To complement the SDS-PAGE and capillary viscometry analyses, SGel and SGelMA samples were analyzed by matrix-assisted laser desorption/ionization time-of flight mass spectroscopy (MALDI-TOF MS) as described before by Liu et al., 2012 [75 (link)]. Samples were mixed with 5 µL of trifluoroacetic acid (TFA) 0.1% v/v and 5 µL of matrix solution (10 mg/mL of synaptic acid in a 50:50 acetonitrile mixture). Then, 1 µL of this mixture was applied to a target plate. Mass spectra were obtained using MALDI-TOF MS Autoflex Speed (Bruker Daltonics, Bremen, Germany) equipped with a smart beam (334 nm) source. Spectra were obtained using a positive and lineal mode with an accelerating voltage of 20 kV. Each spectrum was collected as an average of 1200 laser shots with sufficient energy to produce good spectra.
Autoflex speed maldi tof ms
Manufactured by Bruker
Sourced in Germany, United States
The Autoflex Speed MALDI-TOF-MS is a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF-MS) produced by Bruker. It is designed for the analysis of various types of samples, including biomolecules, polymers, and small molecules. The Autoflex Speed MALDI-TOF-MS utilizes a high-performance laser and a time-of-flight mass analyzer to provide accurate mass measurements and high sensitivity.
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10 protocols using autoflex speed maldi tof ms
1
MALDI-TOF MS Analysis of Hydrogel Samples
2
MALDI-TOF MS for Fungal Identification
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Fresh colony yeast was applied directly to a sample plate of the equipment, the samples were coated with a saturated solution of α-cyano acid 4-hydroxy cinnamic diluted in 50% ACN with 2.5% TFA. The Mass spectra were recorded using a MALDI-TOF MS Autoflex Speed (Bruker Daltonics, Bremen, Germany) equipped with an intelligent beam laser source (334 nm). The analyses were analyzed in linear mode with positive polarity, acceleration voltage of 20 kV and delay extraction of 220 ns. Each spectrum was collected as an average of 1200 laser shots with enough energy to produce good spectra without saturation in the range of m/z from 2000 to 20,000. Before analysis, the equipment was externally calibrated with the protein calibration standard I (Bruker Daltonics, Bremen, Germany, insulin, ubiquitin, cytochrome C and myoglobin) with FlexControl 1.4 software (Bruker Daltonics, Bremen, Germany). The analyses were analyzed with the MALDI Biotyper Compass 4.1 software (Bruker Daltonics, Bremen, Germany) in the range of m/z 3000–15,000 compared with a library of 1301 spectra of fungal identifications.
3
Yeast Colony MALDI-TOF-MS Identification
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Samples of yeast were incubated in the dark at 37 °C for 48 h, then colony samples were applied directly to a sample plate of the equipment, following the procedures as described by Gobom et al., 2011 [34 (link)], where the sample was coated with a saturated solution of α-cyano acid 4-hydroxy cinnamic diluted in 50% ACN with 2.5% TFA. The mass spectra were used using a MALDI-TOF-MS Autoflex Speed (Bruker Daltonics, Bremen, Germany) equipped with an intelligent beam laser source (334 nm). Analysis was carried out in the linear mode with positive polarity, an acceleration voltage of 20 kV and a delay extraction of 220 ns. Each spectrum was collected as an average of 1200 laser shots with enough energy to produce good spectra without saturation in the range of m/z from 2000 to 20,000. Before analysis, the equipment was externally calibrated with the protein calibration standard I (Bruker Daltonics, Bremen, Germany; insulin, ubiquitin, cytochrome C and myoglobin) with FlexControl 1.4 software (Bruker Daltonics, Bremen, Germany). Analysis was carried out with the MALDI Biotyper Compass 4.1 software (Bruker Daltonics, Bremen, Germany) in the range of m/z 3000–15,000 compared to a library of 1301 spectra of fungal identifications.
4
Phosphorylation Ratio Analysis by MALDI-TOF-MS
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α-Cyano-4-hydroxycinnamic acid matrix (CHCA) at 10 mg/mL was prepared in 50% water/acetonitrile and 0.1% trifluoroacetic acid. First, the matrix and samples were mixed at a ratio of 100:1. Then, 1 μL of the analyte/matrix mixture was applied to the MALDI plate and allowed to dry to induce crystallization. Analyses were conducted using a MALDI-TOF-MS autoflex speed (Bruker, Billerica, MA, USA) in the positive ion linear mode. All the spectra were analyzed using flexAnalysis (Applied Biosystems, Waltham, MA, USA). The phosphorylation ratio, defined as the ratio of the ion intensity of the phosphorylated material to that of the unphosphorylated material, was calculated using the following formula: [phosphorylated peptide intensity/(phosphorylated peptide intensity + non-phosphorylated peptide intensity) × 100].
