Cells from each experimental group were cultured independently, in triplicates, to obtain total lysate samples. For each sample, 2 × 106 cells were harvested by trypsin digestion and proteins were extracted in 8 M urea, 50 mM ammonium bicarbonate buffer, and subsequently digested with trypsin. Briefly, cysteine disulfide bonds were reduced with 5 mM Tris(2-carboxyethyl) phosphine (TCEP) at 30 °C for 60 min, followed by cysteine alkylation with 15 mM iodoacetamide (IAA) in the dark, at room temperature for 30 min. Following alkylation, urea was diluted to 1 M urea using 50 mM ammonium bicarbonate, and proteins were finally subjected to overnight digestion with mass spec grade Trypsin/Lys-C mix (Promega, Madison, WI). The digested proteins were desalted using AssayMap C18 cartridges mounted on a BRAVO liquid handling system (Agilent, Columbia, MD), and the organic solvent was removed in a SpeedVac concentrator prior to LC-MS/MS analysis.
Assaymap c18 cartridges
Manufactured by Agilent Technologies
Sourced in United States
The AssayMap C18 cartridges are analytical tools designed for sample preparation in liquid chromatography (LC) and mass spectrometry (MS) applications. These cartridges utilize a C18 stationary phase to facilitate the extraction and purification of analytes from complex matrices.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay, by Thermo Fisher Scientific (4 mentions)
Trypsin lys c mix, by Promega (2 mentions)
Bravo liquid handling system, by Agilent Technologies (1 mentions)
Mass spec grade trypsin lys c mix, by Promega (1 mentions)
Savant spd131dda, by Thermo Fisher Scientific (1 mentions)
Bravo liquid handling platform, by Agilent Technologies (1 mentions)
Assaymap bravo, by Agilent Technologies (1 mentions)
Assaymap bravo platform, by Agilent Technologies (1 mentions)
Assaymap streptavidin cartridges, by Agilent Technologies (1 mentions)
Rapid digestion buffer kit, by Promega (1 mentions)
Mass spectrometry grade trypsin lys c rapid digestion enzyme, by Promega (1 mentions)
7 protocols using assaymap c18 cartridges
1
Protein Extraction and Digestion for Mass Spectrometry
2
Protein Purification and Digestion Protocol
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Beads were thawed and resuspended with 8 M urea, 50 mM ammonium bicarbonate, and cysteine disulfide bonds were reduced with 10 mM tris(2-carboxyethyl)phosphine (TCEP) at 30 °C for 60 min and cysteines were then alkylated with 30 mM iodoacetamide (IAA) in the dark at room temperature for 30 min. Following alkylation, urea was diluted to 1 M urea, and proteins were subjected to overnight digestion with mass spec grade Trypsin/Lys-C mix (Promega, Madison, WI, USA). Finally, beads were pulled down and the solution with peptides collected into a new tube. The beads were then washed once with 50 mM ammonium bicarbonate to increase peptide recovery.
Following digestion, samples were acidified with formic acid (FA) and subsequently desalted using AssayMap C18 cartridges (Agilent, Santa Clara, CA, USA) mounted on an Agilent AssayMap BRAVO liquid handling system. Briefly, C18 cartridges were first conditioned with 100% acetonitrile (ACN), followed 0.1% FA. Sample was then loaded onto the conditioned C18 cartridge, washed with 0.1% FA, and eluted with 60% ACN, 0.1% FA. Finally, the organic solvent was removed in a SpeedVac concentrator prior to LC-MS/MS analysis.
Following digestion, samples were acidified with formic acid (FA) and subsequently desalted using AssayMap C18 cartridges (Agilent, Santa Clara, CA, USA) mounted on an Agilent AssayMap BRAVO liquid handling system. Briefly, C18 cartridges were first conditioned with 100% acetonitrile (ACN), followed 0.1% FA. Sample was then loaded onto the conditioned C18 cartridge, washed with 0.1% FA, and eluted with 60% ACN, 0.1% FA. Finally, the organic solvent was removed in a SpeedVac concentrator prior to LC-MS/MS analysis.
3
Peptide clean-up and quantification
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Peptide clean-up was achieved using a Bravo AssayMAP Liquid Handling Platform (Agilent). After conditioning and equilibration of the resin, acidified peptide solutions were loaded onto AssayMAP C18 Cartridges (Agilent, 5190-6532), washed using 1% acetonitrile (ACN), 0.1% TFA (aq) and eluted using 70% ACN, 0.1% TFA (aq). Eluted peptides were vacuum centrifuged (Thermo Scientific, Savant SPD131DDA) to dry and resuspended in 40 µL of 2% ACN, 0.05% TFA (aq). For clinical cohort analysis, 6 µL of cleaned peptide solution was added to two injection equivalents of PQ500 SIS mix (Biognosys, Ki-3019-96) using a Bravo Liquid Handling Platform (Agilent).
