CD2 was purified upon secretion from untreated and XBP1s-activated HEK293DAX-CD2 cells and purified CD2 SDS-gel bands were processed for N-glycan analysis as described in the Supplemental Information . MS and MS/MS data were acquired using a 4800 MALDI-TOF/TOF (Applied Biosystems) mass spectrometer. For the MS/MS, the collision energy was set to 1 kV. Ar was used as the collision gas. The 4700 calibration standard kit, Calmix (Applied Biosystems), was used as the external calibrant for the MS mode of both instruments. Human [Glu1] fibrinopeptide B (Sigma) was used as an external calibrant for the MS/MS mode of the MALDI-TOF/TOF instrument. MS and MS/MS data were processed using Data Explorer 4.9 (Applied Biosystems) following the detailed protocol presented in the Supplemental Information .
Calmix
Manufactured by Thermo Fisher Scientific
Sourced in United States
Calmix is a laboratory equipment designed for the preparation of sample mixtures. It uses a controlled agitation mechanism to ensure thorough and consistent blending of components. The core function of Calmix is to homogenize various materials for analysis and testing purposes.
Automatically generated - may contain errors
Lab products found in correlation
Hesi 2 interface, by Thermo Fisher Scientific (3 mentions)
4800 maldi tof tof, by Thermo Fisher Scientific (2 mentions)
Glu1 fibrinopeptide b human, by Merck Group (1 mentions)
Data explorer 4, by Thermo Fisher Scientific (1 mentions)
Methyl iodide, by Merck Group (1 mentions)
Tof tof series explorer, by AB Sciex (1 mentions)
Data explorer software, by AB Sciex (1 mentions)
Tof tof 5800 system, by AB Sciex (1 mentions)
Ultramark 1621, by Thermo Fisher Scientific (1 mentions)
Q exactive plus hybrid quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Q exactive orbitrap, by Thermo Fisher Scientific (1 mentions)
Exactive orbitrap, by Thermo Fisher Scientific (1 mentions)
7 protocols using calmix
1
N-Glycan Analysis of Secreted CD2
2
Permethylation and Mass Spectrometry Analysis of N-Glycans
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Permethylation of glycans was carried out as described previously (45 (link)). Briefly, N-glycans were permethylated by treatment with 0.2 ml of methyl iodide (Merck) in NaOH-DMSO (3 g of NaOH in 2 ml of DMSO) slurry for 15 min at 37 °C with intermittent mixing. Permethylated N-glycans were extracted in chloroform, dried under nitrogen, and purified on a Sep-Pak® Classic C18 column (Waters) using 3 ml each of 10, 50, and 75% acetonitrile in water. Eluted fractions were lyophilized and redissolved in 20 μl of methanol, mixed with equal volumes of Super-DHB (2,5-dihydroxybenzoic acid; 20 mg/ml in 70% methanol), and spotted on a MALDI plate. MS and MS/MS data were acquired in positive ion mode using an AB SCIEX TOF/TOF 5800 system. Calmix (Applied Biosystems) was used as an internal standard for calibration in both modes. MS/MS collision-induced dissociation was carried out with argon gas at a voltage of 1 kV. Data were acquired using a TOF/TOF Series Explorer (AB SCIEX). Data from 10,000 shots, collected from different areas of the spot (laser intensity, 4500 for MS and 5000–6000 for MS/MS), were summed up and analyzed using Data Explorer software (AB SCIEX). The observed peaks were annotated using GlycoWorkbench software.
3
MALDI-TOF Glycan Profiling Protocol
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A few microliters of permethylated N-glycan samples, resuspended in methanol, was mixed with the same volume of matrix solution (10 mg/mL 5-Chloro-2-mercaptobenzothiazole, CMBT in MeOH/H2O 80/20). MALDI TOF and MALDI TOF/TOF mass spectra were recorded in reflectron mode and positive polarity using a 4800 MALDI TOF/TOF™ (Applied Biosystems, Foster City, CA, U.S.A.) instrument, equipped with an Nd:YAG laser at 355-nm and 200 Hz repetition rate. In MS mode, 1200 shots were accumulated for each spectrum, with a resolution greater than 15K and a mass accuracy better than 75 ppm. A 4700-calibration standard kit, calmix (Applied Biosystems, Foster City, CA, U.S.A.), was used as external calibrant for the MS mode, and [Glu1] fibrinopeptide B human was used as external calibrant for the MS/MS mode (1 μL of TOF/TOF Calibration Mixture in 24 μL of CHCA matrix solution).