5
Drug Conjugate Characterization and Analysis
6
MALDI-TOF Mass Spectrometry Protocol
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Matrix-assisted laser desorption/ionization (MALDI) time of flight (TOF) mass spectrometry analysis was performed on a BRUKER Autoflex Speed MALDI TOF MS using dithranol as matrix.
7
Casein Hydrolysis by Chymosin
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To examine the effects of chymosin on the hydrolysis characteristics of individual caseins, individual β-CN, αs-CN, and β-LG samples with 0.03 units of chymosin/mL were hatched at 30 °C for 0 and 3 h. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used to determine the molecular weights of the individual β-CN, αs-CN, and β-LG samples, which were analyzed on a Bruker Autoflex Speed MALDI-TOF-MS (Bruker Daltonics, Billerica, MA, USA) by measuring the mass-to-charge proportion (m/z) of the ionized analyte and recording the number of ions at each m/z value. The spectrum was acquired in linear positive mode at a laser frequency of 200 Hz. A method optimized for the mass range was established using the following parameters: acquisition range: 5000 to 35,000 Da.
8
Conjugation of Maytansine to aCD74 IgG
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Polished aCD74 IgG was diluted to 1 mg/ml in PBS and incubated at room temperature overnight with a three‐fold molar excess (40 μM) of DBCO‐maytansine drug‐linker. Excess un‐reacted drug‐linker was removed by desalting spin colum (Zeba spin desalting plate, 7 K MWCO, Thermo Fisher Scientific) equilibrated with PBS. Product quality was accessed ay analytical SEC and the Drug Antibody Ratio (DAR) was determined by reduced matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF). Briefly, protein was reduced with 10 mM TCEP for 20 min at 37°C, diluted 1:1 with S‐DHB matrix (50 mg/ml in 30% acetonitrile:70% water w 0.1% TFA) and dried on a groud‐steel MALDI target. Spectra were acquired on a Bruker autoflex speed MALDI‐TOF MS and DAR was calculated by peak height.
9
Thermal Polymerization of Precursor 1
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The precursor 1 was pressed into a pill form (Specac compression device, seven tons) and put into glass ampoule. The ampoule was sealed under vacuum after three-pump-thaw cycles (~10−2 mbar) and then heated to 520 K for 72 h. After cooling to room temperature, the crude mixture was analyzed by MALDI-ToF mass spectrometry measurements without further purification. MALDI-ToF MS spectra were recorded on a Bruker Autoflex Speed MALDI-ToF MS (Bruker Daltonics, Bremen, Germany) with a tetracyanoquinodimethane (TCNQ) as the matrix. For the thermal polymerization in a vacuum furnace, a powder sample of the precursor was placed inside a tube furnace. After evacuation of the tube, a gas flow consisting of 100 sccm hydrogen and 500 sccm argon was introduced, raising the pressure in the tube to 1.5 mbar. The furnace was kept for 15 min at room temperature for removing residual moisture and oxygen from the system, and then heated to 450 °C within 14 min and kept at this temperature for 31 min. The sample was cooled down to a room temperature under the gas flow.
10
Polymer Characterization by NMR, GPC, and MALDI-TOF
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1H, 13C, and DOSY NMR experiments were performed at 25 °C on a Varian Mercury NMR 400 spectrometer, where chemical shifts (δ in ppm) were determined with respect to the nondeuterated solvents as a reference. Gel permeation chromatography (GPC) was conducted on the instrument equipped with two columns (Shodex KF-803 and KF-804, Tokyo, Japan), a Waters 515 HPLC pump, and a differential refractive index detector (LabAlliance RI2000, New York, USA). Tetrahydrofuran (THF) mixed with tetrabutylammonium bromide (TBAB) (1 wt%) was utilized as an eluent at a flow rate of 1 mL min−1 at 40 °C [49 (link)]. The calibration curve was established by linear polystyrene standards. MALDI-TOF-MS measurements were conducted on a Bruker Autoflex Speed MALDI-TOF-MS (Bruker Daltonics, Billerica, MA, USA). The polystyrene samples for MALDI-TOF-MS measurements were prepared by mixing the polymer (5.0 mg mL−1), dithranol (DIT) (10 mg mL−1), and trifluoroacetic acid (TFA) (1%) in THF.
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