Analyses were performed using an Ultimate 3000 Rapid Separation liquid chromatographic system (Dionex, The Netherlands) coupled to a hybrid Linear Trap Quadrupole-ORBITRAP Velos mass spectrometer (Thermo Fisher Scientific, San José CA). Peptides were separated on a C18 RP analytical column (3 μm particle size, 100 Å pore size, 75 μm i.d., 50 cm length) with a 240-minute gradient from 99% A (ACN 5%, formic acid 0.1% and H2O 95%) to 40% B (ACN 80%, formic acid 0.085% and H2O 20%). The LTQ-ORBITRAP mass spectrometer acquired data throughout the elution process and operated in a data dependent scheme as follows: full MS scans were acquired with the ORBITRAP, followed by up to 10 LTQ MS/MS CID spectra on most abundant precursors detected in the MS scan, as described in details previously24 (link)31 (link).
Linear trap quadrupole orbitrap velos mass spectrometer
Manufactured by Thermo Fisher Scientific
The Linear Trap Quadrupole-ORBITRAP Velos mass spectrometer is a high-performance hybrid mass analyzer. It combines a linear ion trap with an Orbitrap mass analyzer to provide high resolution, accurate mass measurements, and fast data acquisition rates for a wide range of applications.
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Lab products found in correlation
Easy nlc 2, by Thermo Fisher Scientific (2 mentions)
Proteome discoverer 2, by Thermo Fisher Scientific (1 mentions)
Nanoelectrospray ion source, by Thermo Fisher Scientific (1 mentions)
20 cm fused silica emitter, by New Objective (1 mentions)
Reprosil pur c18 aq 1.9 μm resin, by Dr. Maisch (1 mentions)
Mascot search engine, by Matrix Science (1 mentions)
5 protocols using linear trap quadrupole orbitrap velos mass spectrometer
1
Liquid Chromatography-Mass Spectrometry Proteomics
2
Quantifying Proteome Changes in Stretched Cells
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3 µg protein extract of stretched and unstretched cells were reduced in 20 mM ammoniumbicarbonate by 2.5 mM dithiothreitol (DTT) for 1 h at 60 °C and with 10 mM iodoacetic acid (IAA) for 30 min at 37 °C. Subsequently the probes were digested by trypsin over night at 37 °C. MS-data were acquired on a linear-trap quadrupole Orbitrap Velos mass spectrometer (Thermo Fisher Scientific) equipped with a nanoelectrospray ion source. The resulting data were analyzed using the Proteome Discoverer 2.0 software (Thermo Fisher Scientific). SEQUEST-HT and MASCOT search engines were used for the peptide/protein identification. The database searches used the Uniprot Swiss-Prot database (version: October 2015).
3
Nanoscale Peptide Separation and MS Analysis
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Each of the 21 fractions was then re-suspended in 2% acetonitrile/0.1% TFA acid in water and separated by nanoscale C18 reverse-phase liquid chromatography performed on an EASY-nLC II (Thermo Scientific) coupled to a Linear Trap Quadrupole - Orbitrap Velos mass spectrometer (Thermo Scientific). Elution was carried out using a binary gradient with buffer A: 2% acetonitrile and B: 80% acetonitrile, both containing 0.1% of formic acid. Peptides were subsequently eluted at 200 nl/min flow, into a 20 cm fused silica emitter (New Objective) packed in-house with ReproSil-Pur C18-AQ, 1.9 μm resin (Dr Maisch GmbH). Packed emitter was kept at 35°C by means of a column oven integrated into the nanoelectrospray ion source (Sonation). Peptides were eluted at a flow rate of 200 nl/min using 3 different gradients optimised for set of fractions 1-7 (2,20,41% buffer B), 8-15 (5, 25, 46% buffer B) and 16-21 (7, 28, 50% buffer B). Two-step gradients were used, all with 42 minutes for step one and 13 minutes for step two.All gradients were followed by a washing step (100% B) for 10 minutes followed by a 20 minute re-equilibration step (5%), for a total run time of 85 minutes.
Eluting peptides were electrosprayed into the mass spectrometer using a nanoelectrospray ion source (Thermo Scientific). An Active Background Ion Reduction Device was used to decrease air contaminants signal level.
Eluting peptides were electrosprayed into the mass spectrometer using a nanoelectrospray ion source (Thermo Scientific). An Active Background Ion Reduction Device was used to decrease air contaminants signal level.
4
Proteomic Profiling of Trophoblastic Extracellular Vesicles
5
Nanoscale LC-MS/MS Peptide Separation and Analysis
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The tryptic digest obtained was separated by nanoscale C18 reverse‐phase liquid chromatography performed on an EASY‐nLC II (Thermo Scientific) coupled to a Linear Trap Quadrupole—Orbitrap Velos mass spectrometer (Thermo Fisher). Elution was carried out using a binary gradient with buffer A: 2% acetonitrile and B: 80% acetonitrile, both containing 0.1% of formic acid. Samples were resuspended and loaded with buffer A, on a precolumn NS‐MP‐10 BioSphere C18 5 μm 120 Å– 360/100 μm × 0.2cm of length (NanoSeparations), and washed with 25 µL of buffer A at a maximum pressure of 200 bar. Desalted peptides were subsequently eluted at 200 nL·min−1 flow, into a 20 cm fused silica emitter (New Objective) packed in‐house with ReproSil Gold 200 C18, 3 μm resin (Dr. Maisch GmbH, Ammerbuch, Germany). The gradient used started at 5% of buffer B and was increased to 28% over 42 min and then to 45% over 13 min. Finally, a washing step at 100% of B was carried out over 10 min followed by a 5‐min re‐equilibration at 5% B for a total duration of 70 min. The eluting peptide solutions were automatically (online) electrosprayed into the mass spectrometer via a nanoelectrospray ion source. An Active Background Ion Reduction Device (ABIRD) was used to decrease the contaminant signal level.
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