Separation via nanoLC
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For each spot, a total volume of 5 μl of the solution of tryptic digestion peptides was injected with a flow rate of 300 nL/min in a nanoLC Ultra1D plus (ABSciex © , Framingham, MA, USA). A trap column, the Acclaim PepMap100 (100 µm x 2 cm; C18, 2 µm, 100Å) and an analytical column, the Acclaim PepMap RSLC (75 µm x 15 cm, C18, 5 µm, 100 Å) from Thermo Scientific (Thermo Fisher Scientific Inc © , Waltham, MA, USA) were used following the next gradient: 0-1 min (5% B), 1-50 min
9
Proteomic Analysis of Fractionated Samples
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A 2 µg aliquot of each resulting fraction was subjected to LC-ESI-MS/MS analysis as described elsewhere [24] , using a nanoLC Ultra 1D plus (ABSciex © , Framingham, MA, USA) coupled online to a 5600 Triple TOF mass spectrometer (ABSciex © , Framingham, MA, USA). The MS and MS/MS data obtained were analyzed as a
10
1D-nano LC-ESI-MS/MS Proteomic Analysis
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The digested sample was cleaned up/desalted using Stage-Tips C18 (Merck) and the peptide concentration was determined by Qubit™ Fluorometric Quantitation (Thermo Fisher Scientific). A 1 µg aliquot of each digested sample was subjected to 1D-nano LC-ESI-MS/MS analysis using a nano liquid chromatography system (Eksigent Technologies nanoLC Ultra 1D plus, SCIEX) coupled to a high-speed TripleTOF 5600 mass spectrometer (SCIEX) with a Nanospray III source. Peptides were separated using a 250 min gradient ranging from 2% to 90% mobile phase B (mobile phase A: 2% acetonitrile, 0.1% formic acid; mobile phase B: 100% acetonitrile, 0.1% formic acid).
The data were acquired using an ion spray voltage floating (ISVF) 2300 V, curtain gas (CUR) 35, interface heater temperature (IHT) 150, ion source gas 1 (GS1) 25, and declustering potential (DP) 100 V. All the data were acquired using information-dependent acquisition (IDA) mode (0.25 s MS survey scan in the mass range of 350–1250 Da and 35 MS/MS scans of 100 ms in the mass range of 100–1800 Da) using Analyst TF 1.7 software (SCIEX, USA).
The data were acquired using an ion spray voltage floating (ISVF) 2300 V, curtain gas (CUR) 35, interface heater temperature (IHT) 150, ion source gas 1 (GS1) 25, and declustering potential (DP) 100 V. All the data were acquired using information-dependent acquisition (IDA) mode (0.25 s MS survey scan in the mass range of 350–1250 Da and 35 MS/MS scans of 100 ms in the mass range of 100–1800 Da) using Analyst TF 1.7 software (SCIEX, USA).
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