This was done on a Thermo Scientific Dionex UltiMate™ 3000 RSLC nano system (Sunnyvale, CA, USA). The analytical column was a capillary Acclaim PepMap RSLC (Dionex, Sunnyvale, CA, USA) C18 column (300 μm × 15 cm, 2 μm) that was kept at 30 °C and operated at a flow rate of 4 μL/min. The guard/trap column was a C18 PepMap 100 μ-precolumn (300 μm × 5 mm, 5 μm). Mobile phase A was water with 0.05% TFA and mobile phase B was acetonitrile. The trap column mobile phase was 1% acetonitrile with 20 mM TEAA. The samples were maintained at 5 °C in the autosampler. The Urine Extract (5 μL) was injected and the mobile phase was as follows: 4 min with 20 mM TEAA / 1% acetonitrile at 20 μL/min on the trap column (sent to waste), followed by a linear gradient after switching from 10% to 80% solvent B in 100 min, held at 80% of B for 4 min, returned to 10% B in 1 min, and re-equilibrated at 10% B for 3 min. UV absorbance detection was monitored at 220 nm, 260 nm and 320 nm. Collection of the eluate as 20 sec droplets was done with a Probot Microfraction Collector (Dionex) onto a blank MALDI plate (Opti-TOF 384 Well Insert, 123 × 81 mm, AB SCIEX, Framingham, MA, USA). CCA matrix (0.5 μL of 5 mg/mL in 50% acetonitrile with 7 mM ammonium phosphate dibasic to suppress salt adducts) was added to each spot manually using an Eppendorf Research Pro electronic pipette.
Opti tof 384 well insert
Manufactured by AB Sciex
Sourced in United States
The Opti-TOF 384 Well Insert is a laboratory equipment product designed for use with time-of-flight mass spectrometry (TOF-MS) systems. It provides a platform for automated sample handling and analysis. The core function of this product is to facilitate the efficient processing and introduction of samples into the TOF-MS instrument.
Automatically generated - may contain errors
Lab products found in correlation
Acclaim pepmap rslc, by Thermo Fisher Scientific (1 mentions)
Probot microfraction collector, by Thermo Fisher Scientific (1 mentions)
Ultimate 3000 rslcnano system, by Thermo Fisher Scientific (1 mentions)
Abi 4800 maldi tof tof analyzer, by Thermo Fisher Scientific (1 mentions)
4800 plus maldi tof tof analyzer, by AB Sciex (1 mentions)
Mascot software, by Matrix Science (1 mentions)
Data explorer version 4, by AB Sciex (1 mentions)
5800 maldi tof tof mass spectrometer, by AB Sciex (1 mentions)
Ziptip column, by Merck Group (1 mentions)
4800 maldi tof tof, by AB Sciex (1 mentions)
6 protocols using opti tof 384 well insert
1
Shotgun Proteomics by Nano-HPLC-UV-MALDI
2
Cross-linking and Mass Spectrometry Analysis of NADase and SLO
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NADase, SLO, or NADase plus SLO, each at concentrations of 12 μM in PBS, 1 mM DTT, pH 7.4, was cross-linked with 0.005% glutaraldehyde for 10 min at room temperature. Reactions were stopped with Laemmli buffer and analyzed by SDS-PAGE. For MALDI-TOF mass spectrometry analysis, the reaction volume was 500 μl; reactions were stopped after 10 min with addition of Tris to 100 mM and then concentrated and buffer exchanged into 20 mM Tris, pH 6.8, using a spin concentrator (molecular weight cutoff [MWCO], 10,000; Pierce). The samples were then diluted 1:10 in 0.1% trifluoroacetic acid (TFA) and spotted with α-cyano-4-hydroxycinnamic acid (CHCA) on an Opti-TOF 384-well insert (AB Sciex). Masses were determined using standard protocols (51 (link)) on an ABI 4800 MALDI-TOF/TOF analyzer (Applied Biosystems).
3
MALDI-TOF/TOF Bioactive Peptide Identification
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Peptides from SPH, DSPI and DSPH were detected and acquired by a MALDI-TOF/TOF mass spectrometer (4800 Plus MALDI TOF/TOF Analyzer, SCIEX, Framingham, MA). Each spectrum was the accumulated sum of at least 2000 laser shots within the ion range at m/z 700–5000. The samples were co-crystallized with HCCA matrix (4-hydroxycinnamic-α-cyano acid), in a stainless steel MALDI sample plate (Opti-TOF 384- Well insert, AB SCIEX, Framingham, MA) at room temperature. MS data was acquired in reflector positive mode. Some of the ion peptide peaks were further selected for MS/MS sequencing analysis. Combined Peptide Mass Fingerprint (PMF) + MS/MS analysis was performed with the Mascot software (Matrix Science, UK) using the UniProt protein sequencing database for Glycine max taxonomic selection. Methionine oxidation was selected as a variable modification.
