Samples rested at RT for 20 minutes before heating to 99°C for 5 min. FASP was performed using a 30 kDa molecular weight cutoff filter (VIVACON 500; Sartorius Stedim Biotech GmbH, 37070 Goettingen, Germany) essentially according to the procedure described by Wisniewski et al.52 (link). Fifty µl of each cleared protein extract was mixed with 200 μl of freshly prepared 8 M urea in 100 mM Tris-HCl (pH 8.5) (UA-solution) in the filter unit and centrifuged at 14,000 g for 15 min at 20°C to remove SDS. Any residual SDS was washed out by a second washing step with 200 μl of UA. The proteins were alkylated with 100 μl of 50 mM iodoacetamide in the dark for 30 min at RT. Afterward, three washing steps with 100 μl of UA solution were performed, followed by three washing steps with 100 µl of 50 mM TEAB buffer (Sigma-Aldrich). Proteins were digested with 1.25 µg trypsin overnight at 37 °C. Peptides were recovered using 40 μl of 50 mM TEAB buffer followed by 50 μl of 0.5 M NaCl (Sigma-Aldrich). Peptides were desalted using C18 solid phase extraction spin columns (The Nest Group, Southborough, MA), organic solvent removed in a vacuum concentrator at 45°C and reconstituted in 5% formic acid and stored at -80°C until LC-MS/MS analysis (see Supplementary Methods , also for analyses of mass spectrometry data).
Teab buffer
Manufactured by Merck Group
Sourced in United States
TEAB buffer is a chemical solution commonly used in various laboratory applications. It serves as a buffer to maintain a specific pH range in scientific experiments and analyses. The buffer helps to stabilize the pH of the solutions, ensuring consistent and reliable experimental conditions. TEAB buffer is a widely used reagent in various fields of research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
C18 solid phase extraction spin columns, by Nest Group (6 mentions)
Formic acid, by Thermo Fisher Scientific (6 mentions)
Trypsin, by Promega (5 mentions)
Acetonitrile, by Thermo Fisher Scientific (4 mentions)
C18 resin, by Nest Group (3 mentions)
0.5 m nacl, by Merck Group (2 mentions)
Vivacon 500, by Sartorius (2 mentions)
Trifluoroacetic acid tfa, by Merck Group (2 mentions)
Pierce 660 nm protein assay, by Thermo Fisher Scientific (2 mentions)
Proteome discoverer 2, by Thermo Fisher Scientific (1 mentions)
Tmt label reagent, by Thermo Fisher Scientific (1 mentions)
Easy nlctm 1200 system, by Thermo Fisher Scientific (1 mentions)
Nanospray flex source, by Thermo Fisher Scientific (1 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Iodoacetamide, by Merck Group (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Bca assay, by Beyotime (1 mentions)
Trypsin, by Merck Group (1 mentions)
Tmt 6 plex reagent, by Thermo Fisher Scientific (1 mentions)
Ammonium formate, by Merck Group (1 mentions)
17 protocols using teab buffer
1
Filtered Protein Extraction and Digestion
2
Filtered Protein Extraction and Digestion
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Samples rested at RT for 20 minutes before heating to 99°C for 5 min. FASP was performed using a 30 kDa molecular weight cutoff filter (VIVACON 500; Sartorius Stedim Biotech GmbH, 37070 Goettingen, Germany) essentially according to the procedure described by Wisniewski et al.52 (link). Fifty µl of each cleared protein extract was mixed with 200 μl of freshly prepared 8 M urea in 100 mM Tris-HCl (pH 8.5) (UA-solution) in the filter unit and centrifuged at 14,000 g for 15 min at 20°C to remove SDS. Any residual SDS was washed out by a second washing step with 200 μl of UA. The proteins were alkylated with 100 μl of 50 mM iodoacetamide in the dark for 30 min at RT. Afterward, three washing steps with 100 μl of UA solution were performed, followed by three washing steps with 100 µl of 50 mM TEAB buffer (Sigma-Aldrich). Proteins were digested with 1.25 µg trypsin overnight at 37 °C. Peptides were recovered using 40 μl of 50 mM TEAB buffer followed by 50 μl of 0.5 M NaCl (Sigma-Aldrich). Peptides were desalted using C18 solid phase extraction spin columns (The Nest Group, Southborough, MA), organic solvent removed in a vacuum concentrator at 45°C and reconstituted in 5% formic acid and stored at -80°C until LC-MS/MS analysis (see Supplementary Methods , also for analyses of mass spectrometry data).
