The α-cyano-4-hydroxycinnamic acid (CHCA) matrix solution was prepared at 5.5 mg/mL in 6 mM ammonium phosphate monobasic, 50% acetonitrile, and 0.1% trifluoroacetic acid (Sigma Aldrich). The recovered peptides, 0.72 μL from cytochrome c or 3 μL from C. albicans, were mixed in a 1 : 1 (v/v) ratio with this CHCA matrix solution. All sample-matrix mixtures were spotted at a volume of 0.75 μL on a 384 well Opti-TOF 123 × 83 mm SS MALDI plate (Sciex, MA, USA). The instrument is equipped with a 349 nm OptiBeam On-Axis laser with a pulse rate at 400 Hz. Data acquisition and processing were done using TOF-TOF Series Explorer (Sciex) and Data Explorer. The spectra were acquired in Reflectron positive mode from 500–3500 m/z. Peak lists were created using the following parameters: a peak density of 10 per 25 Da, minimal signal-to-noise (S/N) of 10, minimum area of 50, and a maximum peak per spot of 200. Second fragmentation, MS/MS, was also performed via postsource decay (PSD) using the 1 kV in positive ion acquisition mode.
Tof tof series explorer
Manufactured by AB Sciex
Sourced in United States
The TOF-TOF Series Explorer is a mass spectrometer instrument designed for high-resolution analysis of molecular samples. It utilizes a time-of-flight (TOF) mass analyzer to provide accurate mass measurements. The instrument is capable of performing tandem mass spectrometry (MS/MS) experiments, where the sample is fragmented and the resulting ions are analyzed.
Automatically generated - may contain errors
Lab products found in correlation
Data explorer, by AB Sciex (3 mentions)
Acetonitrile, by Merck Group (1 mentions)
Trifluoroacetic acid, by Merck Group (1 mentions)
5800 maldi tof tof mass spectrometer, by AB Sciex (1 mentions)
Massprep automated digester station, by PerkinElmer (1 mentions)
Calmix, by Thermo Fisher Scientific (1 mentions)
Methyl iodide, by Merck Group (1 mentions)
Data explorer software, by AB Sciex (1 mentions)
Tof tof 5800 system, by AB Sciex (1 mentions)
4 protocols using tof tof series explorer
1
CHCA Matrix-Assisted MALDI-TOF/MS Protocol
2
MALDI-MS Analysis of Amyloid-beta Oligomers
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All values were presented as the mean ± standard error of the mean (S.E.M.) of four independent experiments (n=4). Statistical comparisons between experimental groups were performed using the GraphPad Prism software. A two-way ANOVA with Tukey's HSD test was applied. P < 0.05 was considered to have statistical significance.
Mass Spectrometry. Sinapinic acid (SA) was selected as the MALDI matrix on this work based on preliminary experiments comparing it with the two other most commonly used matrices, alpha-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid. SA matrix was prepared to 10 mg/mL in 50% ACN and 0.05% TFA. The Aβ sample and SA matrix were deposited as sandwiched layers, where 0.75 μL of the Aβ solution was spotted between two layers of SA (0.75 μL). The spots were washed twice with 1 μL water prior to analysis. MALDI-MS sample analyses were performed on an Sciex TOF/TOF 5800 MALDI mass spectrometer. TOF-TOF Series Explorer in positive ion linear mode and Data Explorer were used for data acquisition and processing, respectively (Sciex). Total sum of 200 shots/spot were acquired with a 1 kHz OptiBeam onaxis Nd:YAG laser system. Lowest possible laser intensity was used to minimize dissociation and favor the detection of Aβ oligomers. Other instrument parameters were generally optimized for sensitivity while compromising resolution to an acceptable extent.
Mass Spectrometry. Sinapinic acid (SA) was selected as the MALDI matrix on this work based on preliminary experiments comparing it with the two other most commonly used matrices, alpha-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid. SA matrix was prepared to 10 mg/mL in 50% ACN and 0.05% TFA. The Aβ sample and SA matrix were deposited as sandwiched layers, where 0.75 μL of the Aβ solution was spotted between two layers of SA (0.75 μL). The spots were washed twice with 1 μL water prior to analysis. MALDI-MS sample analyses were performed on an Sciex TOF/TOF 5800 MALDI mass spectrometer. TOF-TOF Series Explorer in positive ion linear mode and Data Explorer were used for data acquisition and processing, respectively (Sciex). Total sum of 200 shots/spot were acquired with a 1 kHz OptiBeam onaxis Nd:YAG laser system. Lowest possible laser intensity was used to minimize dissociation and favor the detection of Aβ oligomers. Other instrument parameters were generally optimized for sensitivity while compromising resolution to an acceptable extent.
3
In-Gel Trypsin Digestion for MALDI-TOF/TOF
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In-gel digestion was performed using MassPREP automated digester station (PerkinElmer, Downers Grove, IL, USA). Gel pieces were de-stained using 50 mM ammonium bicarbonate and 50% acetonitrile, which was followed by protein reduction using 10 mM DTT, alkylation using 55 mM IAA, and tryptic digestion in 50 mM ammonium bicarbonate, pH 8. Peptides were extracted using a solution of 1% formic acid and 2% acetonitrile and lyophilized. Prior to mass spectrometry analysis, dried peptide samples were re-dissolved in a 10% acetonitrile and 0.1% trifluoroacetic acid solution. MALDI matrix, a–cyano–4–hydroxycinnamic acid, was prepared as 5 mg/mL in 6 mM ammonium phosphate monobasic, 50% acetonitrile, 0.1% trifluoroacetic acid and mixed with the sample at a 1:1 ratio (v/v). Mass spectrometry data (Figure S1 ) were obtained using an AB Sciex 5800 MALDI TOF/TOF system (Framingham, MA, USA). Data acquisition and data processing were done using a TOF/TOF Series Explorer a nd Data Explorer (both from AB Sciex, Boston, MA, USA), respectively. The instrument was equipped with a 349 nm Nd:YLF OptiBeam On-Axis laser. The laser pulse rate was 400 Hz. Reflectron positive mode was used. Reflectron mode was externally calibrated at 50 ppm mass tolerance and internally at 10 ppm. Each mass spectrum was collected as a sum of 500 shots.
4
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.
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