Ultrapure-grade 2,2,2-trifluoroethanol, trifluoroacetic acid, iodoacetamide and dithiotreitol (DTT) were from Sigma-Aldrich. High-performance liquid chromatography-grade solvents (methanol, acetonitrile and water) and formic acid were from Fisher Scientific. MS-grade trypsin/Lys-C was from Promega (Madison, WI). Amicon spin filters, 0.5 ml, 3 kDa molecular weight cut-off (MWCO), were from Millipore. Solid-phase extraction C18 tips were from Agilent. A measure of 4 ml of EPS urine was concentrated to ∼500 μl using a spin filter with a molecular weight cutoff of 3 kDa, and proteins were precipitated overnight by the addition of ice-cold 100% methanol. Protein pellets were washed twice with 100% methanol and air-dried. Protein resolubilization was performed by the addition of 50% 2,2,2-trifluoroethanol at 60 °C for 2 h. Following reduction with DTT and alkylation with iodoacetamide, proteins were digested overnight at 37 °C using 2 μg trypsin/Lys-C. The reaction was quenched by the addition of trifluoroacetic acid. Desalting was performed by solid-phase extraction using C18 tips. Solvents were removed by vacuum centrifugation, and peptides were resolubilized in 5% acetonitrile and 0.1% formic acid. Peptide concentrations were determined by the micro-BCA assay kit (Thermo Fisher Scientific).
Ms grade trypsin lys c
Manufactured by Promega
MS-grade trypsin/Lys-C is a proteolytic enzyme designed for mass spectrometry (MS) applications. It is a mixture of trypsin and Lys-C, two commonly used enzymes for protein digestion prior to MS analysis. This product is optimized for high-quality peptide generation to support MS-based proteomics workflows.
Automatically generated - may contain errors
Lab products found in correlation
Amicon spin filter, by Merck Group (1 mentions)
Formic acid, by Thermo Fisher Scientific (1 mentions)
Trifluoroacetic acid, by Merck Group (1 mentions)
Iodoacetamide, by Merck Group (1 mentions)
High performance liquid chromatography grade solvents, by Thermo Fisher Scientific (1 mentions)
Micro bca assay kit, by Thermo Fisher Scientific (1 mentions)
Rapigest sf surfactant, by Waters Corporation (1 mentions)
Ammonium bicarbonate, by Merck Group (1 mentions)
Proteasemax surfactant, by Promega (1 mentions)
5 protocols using ms grade trypsin lys c
1
Proteomics Analysis of Extracellular Vesicle Proteins
2
Respiratory Supercomplexes Proteolytic Digestion
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5 µL of purified cyt. bc1c4c5–cbb3 respiratory SC at 3 mg/mL in GTBS was diluted five-fold into digestion buffer to achieve a final concentration of 5 mM DTT, 50 mM Tris-HCl pH 8.0, and 0.1% RapiGest SF surfactant (Waters). This mixture was heated to 60 °C for 30 min. After cooling the sample to RT, cysteine residues were alkylated by the addition of 15 mM iodoacetamide for 30 min in the dark. The sample was digested with MS-grade trypsin/Lys-C (Promega) with a final ratio of 1:20 (w/w) SC: protease. Digestion was allowed to proceed for 18 h at 37 °C before quenching with 0.5% (v/v) trifluoroacetic acid at 37 °C for 30 min. The sample was subsequently centrifuged at 13,000 ×g for 10 min to remove the RapiGest SF, and the supernatant was placed into a separate microcentrifuge tube, frozen, and stored at −80 °C before MS analysis.
3
Mass Spectrometry Reagent Preparation
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Mobile phases were prepared using LC-MS-grade acetonitrile (ACN) and water (J. T. Baker, Netherlands). Formic acid (FA), and ammonium bicarbonate, were purchased from Sigma-Aldrich (Saint Louis, USA). MS-grade trypsin/Lys-C was purchased from Promega (# V5071, Fitchburg, WI).
