DNA binding proteins were denatured by addition SDS to a final concentration of 5%. Then proteins were reduced and alkylated with 10 mM tris(2-carboxyethyl) phosphine and 30 mM iodoacetamide. Afterward, proteins were precipitated in suspension traps (S-Trap micro, Protifi), and sample clean-up was performed according to manufacturer’s instructions. Proteins were digested in the S-Trap in presence of 2 μg Trypsin/Lys-C (Promega) for overnight at 37°C. Eluted peptides were dried and resuspended in 1% formic acid. Mass spectrometry was performed on an orbitrap Fusion Tribrid instrument (Thermo Fisher) equipped with an Easy-nLC 1000 HPLC system, a 75 μm by 2 cm PepMap C18 trapping column, a 75 μm by 50 cm PepMap RSLC C18 analytical column, and an Easy-Spray ion source. Peptides were separated by 1-h gradient with 0.1% formic acid and acetonitrile (3–30%). Precursor ion scans were acquired in the orbitrap and CID fragments were acquired in the linear ion trap. Data analysis was performed using MaxQuant software (version 1.6.17.0) with the human Uniprot reference proteome database (downloaded on 9/8/21).
S trap micro
Manufactured by Protifi
The S-Trap Micro is a lab equipment product designed for protein sample preparation. It functions as a protein extraction and clean-up tool, allowing for efficient protein isolation and purification prior to mass spectrometry analysis.
Automatically generated - may contain errors
Lab products found in correlation
Speedvac, by Labconco (3 mentions)
Fusion tribrid, by Thermo Fisher Scientific (1 mentions)
Easy nlc 1000 hplc system, by Thermo Fisher Scientific (1 mentions)
Trypsin lys c, by Promega (1 mentions)
Methanol, by Thermo Fisher Scientific (1 mentions)
Lys c, by Fujifilm (1 mentions)
Chloroform methanol, by Thermo Fisher Scientific (1 mentions)
Sodium dodecyl sulfate (sds), by Thermo Fisher Scientific (1 mentions)
Phosphoric acid, by Merck Group (1 mentions)
Iodoacetamide, by Merck Group (1 mentions)
Irt peptide, by Biognosys (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Trypsin, by Promega (1 mentions)
Quantitative colorimetric assay, by Thermo Fisher Scientific (1 mentions)
6 protocols using s trap micro
1
Proteomic Profiling of Denatured Proteins
2
Proteome Preparation via S-Trap Digestion
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Samples were solubilized and digested per the S-Trap Micro (Protifi) manufacturer’s protocol2 . Briefly, samples were solubilized in 50 μL of extraction buffer containing 5% sodium dodecyl sulfate (SDS, Affymetrix), 50mM TEAB (pH 8.5, Sigma), and protease inhibitor cocktail (Roche cOmplete, EDTA free), reduced in 5mM TCEP (Thermo), alkylated in 20mM iodoacetamide (Sigma), then acidified with phosphoric acid (Aldrich) to a final concentration of 1.2%. Samples were diluted with 90% methanol (Fisher) in 100 mM TEAB, then loaded onto an S-trap column and washed three times with 50/50 chloroform/methanol (Fisher) followed by three washes of 90% methanol in 100 mM TEAB. A 1:10 ratio (enzyme: protein) of Trypsin (Promega) and LysC (Wako) suspended in 20μL 50mM TEAB was added, and samples were digested for 1.5 hours at 47 °C in a humidity chamber. After incubation, peptides were eluted with an additional 40 μL of 50 mM TEAB, followed by 40 μL of 0.1% trifluoroacetic acid (TFA) (Pierce) in water, and finally 40 μL of 50/50 acetonitrile:water (Fisher) in 0.1% TFA. Eluates were combined and desalted directly using Phoenix peptide cleanup kit (PreOmics) per manufactures protocol, dried by vacuum centrifugation and reconstituted in 0.1% TFA containing iRT peptides (Biognosys, Schlieren, Switzerland).
3
Proteomic Analysis of Liver and Brain Microsomes
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Liver and brain microsomes from mice, cynomolgus macaques, and humans were prepared for proteomic analyses using S-Trap spin columns (S-Trap Micro, PN: C02-Micro-10; ProtiFi, Farmingdale, NY) according to the manufacturer’s protocol, where peptides were generated via reduction, alkylation, acidification, and tryptic digestion. Peptides were then dried down, reconstituted in water 0.1% formic acid, and quantified using a quantitative colorimetric assay (PN: 23275; Thermo Fisher) according to the manufacturer’s protocol. To improve the depth of proteins identified for the targeted P450 and UGT methods, 100 μg of the brain microsome peptides were also separated into eight fractions using high pH reversed-phase chromatography (PN 84868; Thermo Fisher) according to the manufacturer’s protocol.
