The samples used were immobilized metal affinity-chromatography-enriched phosphopeptides from human cell lines (HCT116) or mouse brain tissue,21 (link) as processed using the SL-TMT protocol.7 (link) Phosphopeptides were enriched using the High-Select Fe-NTA Phosphopeptide Enrichment Kit8 (link) and desalted (C18 SepPak, Waters). Enriched phosphopeptides were labeled as described previously with TMT or TMTpro0.7 (link) For TMT10, the labeled peptides were mixed 1:1 across all channels, and desalted (C18 SepPak, Waters) prior to use. Further details are available in the Supporting Information .
Sep pak c18
Manufactured by Waters Corporation
Sourced in United States, United Kingdom, Sweden
The Sep-Pak C18 is a solid-phase extraction (SPE) cartridge designed for sample preparation and purification. It contains a C18 reversed-phase sorbent material that is commonly used for the extraction and clean-up of a wide range of analytes from various sample matrices.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin, by Promega (7 mentions)
Sequence grade modified trypsin, by Promega (6 mentions)
Sterile filter, by Merck Group (5 mentions)
Sequencing grade trypsin, by Promega (5 mentions)
Oasis mcx, by Waters Corporation (4 mentions)
Hplc grade acetonitrile, by Thermo Fisher Scientific (3 mentions)
Lys c, by Fujifilm (3 mentions)
Pngase f, by Roche (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
392 dna rna synthesizer, by Glen Research (3 mentions)
Ultimate 3000, by Thermo Fisher Scientific (3 mentions)
Lc 2010a ht system, by Bioscan (2 mentions)
Tracerlab fxc pro synthesis module, by GE Healthcare (2 mentions)
8 plex itraq reagent, by AB Sciex (2 mentions)
Trypsin gold, by Promega (2 mentions)
0.22 μm membrane filter, by Merck Group (2 mentions)
30 kda filter unit, by Merck Group (2 mentions)
Millex gv, by Merck Group (2 mentions)
Lc ms system, by Agilent Technologies (2 mentions)
Pd c 10 palladium on c, by Merck Group (2 mentions)
131 protocols using sep pak c18
1
Phosphopeptide Enrichment and Labeling Protocol
2
Radiolabeled Compound Production
3
Crosslinking and Mass Spectrometry Analysis
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Eluted proteins in 1 mL of 20 mM HEPES pH 8.5, 200 mM NaCl, and either 2 mM CaCl2 or 2 mM EDTA were crosslinked with 10 mM Biotin-Aspartate Proline-PIR n-hydroxyphthalimide (BDP-NHP) (Weisbrod et al., 2013 (link)). As necessary, the pH was adjusted to ~8.0 with 100 μl of 200 mM HEPES pH 8.5. The reaction was allowed to continue for 1 hr at room temperature. Crosslinked proteins were denatured by the addition of urea buffer (8 M urea, 100 mM Tris-Cl pH 8.0), reduced (5 mM dithiothreitol, 30 min, 55˚C), and alkylated (15 mM iodoacetamide, 1 hr, dark, room temperature). Crosslinked proteins were then digested with sequencing grade trypsin (Promega) overnight at 37˚C. Resulting peptides were desalted with C18-SepPaks (Waters) and dried by vacuum centrifugation. Crosslinked peptides were injected onto an in-house pulled C8 column (3 μm, 200 Å, Magic) and analyzed by Real-time analysis of crosslinked peptide technology (ReACT) (Weisbrod et al., 2013 (link)). Spectra generated from ReACT were searched against a target-decoy database using SEQUEST (Eng et al., 1994 (link)). The complete set of observed peptides is presented in Supplementary file 1 along with their Expect scores and PPM error for the best-scoring relationships observed for each peptide pair. Crosslinked sites were mapped to proteins using xiNet (Combe et al., 2015 (link)).
4
Proteome Analysis by Mass Spectrometry
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Mass spectrometry was performed as described previously [15 (link)]. Protein pellet was denatured in 50 μL of 8 M Urea/0.4 M Ammonium Bicarbonate followed by reduction in 2 μL of 100 mM DTT. The digests were acidified to 0.5% trifluoroacetic acid (TFA), and the peptides were then desalted on C18 Sep-Paks (Waters). The pooled extracts were dried in a vacuum concentrator and resuspended in 30 uL of 5% ACN/0.1% FA for LC-MS analysis. Peptides were analyzed by LC-MS/MS using a Dionex UltiMate 3000 Rapid Separation LC (RSLC) system and a linear ion trap-Orbitrap hybrid Elite mass spectrometer (Thermo Fisher Scientific Inc, San Jose, CA).