4
Sequential Immunoprecipitation for NfL Analysis
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A three-step, sequential immunoprecipitation was used to characterize NfL in brain lysate
and CSF. Antibodies targeting Coil 1A/1B of the rod domain (HJ30.13), Coil 2B of the rod
domain (HJ30.4) and the tail region (HJ30.11) were used. Frozen brain lysates were thawed
and a 450 µl aliquot of the thawed brain lysate was diluted 1:1000 with 1% HSA. Frozen CSF
samples were thawed at room temperature, and 450 µl of the thawed CSF was transferred to a
new 1.6 ml new tube for immunoprecipitation.
Both brain and CSF samples were immunoprecipitated as described above for native CSF
using 30 µl of a 30% (i.e. 3 mg/ml) slurry of an antibody-conjugated bead preparation of
HJ30.13 (Coil 1A/1B antibody). Washed beads were stored on ice until all samples were
ready for on-bead digestion. In the second step, 20 µl of 0.5 ng/ml NfL ISTD in 50 mM
TEABC was added, and NfL was immunoprecipitated a second time by adding 30 µl of a 30%
(i.e. 3 mg/ml) slurry of an antibody-conjugated bead preparation of HJ30.4 (Coil 2B
antibody). The remaining steps were identical to the first immunoprecipitation. Ten
nanograms of ISTD in 50 mM TEABC were again added to the post-IP supernatant prior to the
third sequential immunoprecipitation, which was performed with HJ30.11 (tail antibody).
Bound NfL was digested on beads with 400 ng MS-grade trypsin/Lys-C (Promega) for 16 h at
37°C and samples were extracted as described above.
and CSF. Antibodies targeting Coil 1A/1B of the rod domain (HJ30.13), Coil 2B of the rod
domain (HJ30.4) and the tail region (HJ30.11) were used. Frozen brain lysates were thawed
and a 450 µl aliquot of the thawed brain lysate was diluted 1:1000 with 1% HSA. Frozen CSF
samples were thawed at room temperature, and 450 µl of the thawed CSF was transferred to a
new 1.6 ml new tube for immunoprecipitation.
Both brain and CSF samples were immunoprecipitated as described above for native CSF
using 30 µl of a 30% (i.e. 3 mg/ml) slurry of an antibody-conjugated bead preparation of
HJ30.13 (Coil 1A/1B antibody). Washed beads were stored on ice until all samples were
ready for on-bead digestion. In the second step, 20 µl of 0.5 ng/ml NfL ISTD in 50 mM
TEABC was added, and NfL was immunoprecipitated a second time by adding 30 µl of a 30%
(i.e. 3 mg/ml) slurry of an antibody-conjugated bead preparation of HJ30.4 (Coil 2B
antibody). The remaining steps were identical to the first immunoprecipitation. Ten
nanograms of ISTD in 50 mM TEABC were again added to the post-IP supernatant prior to the
third sequential immunoprecipitation, which was performed with HJ30.11 (tail antibody).
Bound NfL was digested on beads with 400 ng MS-grade trypsin/Lys-C (Promega) for 16 h at
37°C and samples were extracted as described above.
5
Protein Denaturation, Reduction, and Tryptic Digestion
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Protein samples were denatured by the addition of a final concentration of 6 M urea and 2 M thiourea and reduced by the addition of a final concentration of 10 mM dithiothreitol (DTT) followed by incubation at 37 °C for 1 h, 240 rpm. The samples were then cooled down to room temperature before being alkylated by the addition of a final concentration of 50 mM iodoacetamide followed by incubation in the dark for 30 min. Pre-chilled (−20 °C) acetone (10× volume) was used to precipitate the samples overnight at −20 °C. Samples were centrifuged at 14,000g for 40 min at 4 °C, and the supernatant was subsequently discarded. Protein pellets were dried using a speed vac, resuspended in 0.1 M TEAB buffer, pH 8.0, containing 0.02% ProteaseMax surfactant (in the case for lipoprotein samples) and MS grade Trypsin/Lys-C (Promega Cooperation) (1:25 enzyme: protein) and digested overnight at 37 °C, 240 rpm. Digestion was stopped by acidification with TFA. Peptides were then purified robotically, using C18 cartridges (Bravo AssayMAP, Agilent Technologies).
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