To determine the bioactivity, all the identified peptides were searched against a database available online, BIOPEP (Minkiewicz et al., 2008 (link)). For SPH peptides which coincided with those reported in our previous study (Coscueta et al., 2016 (link)), the frequency of bioactive fragments occurrence in peptide sequence (A) was analysed: where a is the number of fragments with a given activity in a peptide sequence, and N is the number of amino acid residues of the peptide (Dziuba, Iwaniak, & Minkiewicz, 2003 ).
To determine the bioactivity, all the identified peptides were searched against a database available online, BIOPEP (Minkiewicz et al., 2008 (link)). For SPH peptides which coincided with those reported in our previous study (Coscueta et al., 2016 (link)), the frequency of bioactive fragments occurrence in peptide sequence (A) was analysed: where a is the number of fragments with a given activity in a peptide sequence, and N is the number of amino acid residues of the peptide (Dziuba, Iwaniak, & Minkiewicz, 2003 ).
4
MALDI-TOF Mass Spectrometry of Peptide-Bound Samples
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The SA MALDI matrix was prepared by dissolving 4 mg in 1 mL of a solution prepared with 50% of ACN in 0.1% TFA. The solution was then sonicated for 1 min and then centrifuged for 5 min at 2000× g. NF samples were prepared by a dry-droplet method. In addition, 1 μL of the solution containing the bound peptides was mixed with 4 μL of SA solution prepared as described above and 1 μL of the obtained mixture was spotted on the MALDI target plate (Opti-TOF 384-Well Insert, ABSciex, Framingham, MA, USA). It was allowed to dry at room temperature before insertion into the mass spectrometer. MALDI-TOF mass spectra were recorded on AB SCIEX MALDI-TOF/TOF 5800 mass spectrometer (ABSciex, Framingham, MA, USA). For MALDI MS measurements in SA, the following settings were applied: bin size was set at 4 ns, final detector voltage was 2.070 kV with multiplier value at 0.75, and 2000 laser shots were accumulated for each spectrum. External mass calibration was performed using the 5800 Mass Standards kit (AB SCIEX, Framingham, MA, USA) containing insulin bovine (MH+ 5734.59), thioredoxin (MH+ 11,674.48), and horse apomyoglobin (MH+ 16,952.56). Data Explorer version 4.11 software (AB SCIEX, Framingham, MA, USA) was used for data acquisition and data processing.
5
Protein Spot Identification and Analysis
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Identification of protein spots of interest was performed as described previously (Jayapalan et al., 2012 (link); Jayapalan et al., 2013 (link)). Further, spots were carefully excised from 2DE gels and destained with potassium ferricyanide (15 mM) and sodium thiosulfate (50 mM) for 20 min at about 20 °C. The proteins were reduced with 10 mM DTT (10 mM) and ammonium bicarbonate (100 mM) for 25 min, and alkylated with 55 mM iodoacetamide (55 mM) for 15 min, at 60 °C and in the dark, respectively. This was followed by subsequent washings with 50 and 100% acetonitrile (ACN, 50 and 100%) in 100 mM ammonium bicarbonate (100 mM), and dehydration of the gel plugs using vacuum centrifugation. The spots were digested with trypsin (6 ng/µl in ammonium bicarbonate solution (50 mM)) at 37 °C, for about 12 h. Peptides were extracted from the gels using ACN (50 and 100 %) subsequently. Extracted peptides were lyophilized, treated with formic acid (0.1%) and desalted using ZipTip columns containing C18 reversed phase media (Millipore, Madison, USA). The sample peptide was mixed with α-cyano-4-hydroxycinamic acid (5 mg/ml) at a ratio of 1:1, and 0.7 µl of the mixture was immediately spotted onto an OptiToF 384-well insert and analyzed using 5,800 MALDI ToF/ToF analyser (ABSciex, Toronto, Canada).
6
Bac7 (1-35) Identification by Mass Spectrometry
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The 30 μg of His6-PrS2-Bac7 (1–35) was digested with 4 μg of factor Xa in a 50 μl mixture containing 20 mM Tris–HCl (pH 8.0). 50 mM NaCl and 2 mM CaCl2. The reaction mixture was then incubated for 4 h at 37 °C, and the cleavage product was analyzed by 19 % SDS-PAGE followed by Coomassie blue staining. In order to confirm Bac7 (1–35) by mass spectrometry, His6-PrS2-Bac7 (1–35) was cleaved by factor Xa protease and the reaction mixture was diluted 25 times by the matrix solution containing sinapinic acid (10 mg/ml) in 0.1 % trifloroacetic acid and 50 % acetonitrile) and spotted on to a target plate (Opti-TOF 384 well insert, ABSciex) and air dried, followed by mass spectrometric analysis by a MALDI-TOF (4800 MALDI-TOF/TOF, ABSciex) using the positive mid-mass linear mode from 2 to 30 kDa.
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