The lysates of the equivalent to 4 × 108 purified sEV were processed according to the sensitive Sp3 protocol [19 (link)]. The cysteine residues were reduced in 100 mM DTT and alkylated in 100 mM iodoacetamide (Acros Organics). 20 ug of beads (1:1 mixture of hydrophilic and hydrophobic SeraMag carboxylate-modified beads, GE Life Sciences) were added to each sample in 50% ethanol. Protein clean-up was performed on a magnetic rack. The beads were washed two times with 80% ethanol and once with 100% acetonitrile (Fisher Chemical). The captured beads proteins were digested overnight at 37 °C under vigorous shaking (1200 rpm, Eppendorf Thermomixer) with 0.5 ug Trypsin/LysC (MS grade, Promega) prepared in 25 mM Ammonium bicarbonate. Next day, the supernatants were collected and the peptides were purified using a modified Sp3 clean-up protocol and finally solubilized in the mobile phase A (0.1% Formic acid in water), sonicated, and the peptide concentration was determined through absorbance at 280 nm measurement using a nanodrop instrument.
Trypsin lysc ms grade
Manufactured by Promega
Sourced in Austria
Trypsin/LysC (MS grade) is a laboratory reagent used for the digestion and processing of proteins for mass spectrometry analysis. It contains a mixture of the proteolytic enzymes trypsin and LysC, which are commonly used to cleave proteins into smaller peptides for identification and characterization.
Automatically generated - may contain errors
Lab products found in correlation
Iodoacetamide, by Thermo Fisher Scientific (3 mentions)
Seramag carboxylate modified beads, by GE Healthcare (3 mentions)
Acetonitrile, by Thermo Fisher Scientific (3 mentions)
Iodacetamid, by Merck Group (2 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
6 protocols using trypsin lysc ms grade
1
Sensitive Sp3 Protein Cleanup and Digestion
2
Sensitive Sp3 Protein Digestion and Cleanup
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The lysed samples were processed according to the sensitive Sp3 protocol [32 (link)]. The cysteine residues were reduced in 100 mM DTT and alkylated in 200 mM iodoacetamide (Acros Organics). 20 ug of beads (1:1 mixture of hydrophilic and hydrophobic SeraMag carboxylate-modified beads, GE Life Sciences) were added to each sample in 50% ethanol. Protein clean-up was performed on a magnetic rack. The beads were washed two times with 80% ethanol and once with 100% acetonitrile (Fisher Chemical). The captured on-beads proteins were digested overnight at 37 °C under vigorous shaking (1200 rpm, Eppendorf Thermomixer) with 0.5 ug Trypsin/LysC (MS grade, Promega) prepared in 25 mM ammonium bicarbonate. Next day, the supernatants were collected, and the peptides were purified using a modified Sp3 clean up protocol and finally solubilized in the mobile phase A (0.1% Formic acid in water), sonicated and the peptide concentration was determined through absorbance at 280-nm measurement using a nanodrop instrument.
3
Filter-Assisted Protein Digest Protocol
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The proteins were used for a filter-assisted protein digest, as described previously [25 (link)]. Briefly, the isolated proteins were centrifuged at 4536×g for 30 min and the protein pellet dried. After dissolving in sample buffer, the protein concentration was determined with a Bradford assay and 20 µg of total protein was used for the digestion. After reduction with dithiothreitol and alkylation with iodacetamid (both Sigma-Aldrich, Vienna, Austria), proteins were digested with Trypsin/Lys-C (MS grade; Promega Corporation, Madison, WI, USA) and dried via vacuum centrifugation.
4
Filter-Assisted Protein Digestion Protocol
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The proteins were used for a filter-assisted protein digest as described previously (15 (link)). In short, the precipitated proteins were centrifuged at 4536 × g for 30 min, the supernatant discarded and the protein pellet dried. After dissolving in sample buffer the protein concentration was determined and 20 μg of total protein was used for the digestion. After reduction with dithiothreitol and alkylation with iodacetamid (both Sigma-Aldrich) proteins were digested with Trypsin/Lys-C (MS grade; Promega Corporation, Madison, WI) and dried via vacuum centrifugation.
5
Sensitive Sp3-based Protein Extraction
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The protein extracts, obtained from three representative collection sites in Naxos and three in Lakoma, with three biological replicates each one at harvest and at post-harvest, were processed according to the sensitive Sp3 protocol. The cysteine residues were reduced in 100 mM DTT and alkylated in 200 mM iodoacetamide (Acros Organics). 20 μg of beads (1:1 mixture of hydrophilic and hydrophobic SeraMag carboxylate-modified beads, GE Life Sciences) were added to each sample in 50% ethanol. Protein clean-up was performed on a magnetic rack. The beads were washed twice with 80% ethanol and once with 100% acetonitrile (Fisher Chemical). The captured-on beads proteins were digested overnight at 37°C under vigorous shaking (1,200 rpm, Eppendorf Thermomixer) with 1 μg Trypsin/LysC (MS grade, Promega) prepared in 25 mM Ammonium bicarbonate. The next day, the supernatants were collected, and the peptides were purified using a modified Sp3 clean up protocol and finally solubilized in the mobile phase A (0.1% Formic acid in water), sonicated and the peptide concentration was determined through absorbance at 280 nm measurement using a nanodrop instrument.
6
Proteomic Analysis of Fetal Kidney
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Isolated male fetal kidneys (n = 3 for each group) were homogenized in sample buffer (7.5 M urea, 1.5 M thiourea, 4% CHAPS, 0.05% SDS, 100 mM dithiothreitol (DTT)) using an ultrasonic stick. Protein concentrations were determined by means of a Bradford assay (Bio-Rad-Laboratories). An in-gel digestion protocol was applied as described previously (13 (link), 14 (link)). In brief, 80 μg of each sample was loaded on SDS-PAGE, which allowed us to prefractionate the sample in order to reduce the complexity thereof. After fixation with 50% methanol/10% acetic acid, the gels were washed and sensitized with 0.02% Na2S2O3. Gels were then stained with 0.1% AgNO3 for 10 min, rinsed and developed with 3% Na2CO3/0.05% formaldehyde. Each protein band was then cut into four slices and destained. Upon reduction with DTT and alkylation with iodoacetamide (IAA), the proteins were digested enzymatically using Trypsin/Lys-C (MS grade; Promega Corporation). Following digestion, the peptides were eluted, dried, and stored at −20 °C until LC-MS/MS analysis.
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