4
Disulfide Reduction and Alkylation for Proteomics
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Disulfide bonds were reduced with 5 mM Tris(2-carboxyethyl)phosphine-HCl at 30°C for 60 min, and cysteines were subsequently alkylated (carbamidomethylated) with 15 mM iodoacetamide in the dark at room temperature for 30 min. Proteins were then precipitated with nine volumes of methanol, pelleted, and resuspended in 1 M urea and 50 mM ammonium bicarbonate. Following precipitation, protein concentration was determined using a bicinchoninic acid protein assay. A total of 0.2 mg of protein was subjected to overnight digestion with 8.0 μg of mass spec grade Trypsin/Lys-C mix (Promega). Following digestion, samples were acidified with formic acid (FA), and subsequently, 150 μg peptides were desalted using AssayMap C18 cartridges mounted on an Agilent AssayMap BRAVO liquid handling system. C18 cartridges were first conditioned with 100% acetonitrile (ACN), followed by 0.1% FA. The samples were then loaded onto the conditioned C18 cartridge, washed with 0.1% FA, and eluted with 60% MeCN and 0.1% FA. Finally, the organic solvent was removed in a SpeedVac concentrator prior to LC–MS/MS analysis.
5
Protein Sample Preparation for Mass Spectrometry
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Supernatant medium was buffer exchanged using 3.5‐kDa Amicon filter (Millipore) to 8 M urea, 50 mM ammonium bicarbonate buffer. While in the filter, proteins were reduced with 5 mM tris(2‐carboxyethyl)phosphine (TCEP) at 30°C for 60 min and subsequently alkylated with 15 mM iodoacetamide (IAA) in the dark at room temperature for 30 min. The buffer was then exchanged again to 1 M urea, 50 mM ammonium bicarbonate; the sample was recovered from the Amicon tube into a new microfuge tube, and protein concentration was determined using bicinchoninic acid (BCA) protein assay (Thermo Scientific). Proteins were subjected to overnight digestion with mass spec grade Trypsin/Lys‐C mix (1:25 enzyme/substrate ratio). Following digestion, samples were acidified with formic acid (FA) and subsequently desalted using AssayMap C18 cartridges mounted on an Agilent AssayMap BRAVO liquid handling system. Cartridges were sequentially conditioned with 100% acetonitrile (ACN) and 0.1% FA; samples were then loaded, washed with 0.1% FA and peptides eluted with 60% ACN, 0.1% FA. Finally, the organic solvent was removed in a SpeedVac concentrator prior to LC‐MS/MS analysis.
6
Cerebral Cortex Protein Extraction
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After dissection, the cerebral cortex tissues were individually homogenized and sonicated on cold HEPES/sucrose buffer containing protease and phosphatase inhibitors. The proteins were extracted using methanol and chloroform and stored at −80 °C until further processing. Protein extracts were resuspended with 8 M urea 50 mM ammonium bicarbonate, and protein concentration was determined using a bicinchoninic acid (BCA) protein assay (Thermo Scientific). Proteins were then digested, acidified with formic acid and subsequently desalted using AssayMap C18 cartridges (Agilent) mounted on an Agilent AssayMap BRAVO liquid handling system. More detailed procedures on the preparation of proteins prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis are described in the Supplementary Methods .
7
Biotinylated Protein Isolation and Tryptic Digestion
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Biotinylated proteins were isolated from the organ homogenates using a Bravo AssayMap platform and AssayMap streptavidin cartridges (Agilent). The cartridges were equilibrated with ammonium bicarbonate (50 mM; pH 8), and biotinylated samples were loaded. Nonbiotinylated proteins were removed by extensive wash with 8 M urea in 50 mM ammonium bicarbonate buffer (pH 8). Cartridges were further washed with rapid digestion buffer (Promega; rapid digestion buffer kit), and bound proteins were subjected to on-column digestion using a mass spectrometry-grade trypsin/Lys-C rapid digestion enzyme (Promega, Madison, WI) at 70°C for 2 h. Released peptides were desalted using AssayMap C18 cartridges (Agilent). Samples were stored at −20°C prior to DIA-SWATH-MS analysis.
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