3
Phosphoproteome Analysis of MAL2 Overexpression
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Lysates of NCI-H23 cells infected with control lentivirus or overexpressing MAL2 were further lysed with sonication at 4 °C for 3 min (80 W, on 1 s and off 1 s) and centrifuged at 12,000×g at 4 °C for 10 min to remove insoluble particles, then centrifuged one more time and the supernatant was collected. The samples were fractionated using sequencing-grade trypsin in 100 mM TEAB buffer (Sigma). Then, the samples were labeled using the TMT label reagent (Thermo scientific) according to the manufacturer’s instructions. Phosphorylated peptides were enriched by using titanium dioxide beads (TiO2). Mass spectrometry analysis was performed by Shanghai OE-biotech (China) with an EASY-nLCTM 1200 system (Thermo, USA) in Q-Exactive mass spectrometer equipped with a Nanospray Flex source (Thermo, USA). The data were processed with Proteome Discoverer™ 2.2 (Thermo, USA) software against the Homo sapiens Uniprot database. The phosphorylatedproteins with an average fold change (FC > 1.2; P < 0.05) in the experimentally treated groups were considered to be the differentially accumulated proteins.
4
Protein Denaturation and Reduction Protocol
5
SDS-PAGE Gel Protein Preparation
6
Plasma Protein Quantification and Trypsin Digestion
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Protein quantification used the Bicinchoninic Acid (BCA) assay (Beyotime, P0012). To preserve the integrity of low‐abundance proteins, we chose not to deplete high‐abundance proteins during sample preparation. Each sample had 100 μg of plasma protein diluted with 8 M urea and 0.1% FA to a final volume of 90 μL. The pH was adjusted to approximately 8.0 with 10 μL of 1 M triethylammonium bicarbonate (TEAB) buffer (Sigma, T7408). To reduce protein disulfide bonds, 2 μL of 0.5 M tris(2‐carboxyethyl) phosphine (TCEP) reagent (Thermo, 77720) was added and incubated at 500 rpm and 37°C for 1 h. After cooling, 4 μL of 1 M chloroacetamide (CAA) was mixed for 40 min. Proteins were precipitated with 700 μL of pre‐cooled acetone at −20°C for at least 4 h, then dissolved and washed with pre‐cooled 90% acetone twice. After evaporating residual acetone, proteins were reconstituted with 100 μL of 100 mM TEAB buffer. Trypsin (Promega, V5113) was added and incubated for 16 h at 37°C. The resulting peptide mixture was desalted, dried, and redissolved in 0.1% FA. Final concentration of 500 ng/μL was achieved.
7
Protein Reduction, Alkylation, and Digestion
8
Protein Digestion and Purification
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One hundred and twenty micrograms of protein samples were added with trypsin for digestion, and then the volume was replenished to 100 μl by 100 mM TEAB buffer (Sigma Corporation), and enzymatic digestion at 37°C. trypsin (Promega) and CaCl2 were added proportionally for a night. Then, they were adjusted to a pH < 3, mixed well, and centrifuged with 12,000g for 5 min at room temperature. The supernatant was slowly desalted through the C18 column and then washed with a cleaning solution, which was prepared with 0.1% formic acid (Thermo Fisher, Inc) and 3% acetonitrile (Thermo Fisher, Inc) three times. After that, moderate eluent (0.1% formic acid, 70% acetonitrile) was added for washing. The filtrate was collected, lyophilized, and stored at a low temperature (−20°C) for later use.
9
Peptide Labeling with TMT Reagent
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Peptides were labeled with 6-plex TMT reagent using manufacturer’s protocol with some modification (Thermo Fisher Scientific). The six peptide samples from each time series were resuspended in 100 μl of 100 mM TEAB buffer (pH 8.0; Sigma-Aldrich) and a unit of each TMT reagent was resuspended in 40 μl of acetonitrile. Subsequently, the prepared TMT reagent was transferred to the peptide sample and then vortexed. The samples were incubated for 2 h at room temperature (RT). The labelled peptide samples from each time series were pooled and concentrated by vacuum centrifugation. The labelled sample was resuspended 100 μl with 10 mM ammonium formate (Sigma-Aldrich) in water (pH 10).
10
Protein Extraction, Reduction, and Tryptic Digestion
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Approximately 1 mg of total soluble protein extract was reduced with 5 mM dithiothreitol for 2 h at 37°C, cooled and then proteins were alkylated with 14 mM iodoacetamide for 30 min at room temperature in the dark. Unreacted iodoacetamide was quenched by increasing dithiothreitol concentration to 10 mM with a further incubation for 15 min at room temperature in the dark. Proteins were diluted to 1.5 M urea with 50 mM triethylammonium bicarbonate (TEAB) buffer (Sigma-Aldrich) and incubated at 50:1 ratio with sequencing-grade modified trypsin (Promega, Madison, WI, USA) overnight at 37°C with gentle agitation. Protein digestion was stopped by acidification of the mixture to pH 2.0 with trifluoroacetic acid. Acidification serves to precipitate lipids that would interfere with downstream purification and importantly to prepare the samples for desalting which requires peptide mixture to be acidic (Hsu et al., 2009 (link)). Peptides were then purified using Sep-Pak Vac tC18 100 mg cartridge (Waters, Milford, MA, USA), as described previously (Groen et al., 2013 (link)), and completely dried in a Speed Vac concentrator (Thermo Scientific, Bremen, Germany).
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