To detect Tyr-717 phosphorylation after oxidative stress via mass-spectrometry, we performed a pull-down of GST-tagged PKD2 from HEK293 cells stimulated with H2O2 or left unstimulated. GST-PKD2 was eluted from glutathione-sepharose beads and subjected to TCA/aceton precipitation and trypsin digestion (10 µg modified trypsin (Promega) in 200 mM AmBic, 5% CH3CN, 0.1% RapiGest). Digested proteins were subjected to desalting on C18 Micro Spin Columns (Harvard Apparatus) and equal fractions were loaded on a hybrid quadrupole-orbitrap nano LC-MS system (QExactive, Thermo Fisher Scientific) using a data dependent analysis method. Number of ‘queries matched’ for the PKD2 protein was compared between conditions, and equal amounts of protein were subjected to phospho-enrichment on 50 µl IMAC bead suspension (Phos-Select, Sigma). The eluates were again desalted by C18 Micro Spin Columns and loaded on a QExactive LC-MS system using a targeted analysis method (tMS2). Data analysis was executed by using the MASCOT (Matrix Science) search engine together with the Proteome Discoverer 1.4 PhosphoRS 3.0 workflow. Data mining was done with XCalibur 3.0.63 Qual Browser and MZmine software. The phosphorylation site assignment was manually verified.
C18 micro spin columns
Manufactured by Harvard Apparatus
Sourced in United States
C18 Micro Spin Columns are small, disposable chromatography columns designed for sample preparation and purification. They contain a silica-based C18 stationary phase, which is commonly used for the reversephase separation of compounds. These columns can be used to extract, concentrate, or desalt various analytes from complex mixtures prior to analysis.
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Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Modified trypsin, by Promega (1 mentions)
Phos select, by Merck Group (1 mentions)
Q exactive, by Thermo Fisher Scientific (1 mentions)
Mascot, by Matrix Science (1 mentions)
Esi ion max source, by Thermo Fisher Scientific (1 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Ltq orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Magic c18 aq 3 μm, by Bruker (1 mentions)
Microcon, by Merck Group (1 mentions)
Rc dc protein assay, by Bio-Rad (1 mentions)
Q exactive orbitrap ms, by Thermo Fisher Scientific (1 mentions)
Ultimate 3000 nano uplc system, by Thermo Fisher Scientific (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Ultracel ym 30, by Merck Group (1 mentions)
S2 focused ultrasonicator, by Covaris (1 mentions)
Tmt six plex reagents, by Thermo Fisher Scientific (1 mentions)
Speedvac concentrator plus, by Eppendorf (1 mentions)
Peptide n glycosidase f, by Merck Group (1 mentions)
Naoh beads, by Merck Group (1 mentions)
11 protocols using c18 micro spin columns
1
Detecting Tyr-717 Phosphorylation in PKD2
2
Characterization of L19-IFNγ Fusion Proteins
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100 μg of each fusion protein was reduced in denaturing conditions by incubation in 50 mM tris (2-carboxyethyl)phosphine (TCEP) and 4 M guanidinium chloride for 1 h at room temperature. The samples were then desalted using C18 Micro Spin Columns (Harvard Apparatus). The eluate from C18 purification was adjusted to 48% CH3CN/0.2% HCOOH and then directly injected into a Q Exactive mass spectrometer (Thermo Scientific) equipped with an ESI Ion Max source (Thermo Fisher). Source parameters were set as polarity positive ion mode; resolution (FWHM at 200 m/z) 70,000; microscan 10; S-lens RF level 55; spray voltage 3.5 kV; and scan range 100–1500 m/z. By applying an in-source CID offset voltage of 80 eV fragmentation of L19-IFNg variants was induced. The resulting fragmented spectra were screened for the presence of y ions, which contain C-terminal sequence information and they were manually annotated allowing a mass error of 10 ppm.
3
RIPK1 Interactors Identification by Mass Spectrometry
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Prior to mass spectrometry analysis of RIPK1 interactors, eluted protein complexes were digested with Trypsin and peptides were purified using C18 Microspin columns (Harvard Apparatus) according to the manufactures instruction. LC-MS/MS analysis was performed on a dual pressure LTQ-Orbitrap mass spectrometer (Thermo Scientific), which was connected to an electrospray ion source (Thermo Scientific). Peptide separation was carried out using an easy nano-LC systems (Proxeon Biosystems) equipped with an RP-HPLC column packed with C18 resin (Magic C18 AQ 3 μm; Michrom BioResources). A 0.3 μl/min linear gradient from 96% solvent A (0.15% formic acid, 2% acetonitrile) and 4% solvent B (98% acetonitrile, 0.15% formic acid) to 40% solvent B over 40 min. The data acquisition mode was set to obtain one high-resolution MS scan in the FT part of the mass spectrometer at a resolution of 60,000 FWHM followed by MS/MS scans in the linear ion trap of the 20 most intense ions. Raw files were converted to the mzXML format, and searched against the human swissprot protein database. Further data processing including SAINT was carried out as described previously (Choi et al., 2011 (link)).
