For SILAC, SH-SY-5Y cells stably expressing GFP or a GFP-tagged construct of the protein of interest were cultured for at least six doublings in SILAC DMEM (89985; Thermo Fisher Scientific) supplemented with 10% dialyzed FBS (F0392; Sigma-Aldrich). Cells expressing GFP were grown in media containing light amino acids (R0K0), whereas cells expressing the GFP-tagged protein of interest were grown in medium (R10K8 or R6K4). Amino acids R10,R6, R0, and K0 were obtained from Sigma-Aldrich, whereas K4 was from Thermo Fisher Scientific. Cells where lysed in immunoprecipitation buffer (50 mM Tris-HCl, 0.5% NP-40, and Roche protease inhibitor cocktail) and subjected to GFP trap (ChromoTek). Precipitates were pooled and separated on NuPAGE 4–12% precast gels (Invitrogen) before liquid chromatography–tandem mass spectrometry analysis on an Orbitrap Velos mass spectrometer (Thermo Fisher Scientific).
Orbitrap velos mass spectrometer
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The Orbitrap Velos mass spectrometer is an analytical instrument used for the detection and identification of chemical compounds. It utilizes high-resolution mass spectrometry to provide accurate mass measurements and detailed structural information about molecules. The core function of the Orbitrap Velos is to perform sensitive and high-performance analysis of a wide range of samples.
Automatically generated - may contain errors
Lab products found in correlation
Nanoacquity, by Waters Corporation (4 mentions)
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Silac dmem, by Thermo Fisher Scientific (3 mentions)
Nupage 4 12 precast gels, by Thermo Fisher Scientific (3 mentions)
Gfp trap beads, by Proteintech (3 mentions)
Trypsin, by Promega (3 mentions)
Mascot search, by Matrix Science (3 mentions)
Gfp trap, by Proteintech (2 mentions)
Nanoacquity uplc, by Waters Corporation (2 mentions)
Pepmap c18, by Thermo Fisher Scientific (2 mentions)
Ltq orbitrap velos, by Thermo Fisher Scientific (2 mentions)
Nanoacquity beh c18, by Waters Corporation (2 mentions)
Mascot distiller, by Matrix Science (2 mentions)
Rslcnano hplc, by Thermo Fisher Scientific (2 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (2 mentions)
Triversa nanomate, by Advion (2 mentions)
Picofrit analytical column, by New Objective (1 mentions)
Sds page, by Merck Group (1 mentions)
Pageblue protein staining solution, by Thermo Fisher Scientific (1 mentions)
Ultimate 3000 rslc nanoflow system, by Thermo Fisher Scientific (1 mentions)
54 protocols using orbitrap velos mass spectrometer
1
SILAC-based GFP Interactome Profiling
2
SILAC-based Mass Spectrometry of GFP-tagged Proteins
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For SILAC, hTERT RPE1 cells lentivirally transduced to stably express GFP or a GFP-tagged construct of the protein of interest were cultured for at least six doublings in SILAC DMEM (89985; Thermo Fisher Scientific) supplemented with 10% dialyzed FBS (F0392; Sigma-Aldrich). Cells expressing GFP were grown in media containing light amino acids (R0K0), whereas cells expressing the GFP-tagged protein of interest were grown in medium (R6K4). Amino acids R6, R0, and K0 were obtained from Sigma-Aldrich, whereas K4 was from Thermo Fisher Scientific. Cells where lysed in immunoprecipitation buffer (50 mM Tris-HCl, 0.5% NP-40, and Roche protease inhibitor cocktail) and subjected to GFP trap (ChromoTek). Precipitates were pooled and separated on NuPAGE 4–12% precast gels (Invitrogen) before liquid chromatography–tandem mass spectrometry analysis on an Orbitrap Velos mass spectrometer (Thermo Fisher Scientific).
3
Proteomic Analysis of Peptides by LC-MS/MS
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Samples were reconstituted in 67.5 µL of 0.1% formic acid. An aliquot of 4.5 μL was analysed on an ETD enabled Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Waltham, MA, US) connected to an EASY-nLC system (Proxeon Biosystem, West Palm Beach, FL, US) through a Proxeon nanoelectrospray ion source. Peptides were separated by a 2–90% acetonitrile gradient in 0.1% formic acid using a PicoFrit analytical column (20 cm × ID75 μm, 5-μm particle size, New Objective) at a flow rate of 300 nL min−1 for 85 minutes. The nanoelectrospray voltage was set to 2.2 kV, and the source temperature was 275 °C. All instrumental methods were set up in the data-dependent acquisition mode (DDA). Full scan MS spectra (300–1600 m/z) were acquired in the Orbitrap analyser after accumulation to a target value of 1 × 106. The resolution in the Orbitrap was set to r = 60.000, and the 20 most intense peptide ions with charge state ≥2 were sequentially isolated to a target value of 5.000 and fragmented in the linear ion trap using low-energy CID (normalised collision energy of 35%). The signal threshold for triggering an MS/MS event was set to 1.000 counts. Dynamic exclusion was enabled with an exclusion size list of 500, an exclusion duration of 60 s, and a repeat count of 1. An activation of q = 0.25 and an activation time of 10 ms were used71 (link).
