Co-immunoprecipitation was performed using 293T, MNNG/HOS, and U2OS cells. Equal amounts of protein (3000 μg) were incubated with IgG, MCM2, or MCM3 antibodies at 4°C overnight and then the mixtures were incubated with protein A/G magnetic beads at 4°C for 3 h. The beads were washed using phosphate-buffered saline containing 1% Triton X-100 and eluted using 2× protein loading buffer at 100°C for 10 min. IgG-, MCM2-, or MCM3-bound proteins were resolved by SDS–PAGE and stained using the Silver Staining Kit (Beyotime Biotechnology, Shanghai, China). For the mass spectrometry analysis, MCM2-bound or MCM3-bound proteins were resolved by SDS–PAGE and stained with Coomassie blue R250 (Solarbio, Beijing, China). After destaining, reduction, and trypsin digestion for 12 h, the peptides were extracted using acetonitrile. The peptides were analyzed using a NanoLC System (NanoLC-2D Ultra; Eksigent, Dublin, CA, USA) equipped with a Triple TOF 5600 mass spectrometer (AB SCIEX, Framingham, MA, USA). Protein identification was performed using ProteinPilot4.1 (AB SCIEX). For this study, a strict unused confidence cutoff of >1.3 and ≥2 peptides were used for protein identification.
Nanolc ultra 2d
Manufactured by AB Sciex
Sourced in United States
The NanoLC Ultra 2D is a liquid chromatography system designed for high-performance and high-resolution separation of complex samples. It features a dual-pump configuration for versatile solvent delivery, enabling efficient separation and fractionation of analytes. The system is suitable for a wide range of applications, including proteomics, metabolomics, and environmental analysis.
Automatically generated - may contain errors
Lab products found in correlation
Tripletof 5600 mass spectrometer, by AB Sciex (3 mentions)
Proteinpilot 4, by AB Sciex (3 mentions)
Silver staining kit, by Beyotime (2 mentions)
Coomassie blue r250, by Solarbio (2 mentions)
C18 cl 120, by AB Sciex (2 mentions)
Tripletof 5600, by AB Sciex (2 mentions)
Nanospray 3, by AB Sciex (2 mentions)
Ip lysis buffer, by Beyotime (1 mentions)
Chromxp c18 lc 3 μm, by AB Sciex (1 mentions)
Itraq reagent 4 plex kit, by Thermo Fisher Scientific (1 mentions)
Chromxp c18 cl 3 μm, by AB Sciex (1 mentions)
Formic acid, by Merck Group (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Nanolc system, by AB Sciex (1 mentions)
Analyst tf 1, by AB Sciex (1 mentions)
Acclaim pepmap rslc c18 column, by Thermo Fisher Scientific (1 mentions)
Nanolc as 2 autosampler, by AB Sciex (1 mentions)
Tripletof 5600 electrospray tandem mass spectrometer, by AB Sciex (1 mentions)
Acclaim pepmap 100 c18 trap, by Thermo Fisher Scientific (1 mentions)
Picofrit column, by New Objective (1 mentions)
15 protocols using nanolc ultra 2d
1
MCM2/MCM3 Interactome Profiling
2
TC2N Interactome Profiling in Breast Cancer
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Total extracts of MCF7 and MB-231 with or without TC2N overexpression were lysed with IP lysis buffer (Beyotime, China). The co-IP analyses were performed using a Co-Immunoprecipitation Kit (Pierce, US) according to the manufacturer’s protocol. Subsequent WB analyses were performed as described above. The experiment was repeated thrice.
For proteomic analysis, the cell lysates were further analyzed by using a NanoLC system (NanoLC-2D Ultra, Eksigent) as previously described28 (link). Based on the Swiss-Prot Homo sapiens protein databases, which were released in November 2012 (84,736 proteins) and May 2013 (88,631 proteins), the acquired proteins were identified by using ProteinPilot 4.5 software (AB SCIEX, USA). All of these proteins are listed in TableS2 .