4
Proteomic Sample Preparation using FASP
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Proteins eluted after SBP pull-down were processed using the filter-aided sample preparation protocol method (FASP) [46 (link)] according to a workflow described in Gómez-Herranz et al. [43 (link)]. Briefly, protein concentration was determined using the RC-DC protein assay (Bio-rad, California, CA, USA). Approximately 100 µg of protein dissolved in 20 mM HEPES pH 8, 2 mM DTT, and 8 M Urea was added to a 10 kDa spin filter column (Microcon, Merck-Millipore, Burlington, MA, USA), on-filter reduced, alkylated, and trypsin-digested to peptides. Tryptic peptides were then desalted using C18 microspin columns (Harvard Apparatus, Cambridge, MA, USA) [43 (link)].
5
Phosphoproteomic Analysis of Recombinant PKD
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20 μg of recombinant PKD, incubated with and without ATP for 60 min, were precipitated 37 and digested with trypsin (0.4 μg, overnight, 37 °C). The resulting peptide mixture was desalted on C18 Micro Spin Columns (Harvard Apparatus) before being subjected to anti‐pTyr IP (PY99, Santa Cruz Biotechnology, Dallas, TX, USA) in TBS/1% n‐octyl glucoside (overnight, 4 °C). Beads were eluted with 50% acetonitrile/1% formic acid (10 min, RT). Subsequently, samples were prepared for MS by using C18 ZipTips. The resulting peptide mixture was submitted to high resolution LC‐MS/MS using an Ultimate 3000 nano UPLC system interfaced with an Orbitrap Q‐Exactive MS via an EASY‐spray (C18, 15 cm) column (Thermo Fisher Scientific). The Q‐Exactive MS was operated in data‐dependent mode selecting the top ten precursors for MS/MS. Protein identifications were obtained from the mascot (Matrix science, version 2.2.2) search engine using UniProt/SwissProt (Homo sapiens, 20202 entries) as a database, allowing up to three missed tryptic cleavages, phosphorylation of STY and oxidation of Met as variable modifications. Peptide abundances were determined using the progenesis qi software package (Nonlinear Dynamics, Newcastle upon Tyne, UK).
6
CoCl2-induced Proteome Profiling
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R28 cells (2 × 105) were treated with CoCl2 (200 μM) for 9 h. Then, they were treated with hPMSC exosomes (12 μg/mL). After 24 h, the cells were harvested. Each cell pellet then was mixed with 1 mL of lysis solution (8 M Urea, 0.1 M Tris-HCl buffer, pH 8.5) and 40 μL of protease inhibitor cocktail (× 25 stock solution) in glass tubes. Cell lysis was performed using a Covaris S2 Focused-Ultrasonicator (Covaris, Woburn, MA, USA) for 8 min. The protein concentrations in the samples were determined using Pierce BCA Protein Assay Kits (Thermo Fisher Scientific). Filter-aided sample preparation (FASP) was performed using Ultracel® YM-30 centrifugal filters (Merck Millipore, Germany), as previously reported [24 (link)]. In brief, protein (100 μg) was reduced with TCEP (37 °C, 30 min), alkylated with IAA (25 °C, 30 min, in the dark), and digested with trypsin (37 °C, 18 h, enzyme to protein ratio = 1:50). After digestion, the peptide mixtures were collected. FA was added to inactivate trypsin. The samples were then desalted using C18 Micro spin columns (Harvard Apparatus, MA, USA), vacuum-dried (1800 rpm, 3 h, ScanSpeed 40 centrifugal evaporator), and reconstituted in 0.1% FA/water (solvent A) prior to analysis.
7
Automated and Manual Peptide Cleanup
8
Desalting of Cross-Linked Peptides
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C18 Micro Spin Columns (Harvard Apparatus) were used to desalt cross-linked tryptic peptides using a protocol described in Bouchal et al.70 (link) with minor changes. Briefly, C18 spin columns were activated by three washes of 0.1% formic acid (FA) in acetonitrile (AcN) (Merck) and two washes of 0.1% FA. The column was hydrated for 15 min by 0.1% FA. The sample was loaded into the spin column and washed three times with 0.1% FA. Desalted peptides were eluted by a step gradient using 50%, 80%, and 100% AcN in 0.1% FA consecutively. Samples were dried in SpeedVac Concentrator plus (Eppendorf) until no residual liquid was present. Eluted peptides were dissolved in 100 µl of 0.08% trifluoroacetic acid in 2.5% AcN and concentration was measured on NanoDrop 2000 (Thermo Scientific). Approximately 1 µg of cross-linked peptides from each sample was injected into the LC–MS/MS system.
9
Glycoproteomics Analysis of Ribonuclease B
10
SPANX-A Interactome Profiling in Melanoma
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For the SPANX-A interactome, A375 melanoma cells were transfected with the YFP-SPANX-A plasmid as described above. After protein extraction using Co-IP buffer (100 mM NaCl, 20 mM Tris–HCl, 1% NP-40, and complete protease inhibitor cocktail (Complete tablets, Roche)), the pull down was performed in quadruplicate with GFP-Trap_Magnetic Agarose (Chromotek, Germany) following the manufacturer’s instructions. The precipitated protein complexes were independently recovered, washed and eluted with guanidinium hydrochloride 8 M pH 8 at 70 °C for 15 min. The eluted proteins were then reduced, alkylated and subjected to in-solution digestion with trypsin (Roche Diagnostics) overnight at 37 °C. The resulting peptides were desalted using C18 Micro SpinColumns (Harvard apparatus), dried in a speed-vac centrifuge (Thermo Scientific) and resuspended in 0.1% FA before LC–MS/MS analysis.
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