4
Protein Digestion and Labeling for Mass Spectrometry
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Samples were diluted to 1 mg/mL in 5% SDS, 100 mM TEAB, and 25 μg of protein from each sample was reduced with dithiothreitol to 2 mM, followed by incubation at 55°C for 60 minutes. Iodoacetamide was added to 10 mM and incubated in the dark at room temperature for 30 minutes to alkylate proteins. Phosphoric acid was added to 1.2%, followed by six volumes of 90% methanol, 100 mM TEAB. The resulting solution was added to S-Trap micros (Protifi), and centrifuged at 4,000 × g for 1 minute. The S-Traps containing trapped protein were washed twice by centrifuging through 90% methanol, 100 mM TEAB. 1 μg of trypsin was brought up in 20 μL of 100 mM TEAB and added to the S-Trap, followed by an additional 20 μL of TEAB to ensure the sample did not dry out. The cap to the S-Trap was loosely screwed on but not tightened to ensure the solution was not pushed out of the S-Trap during digestion. Samples were placed in a humidity chamber at 37°C overnight. The next morning, the S-Trap was centrifuged at 4,000 × g for 1 minute to collect the digested peptides. Sequential additions of 0.1% TFA in acetonitrile and 0.1% TFA in 50% acetonitrile were added to the S-trap, centrifuged, and pooled. Samples were frozen and dried down in a Speed Vac (Labconco) prior to TMTpro labeling.
5
Protein Digestion and TMT Labeling
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Samples were diluted to 1 mg/ml in 5% SDS, 100 mM TEAB, and 25 μg of protein from each sample was reduced with dithiothreitol to 2 mM, followed by incubation at 55°C for 60 min. Iodoacetamide was added to 10 mM and incubated in the dark at room temperature for 30 min to alkylate the proteins. Phosphoric acid was added to 1.2%, followed by six volumes of 90% methanol, 100 mM TEAB. The resulting solution was added to S‐Trap micros (Protifi), and centrifuged at 4,000 g for 1 min. The S‐Traps containing trapped protein were washed twice by centrifuging through 90% methanol, 100 mM TEAB. 1 μg of trypsin was brought up in 20 μl of 100 mM TEAB and added to the S‐Trap, followed by an additional 20 μl of TEAB to ensure the sample did not dry out. The cap to the S‐Trap was loosely screwed on but not tightened to ensure the solution was not pushed out of the S‐Trap during digestion. Samples were placed in a humidity chamber at 37°C overnight. The next morning, the S‐Trap was centrifuged at 4,000 g for 1 min to collect the digested peptides. Sequential additions of 0.1% TFA in acetonitrile and 0.1% TFA in 50% acetonitrile were added to the S‐trap, centrifuged, and pooled. Samples were frozen and dried down in a Speed Vac (Labconco) prior to TMTpro labeling.
6
Protein Reduction, Alkylation, and Tryptic Digestion
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Samples were diluted to 1 mg/ml in 5% SDS and 100 mM TEAB, and 25 µg of protein from each sample was reduced with dithiothreitol to 2 mM, followed by incubation at 55°C for 60 min. Iodoacetamide was added to 10 mM and incubated in the dark at room temperature for 30 min to alkylate proteins. Phosphoric acid was added to 1.2%, followed by six vol of 90% methanol and 100 mM TEAB. The resulting solution was added to S-Trap micros (Protifi) and centrifuged at 4,000 × g for 1 min. The S-Traps containing trapped protein were washed twice by centrifuging with 90% methanol and 100 mM TEAB. 1 µg of trypsin was brought up in 20 μl of 100 mM TEAB and added to the S-Trap, followed by an additional 20 μl of TEAB to ensure the sample did not dry out. The cap to the S-Trap was loosely screwed on but not tightened to ensure the solution was not pushed out of the S-Trap during digestion. Samples were placed in a humidity chamber at 37°C overnight. The next morning, the S-Trap was centrifuged at 4,000 × g for 1 min to collect the digested peptides. Sequential additions of 0.1% TFA in acetonitrile and 0.1% TFA in 50% acetonitrile were added to the S-trap, centrifuged, and pooled. Samples were frozen and dried down in a Speed Vac (Labconco) prior to TMTpro labeling.
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