5
Radiolabeled Compound Synthesis and Purification
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Reagents and solvents were purchased from Aldrich Chemical or Fisher Scientific and were used without further purification unless noted. Chromatography columns for HPLC analysis and purification were purchased from Phenomenex or Waters. High performance liquid chromatography (HPLC) was performed using a Shimadzu LC-2010A HT system equipped with a Bioscan B- FC-1000 radiation detector. Sodium chloride, 0.9% USP and sterile water for Injection, USP were purchased from Hospira; Dehydrated Alcohol for Injection, USP was obtained from Akorn Inc. Sterile filters were acquired from Millipore; 10 cc sterile vials were obtained from Hollister-Stier; C18 sep-paks were purchased from Waters Corporation and were flushed with 10 ml of ethanol followed by 10 ml of sterile water prior to use. BCPP-EF reference standard 1 and labeling precursor 15 were synthesized in house. Detailed procedures are provided in the Supplementary Material .
6
Mass Spectrometry-Based Protein Analysis
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Mass spectrometry was performed as described previously. Protein pellet was denatured in 50 μl of 8 M urea/0.4 M ammonium bicarbonate followed by reduction in 2 μl of 100 mM dithiothreitol. The digests were acidified to 0.5% trifluoroacetic acid and the peptides were then desalted on C18 Sep-Paks (Waters). The pooled extracts were dried in a vacuum concentrator and resuspended in 30 μl of 5% ACN/0.1% FA for liquid chromatography (LC) mass spectrometry analysis. Peptides were analyzed by LC–tandem mass spectrometry using a Dionex UltiMate 3000 Rapid Separation LC (RSLC) system and a linear ion trap-Orbitrap hybrid Elite mass spectrometer (Thermo Fisher Scientific, San Jose, CA).
7
Protein Digestion and Peptide Preparation
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Protein pellet was denatured in 50 μL of 8 M Urea/0.4 M Ammonium Bicarbonate followed by reduction in 2 μL of 100 mM DTT. Protein was alkylated with 18 mM iodoacetamide for 30 min at room temperature in the dark. Samples were diluted with four volumes of water to bring urea concentration to 1.8 M. Sequencing-grade trypsin (Promega) was added at 1:100 (enzyme: substrate) and incubated at 37 °C overnight. The digests were acidified to 0.5% trifluoroacetic acid (TFA), and the peptides were desalted on C18 Sep-Paks (Waters). Peptides were eluted with 2X 50 μL of 80% ACN/0.1% TFA to ensure complete recovery. The pooled extracts were dried in a vacuum concentrator and resuspended in 30 μL of 5% ACN/0.1% FA for LC-MS analysis.
8
Quantitative Proteomics Sample Preparation
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Cells were lysed using a procedure previously described [44 (link)]. The Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Waltham, MA, USA) was used to quantify the total protein amount per sample. For all eight samples, 100 μg of protein was used for bottom-up proteomics protein digestion as previously described [44 (link),45 (link)]. Peptides were desalted with 10 Oasis HLB Cartridges (Waters, Milford, MA, USA) and labeled (iTRAQ 8-plex reagents, AB Sciex, Farmington, MA, USA). Samples were next combined, dried under vacuum, and desalted again with 100 mg C18 Sep-Paks (Waters). The sample was then resuspended in 120 μL of 10 mM KH2PO4 in 20% ACN, pH 2.85 (Buffer A) for strong cation exchange (SCX) liquid chromatography fractionation.
9
Preparation and Purification of Radiopharmaceuticals
10
Radiolabeled Compound Purification Protocol
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Unless otherwise stated, reagents and solvents were commercially available and used without further purification: sodium chloride, 0.9% USP, and sterile water for injection, USP, were purchased from Hospira; ethanol was purchased from American Regent; HPLC grade acetonitrile was purchased from Fisher Scientific. Other synthesis components were obtained as follows: sterile filters were obtained from Millipore; sterile product vials were purchased from Hollister-Stier; C18 Sep-Paks were purchased from Waters Corporation. C18 Sep-Paks were flushed with 10 mL of ethanol followed by 10 mL of water prior to use. Radio-HPLC was performed using a Shimadzu LC-2010A HT system equipped with a Bioscan B-FC-1000 radiation detector.
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