For proteomic analysis, the cell lysates were further analyzed by using a NanoLC system (NanoLC-2D Ultra, Eksigent) as previously described28 (link). Based on the Swiss-Prot Homo sapiens protein databases, which were released in November 2012 (84,736 proteins) and May 2013 (88,631 proteins), the acquired proteins were identified by using ProteinPilot 4.5 software (AB SCIEX, USA). All of these proteins are listed in Table
3
Shotgun Proteomics of Complex Samples
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A NanoLC system (NanoLC-2D Ultra, Eksigent, Dublin, CA) equipped with a Triple TOF 5600 mass spectrometer (AB SCIEX, USA) was used for analysis. Peptides were trapped on a NanoLC pre-column (Chromxp C18-LC-3 μm, size 0.35 × 0.5 mm, Eksigent, Dublin, CA) and then eluted onto an analytical column (C18-CL-120, size 0.075 × 150 mm, Eksigent, Dublin, CA). The NanoLC gradient was 5–35% Buffer B (98% ACN, 2% H2O, 0.1% FA) over 120 min at a flow rate of 300 nL/min. Full-scan MS was performed in positive ion mode with a nano-ion spray voltage of 2.2 kV. Survey scans were acquired from 350 to 1500 (m/z) with up to 40 precursors selected for MS/MS (m/z 100–1500). The collision energy (CE) for collision-induced dissociation was automatically controlled using an Information-Dependent Acquisition CE parameter script to achieve optimum fragmentation efficiency. The mass spectrometer was calibrated using beta galactosidase tryptic peptides.
4
Quantitative Proteomic Profiling of Tumor Samples
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The global protein expression profile was determined using the iTRAQ quantitative proteomic approach. Protein preparation, iTRAQ labeling, strong cation exchange (SCX), nanoLC-MS/MS analysis, and protein identification and quantitation were performed following the procedures detailed in a previous study60 (link). Briefly, the tumor protein lysates from 6 randomly selected SE or EE mice were pooled, and then labeled with the iTRAQ labeling reagents 114 and 116 (iTRAQ Reagent 4-Plex Kit, Applied Biosystems), respectively. The iTRAQ-labeled samples were fractionated by SCX chromatography, and subsequently analyzed using the NanoLC system (NanoLC-2D Ultra, Eksigent, Dublin, CA, USA). Protein identifications and quantitations were performed with the ProteinPilot software (version 4.1; AB SCIEX, Foster City, CA, USA) and the mouse UniProtKB/Swiss-Prot proteome database. The FDR for the peptide identifications was estimated using a decoy database search strategy. We used a strict unused protein score cutoff of > 1.3 (confidence > 95%) for the protein identifications. Only those proteins identified from at least two peptides were included for the quantitative assay. The proteins were considered to be differentially expressed if their iTRAQ ratios were > 1.5 or < 0.67 in the comparison of EE versus SE.
5
Peptide Separation via Nano-LC
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Chromatographic separation was performed on a NanoLC system (NanoLC-2D Ultra, Eksigent). Peptides were trapped on a NanoLC pre-column (ChromXP C18-CL-3 μm, I.D. 0.35 × 0.5 mm Eksigent) and then eluted onto an analytical column (C18-CL-120, I.D. 0.075 × 150 mm, Eksigent). A 1,000-ng sample was loaded, trapped and desalted at 3 μl/min for 37 min with 100% mobile phase A [2% acetonitrile (Sigma-Aldrich) in 0.1% formic acid (Sigma-Aldrich)]. Peptides were separated at a flowrate of 250 nl/min using a stepwise gradient of buffer B [98% acetonitrile (Sigma-Aldrich) in 0.1% formic acid (Sigma-Aldrich)] from 5% to 10% B in the first 10 min and from 10% to 40% in the following 60 min, from 40% to 50% in the next 10 min. The column was washed after the gradient with 90% B for 10 min before re-equilibrating to the initial chromatographic conditions for 10 min.
6
Affinity Purification and Mass Spectrometry
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Co‐IP was performed in MNNG, MG63, and U2OS cells. Equal amounts of protein (3000 μg) were incubated with antibodies at 4 °C overnight, and the mixture was incubated with protein A/G magnetic beads at 4 °C for 3 h. The beads were washed using phosphate‐buffered saline containing 1‰ Triton X‐100 and eluted using 2× protein loading buffer at 100 °C for 10 min. For silver staining, IgG‐bound or CNOT1‐bound proteins were resolved by SDS/PAGE and stained with a silver staining kit (Beyotime Biotechnology, Shanghai, China). For mass spectrometry analysis, CNOT1‐bound proteins were resolved by SDS/PAGE and stained with Coomassie Blue R250 (Solarbio, Beijing, China). After destaining, reduction, and trypsin digestion for 12 h, the peptides were extracted using acetonitrile. The peptides were analyzed using a NanoLC system (NanoLC‐2D Ultra; Eksigent, Dublin, CA, USA) equipped with a Triple TOF 5600 mass spectrometer (AB SCIEX). Protein identification was performed with proteinpilot4.1 software (AB SCIEX). For this study, a strict unused confidence cutoff > 1.3 and peptides ≥ 2 were used for protein identification.
