CSF and serum IgG (0.5 to 2.0 ml) was applied to protein A sepharose columns (GE Healthcare, Pittsburgh, PA, USA) and purified according to the manufacturer’s instructions. Protein concentrations were determined by bicinchoninic acid (BCA) Protein-Assay (Pierce, Thermo Fisher, Waltham, MA, USA), and the purity analyzed by sodium dodecyl sulfate polyacrylamide electrophoresis. Excised heavy- and light-chain gel pieces were destained in ammonium bicarbonate/50% acetonitrile (ACN) and dehydrated in 100% acetonitrile. Disulfide bonds were reduced by dithiothreitol, and cysteine residues were alkylated with iodoacetamide. Heavy-chain proteins were digested with trypsin, light chains with trypsin and S. aureus V8 protease (Glu-C). Following digestion, the tryptic mixtures were extracted in 1% formic acid/50% acetonitrile). Samples were analyzed on a linear trap quadropole (LTQ) Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) coupled to an Eksigent nanoLC-2D system (Framingham, MA, USA) through a nanoelectrospray LC-MS interface using a 90-minute gradient from 6 to 40% ACN. Peptide fragmentation was performed in a higher energy collisional dissociation cell with normalized collision energy of 40%, and tandem mass spectra were acquired in the Orbitrap mass analyzer. Data acquisition was performed using Xcalibur software (version 2.0.6; Waltham, MA, USA).
Nanolc 2d system
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The NanoLC-2D system is a liquid chromatography instrument designed for the separation and analysis of complex samples at the nanoscale. It features two independent pumps that enable high-resolution gradient formation and precise flow control for efficient separation of analytes. The system is equipped with an autosampler and a UV-Vis detector to provide automated and sensitive detection of separated compounds.
Automatically generated - may contain errors
Lab products found in correlation
Ltq orbitrap velos mass spectrometer, by Thermo Fisher Scientific (3 mentions)
Protein a sepharose column, by GE Healthcare (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
Mascot, by Matrix Science (1 mentions)
Xcalibur software 2, by Thermo Fisher Scientific (1 mentions)
Ltq orbitrap xl mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Xcalibur, by Thermo Fisher Scientific (1 mentions)
Mascot server, by Matrix Science (1 mentions)
4 protocols using nanolc 2d system
1
Immunoglobulin Purification and Mass Spectrometry
2
Mass Spectrometry Analysis of CSF and Serum IgG Peptides
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CSF and serum IgG (0.5–2.0 mL) peptides were analyzed by mass spectrometry as described previously.6 Heavy‐chain proteins were digested with trypsin and the tryptic mixtures were extracted in 1% formic acid/50% acetonitrile (ACN). Samples were analyzed on a linear trap quadropole (LTQ) Orbitrap Velos mass spectrometer (Thermo Fisher Scientific) coupled to an Eksigent nanoLC‐2D system through a nanoelectrospray LC−MS interface using a 90 min gradient from 6% to 40% ACN. Peptide fragmentation was performed in a higher energy collisional dissociation cell with normalized collision energy of 40%, and tandem mass spectra were acquired in the Orbitrap mass analyzer. Data acquisition was performed using Xcalibur software (version 2.0.6). Tandem mass (MS/MS) spectra were converted into mgf files using an in‐house script. Mascot (version 2.2; Matrix Science Inc., London, UK) was used to perform database searches against our database containing translated blood B‐cell VH transcriptome CDR3 parts (+10 amino acids in front and after the CDR3 part). Peptide tolerance was set at ± 15 ppm with an MS/MS tolerance of ± 0.1 Da from spectra. Scaffold (version4, Portland, OR, USA) was used to validate each individual peptide and protein identifications. Peptide identifications were accepted at a ˃95.0% probability, protein identifications at a ˃90.0% probability.
3
Peptide Analysis by Nano-LC-MS/MS
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Peptides were separated using an Eksigent nano-LC 2D™ system coupled to an LTQ-Orbitrap XL mass spectrometer (ThermoFisher Scientific, Bremen, Germany) operated in data-dependent acquisition mode. Survey full-scan MS spectra (m/z 300–2000) were acquired in the Orbitrap in centroid mode, with a resolution of 60,000 at m/z. The spray voltage was set to 2 kV, and the temperature of the heated capillary was 180°C. After the MS1 survey scan, a multistage activation was performed by scanning for neutral losses generated by the three most intense ions. In case of neutral loss, a further stage of CID fragmentation as described above was triggered to fragment the peptide backbone. The Xcalibur software 2.0.7 (ThermoScientific) controlled HPLC, mass spectrometer and the data acquisition. All unassigned charge states and singly charged ions were rejected for fragmentation. The dynamic exclusion list was limited to a maximum of 500 masses with a maximum retention time of minutes and a relative mass window of 10 ppm.
4
Untargeted Proteomic Identification via LC-MS/MS
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