4
High-Resolution Q-Exactive LC-MS Protocol
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The LC system was coupled to a Thermo Scientific Q-Exactive Orbitrap mass spectrometer equipped with a HESI II interface (Thermo Scientific, Hemel Hempstead, United Kingdom). The set up was calibrated (Thermo calmix) in both ionization modes and tuned for the lower m/z range before analysis. Full scan (MS1) data was acquired in positive and negative switching mode in profile mode at 35,000 resolution (at m/z 200) using 1 microscan, an AGC target of 106 cts, a maximum injection time of 250 ms, with spray voltages +3.8 and -3.0 kV, capillary temperature 320°C, heater temperature of 150°C, sheath gas flow rate 40 a.u., auxiliary gas flow rate 5 a.u., sweep gas flow rate 5 a.u, a full scan mass window of 70–1050 m/z, and using m/z 74.0964 (+) and m/z 112.98563 (-) as locking masses. LC-MS/MS fragmentation data of selected samples from Study 1 were also obtained on this setup using similar settings.
5
Quantitative LCMS of Metabolites
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Samples were analyzed in randomized order, with an injection volume of 5 µL. Before each draw, the auto-injector was washed for 2s (10 μL/s) with 10% methanol. Ultra-high performance liquid chromatography was performed on a Thermo Vanquish instrument, using a 1.7 µm Kinetex C18 50 × 2.1 mm column, 100 Å pore size, protected by a SecurityGuard ULTRA C18 Guard Cartridge (Phenomenex), with water + 0.1% formic acid as mobile phase A and acetonitrile + 0.1% formic acid as mobile phase B, at a 0.5 mL/min flow rate. Autosampler temperature was set to 10 °C and column compartment to 40 °C. LC gradient was set according to Table 3 .
MS/MS analysis was performed on a Thermo Fisher Q-Exactive Plus hybrid quadrupole orbitrap mass spectrometer. Ions were produced using electrospray ionization and MS spectra acquired in positive mode only. Calibration was done using Thermo Fisher Calmix containing caffeine, MRFA, Ultramark 1621, and n-butylamine in acetonitrile/MeOH/acetic acid solution. Instrument parameters can be found as follows inTable 4 .
MS/MS analysis was performed on a Thermo Fisher Q-Exactive Plus hybrid quadrupole orbitrap mass spectrometer. Ions were produced using electrospray ionization and MS spectra acquired in positive mode only. Calibration was done using Thermo Fisher Calmix containing caffeine, MRFA, Ultramark 1621, and n-butylamine in acetonitrile/MeOH/acetic acid solution. Instrument parameters can be found as follows in
6
HESI-Orbitrap LC-MS Protocol for Metabolomics
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The LC system was coupled to a Thermo Scientific Q-Exactive Orbitrap mass spectrometer equipped with a HESI II interface (Thermo Scientific, Hemel Hempstead, UK). The set up was calibrated (Thermo calmix) in both ionization modes and tuned for the lower m/z range before analysis. Full scan (MS1) data was acquired in positive and negative switching mode in profile mode at 35,000 resolution (at m/z 200) using 1 microscan, an AGC target of 106 cts, a maximum injection time of 250 ms, with spray voltages + 3.8 and − 3.0 kV, capillary temperature 320 °C, heater temperature of 150 °C, sheath gas flow rate 40 a.u., auxiliary gas flow rate 5 a.u., sweep gas flow rate 5 a.u., a full scan mass window of 70–1050 m/z, and using m/z 74.0964 (+) and m/z 112.98563 (−) as locking masses.
7
Orbitrap-based Metabolomics Analysis Protocol
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The LC system was coupled to a Thermo Scientific Exactive Orbitrap mass spectrometer equipped with a HESI II interface (Thermo Scientific, Hemel Hempstead, United Kingdom). The set up was calibrated (Thermo calmix) in both ionization modes and tuned for the lower m/z range before analysis. Full scan (MS1) data was acquired in positive and negative switching mode in profile mode at 50,000 resolution (at m/z 200) using 1 microscan, an AGC target of 106 cts, a maximum injection time of 250 ms, with spray voltages +4.5 and -3.5 kV, capillary temperature 275°C, heater temperature of 150°C, sheath gas flow rate 40 a.u., auxiliary gas flow rate 5 a.u., sweep gas flow rate 5 a.u, a full scan mass window of 70–1400 m/z, and using m/z 74.0964 (+) and m/z 112.98563 (-) as locking masses.
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