4
SDS-PAGE and Proteomic Analysis
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SDS-PAGE was performed with 4%–20% precast gels in Tris-based buffer system from SERVA Electrophoresis (#43289.01) according to the manufacturer’s instructions. PageBlue Protein Staining Solution (Fermentas, #R0571) was used for coomassie-staining the gels, images were taken with iPhone 6s (Apple) and the contrast was adjusted using ImageJ (version 1.52d). Global identification and quantification of MSU- and zymosan-binding proteins in the presence of different donor sera was done as previously described (25 (link)). In brief, eluted proteins were reduced with DTT, alkylated with acrylamide, and separated using SDS-PAGE (4%–20%, Sigma-Aldrich). Whole lanes were cut into three individual slices and proteins therein were in-gel digested with trypsin. Generated peptides were analyzed using an LC-MS system consisting of an Orbitrap Velos mass spectrometer coupled to an Ultimate 3000 RSLC nanoflow system (Thermo Fisher Scientific). Raw data were analyzed with the Andromeda search engine implemented in MaxQuant software (version 1.5.3.30; www.maxquant.org ). Proteins were identified based on a false discovery rate (FDR) of less than 0.01 on protein and peptide level.
5
Cysteine Modification Analysis of SARS-CoV-2 Proteins
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WT or Omicron RBD protein (5 μM in 20 μl) was mixed with 20 μl buffer A containing TCEP (0.2 mM) or blank and incubated at 37 °C for 30 minutes. IAM solution (25 mM 10 μl) was then added and incubated in dark at room temperature for 30 minutes. Next, DTT (60 mM in 10 μl) solution was added and incubated in dark at room temperature for 60 minutes. Mass spectrometry analysis was performed as described. Briefly, the protein was further digested by trypsin over night. The digested samples were dried and re-suspended in 200 µL 80% acetonitrile/2% formic. The peptides were injected into C18 nano-column on an EASY-nLC (Proxeon) coupled with an Orbitrap Velos mass spectrometer (Thermo Fisher Scientific). A ninety-minute gradient was used for separation at a flow rate of 300nL/minutes. Sixteen MS/MS collision induced dissociation (CID) data-dependent scans were acquired simultaneously with one full scan mass spectra with 60,000 resolution and 1E6 AGC. MaxQuant (version 2.0.3.0) was used to search Uniprot/SwissProt human protein database with 20,600 human proteins modified to contain BSA and trypsin, and with variable modification of methionine oxidation, protein N-terminal acetylation and cysteine carbamidomethylation as variable modifications and cysteine carbamidomethylation as fixed modifications. Up to 1% false discovery rate was used for both peptides and proteins.
6
Comprehensive SILAC-Based Muscle Proteomics
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SILAC samples were generated in our previous study33 (link), which described only a subset of the interacting proteins, with this current study we reveal the full dataset. Briefly, transduced C2C12 myoblasts were expanded in R0K0 or R6K4 isotopically labelled DMEM (Dundee Cell Products) supplemented with 10% (v/v) dialysed FBS (Dundee Cell Products) for at least six passages prior to differentiation to achieve full labeling. Myoblasts were then differentiated in R0K0 or R6K4 isotopically labelled DMEM supplemented with 2% (v/v) dialysed horse serum. Myotubes were harvested in precipitation buffer 50 mM Tris–HCl pH 7.4, 50 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 0.5% NP40, 1 mM DTT, 50 mM NaF, 5 mM Na4P2O7, 1 mM Na3VO4, 0.5 mM PMSF, Calbiochem protease inhibitor cocktail), lysates incubated (4 °C, 1 h) with GFP-trap beads (ChromoTek), precipitates washed four times with precipitation buffer and samples pooled prior to eluting, separation on NuPAGE 4–12% precast gel (Invitrogen) and Nano LC–MS/MS on an Orbitrap Velos mass spectrometer (Thermo Fisher Scientific). Mass spectrometric detection and quantification was performed as previously published88 (link).
7
Proteomic Analysis of Recombinant SPOP Protein
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LC–MS/MS analyses of the Arg-C and Asp-N digests of recombinant SPOP protein were carried out on a nanoACQUITY UPLC (Waters, Milford, MA) coupled to an Orbitrap Velos mass spectrometer (Thermo Scientific). The LC separation was performed on a C18 column with 0.1% FA in H2O/ACN as buffer A/B at a gradient of 1–8% B in 0–2 min, 8–12% B in 2–20 min, 12–30% B in 20–75 min, 30–45% in 75–97 min, and 45–95% B in 97–100 min. The heated capillary temperature and spray voltage was 350 °C and 2.2 kV, respectively. Full MS spectra were recorded at a resolution of 60,000 over the range of m/z 300–2000. Ten parent ions with most abundant intensity were selected for MS/MS using CID with a collision energy of 35V. The resulting raw data were searched against the UniProt human protein database [25 ] using the MSGF+ algorithm [26 (link), 27 (link)]. The parameter was 20 ppm tolerance for precursor ion masses and 0.5 Da tolerance for fragment ions, with static carbamidomethylation on cysteine residues (+57.0215 Da) and dynamic oxidation of methionine residues (+15.9449 Da). The data filtering criteria is MSGF E < 10−8, Q < 0.01 and mass measurement error <20 ppm (±10 ppm).
8
LC-MS/MS Proteomics Workflow
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LC–MS/MS analysis of the proteins was performed using an UltiMate 3000 nano HPLC system (Thermo Scientific, Germering, Germany). Water served as solvent A and acetonitrile served as solvent B (with 0.1% formic acid additions). Aliquots of 200 µg proteolytic peptides obtained via trypsin were loaded onto a trapping column (maintained at 35 °C) for desalting (Thermo Scientific, Dionex, PepMap C18, Sunnyvale, CA, USA) before the peptides were separated by gradient elution on a 500 × 0.075 mm column (0.5 µL/min, held at 50 °C; Reprosil C18-AQ, Dr. Maisch) from 12% to 40% B. The column effluent was directed to an Orbitrap Velos mass spectrometer via a nanoelectrospray ion source (Thermo Scientific, Bremen, Germany). Up to 10 MS/MS spectra from ions of interest (charge states +2 and higher) were data-dependently captured in the instrument’s linear ion trap, while survey scans were detected at a notional resolution of R = 60,000. The total acquisition time of each analysis was 45 min.
9
Proteomics Analysis of Mis-annotated STK19
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For data base analysis, STK19 detected peptides were downloaded from peptide Atlas (Deutsch et al., 2008 (link)) and Proteomics DB (Schmidt et al., 2018 (link)) latest versions. To minimize unspecific mapping, peptides between 7 and 20, found in more than one experiment and with a maximum of one missed cut site were analyzed. Peptides with the same start or end position were merged for plotting. For the analysis of overexpressed mis-annotated STK19, eluted proteins from immunoprecipitations were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), until the running front had migrated approximately 1–2 cm into the gel (10% NuPAGE, Invitrogen, NP0301), and stained with colloidal Coomassie (InstantBlue, Expedeon). After excision of 8 horizontal gel slices per lane, proteins were in-gel digested with trypsin (Promega/Pierce) using a Janus liquid handling system (Perkin Elmer). Tryptic peptides were analyzed by liquid chromatography-mass spectrometry (LC–MS) using an Orbitrap Velos mass spectrometer coupled to an Ultimate 3000 uHPLC equipped with an EASY-Spray nanosource (Thermo Fisher Scientific) and acquired in data-dependent mode. The data were searched against the human Uniprot database using the Andromeda search engine. Raw data were processed using MaxQuant v1.6.0.1 (Cox and Mann, 2008 (link)). Peptide intensities were log2 transformed.
10
Disulfide Bond Peptide Mapping
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Reduced peptide mapping analysis was conducted by enzymatic digestion with trypsin (Roche Life Science, Indianapolis, IN, USA), followed by reduction with dithiothreitol and alkylation with sodium iodoacetic acid (Sigma-Aldrich, St Louis, MO, USA). The resulting cleavage fragments were separated by reverse-phase ultra-HPLC (Agilent 1290, Agilent Technologies; Acquity H-Class, Waters Corp, Milford, MA, USA) using an increasing gradient of acetonitrile in water. The eluted peaks were detected by UV absorbance (214 nm) and peptides identified by online liquid chromatography with tandem mass spectrometry (MS/MS) using an Orbitrap Velos mass spectrometer (Thermo Scientific). For non-reduced peptide mapping, samples were digested with endoprotease Lys-C (Wako Chemicals) under denaturing but non-reducing conditions, and the resulting peptides were analyzed in a similar manner. Peptides containing disulfide bonds were identified by comparison of peptides generated under reducing and non-reducing conditions.
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