7
Shotgun Proteomics by Information-Dependent Acquisition
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Samples were analysed on an AB Sciex 5600 TripleTOF (ABSciex) in information-dependent acquisition (IDA) mode. Peptides were fractionated by liquid chromatography (nanoLC Ultra 2D, Eksigent) on a MicroLC column ChromXPTM C18CL reverse-phase column (300 µm ID×15 cm length, 3 µm particles, 120 Å pore size, Eksigent) at 5 µl min−1 and eluted into the mass spectrometer with an acetonitrile gradient in 0.1 % FA (2 –30 % ACN, in a linear gradient for 30 min), using an electrospray ionization source (DuoSprayTM Source, ABSciex) with a 50 µm internal diameter (ID) stainless steel emitter (New Objective). For information-dependent acquisition (IDA) experiments, the mass spectrometer was set to scanning full spectra (350–1250 m/z) for 250 ms, followed by up to 80 MS/MS scans (100–1500 m/z from a dynamic accumulation time – minimum 30 ms for precursor above the intensity threshold of 1000 – in order to maintain a cycle time of 2.7 s). Candidate ions with a charge state between +2 and +5 and counts above a minimum threshold of 10 counts s–1 were isolated for fragmentation, and one MS/MS spectrum was collected before adding those ions to the exclusion list for 15 s (mass spectrometer operated by Analyst TF 1.6, ABSciex). Rolling collision was used with a collision energy spread of 5.
8
Peptide Separation and Mass Spectrometry
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Peptides were separated on an Acclaim PepMap 100 C18 trap and an Acclaim PepMap RSLC C18 column (ThermoFisher Scientific), using a nanoLC Ultra 2D plus loading pump and nanoLC as-2 autosampler (Eksigent). Peptides were eluted with a gradient of increasing acetonitrile, containing 0.1% formic acid (5–40% acetonitrile in 15 min, 40–95% in a further 5 min, followed by 95% acetonitrile to clean the column, before re-equilibration to 5% acetonitrile). The eluate was sprayed into a TripleTOF 5600 electrospray tandem mass spectrometer (ABSciex) and analysed in Information Dependent Acquisition (IDA) mode, performing 200 msec of MS followed by 100 msec MSMS analyses on the 15 most intense peaks seen by MS.
9
LC-SID-MRM MS Quantitation of CFTR Protein
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The optimization
and data analysis of this work were assisted by the Skyline software.33 (link) The preparation of stable isotope reference 18O(Δ4)-CFTR01 and LC–SID–MRM MS quantitation
were performed following previously reported procedures.25 (link) Some exceptions were those experiments where
CFTR protein quantitation was accomplished in CFBE and BHK-wtCFTR
cells. In those cases, an Eksigent NanoLC-Ultra 2D+ (Redwood
City, CA) was used at a flow rate of 400 nL/min with a self-pack Picofrit
column (New Objective, Woburn, MA) filled with 2.7 μM diameter,
160 Å pore Halo resin (MacMod, Chadds Ford, PA), and the length
of resin bed was around 15 cm. Solvent A was composed of 98.8% H2O, 1.0% acetonitrile, and 0.2% formic acid (v/v), and solvent
B was composed of 98.8% acetonitrile, 1.0% water, and 0.2% formic
acid (v/v). A typical running gradient was 1% B at 0 min →
3% B at 5 min → 30% B at 50 min → 80% B at 59 min →
90% B at 69 min → 1% B at 80 min, with a 10 min equilibration.
Samples were loaded on the trap column at flow rate of 4 μL/min
at 1% solvent B for 10 min. A triple quadruple mass spectrometer was
used, 4000 QTrap from ABSCIEX (Foster City, CA). Transitions for the
native and stable isotope labeled peptides are summarized in Table1 .
and data analysis of this work were assisted by the Skyline software.33 (link) The preparation of stable isotope reference 18O(Δ4)-CFTR01 and LC–SID–MRM MS quantitation
were performed following previously reported procedures.25 (link) Some exceptions were those experiments where
CFTR protein quantitation was accomplished in CFBE and BHK-wtCFTR
cells. In those cases, an Eksigent NanoLC-Ultra 2D+ (Redwood
City, CA) was used at a flow rate of 400 nL/min with a self-pack Picofrit
column (New Objective, Woburn, MA) filled with 2.7 μM diameter,
160 Å pore Halo resin (MacMod, Chadds Ford, PA), and the length
of resin bed was around 15 cm. Solvent A was composed of 98.8% H2O, 1.0% acetonitrile, and 0.2% formic acid (v/v), and solvent
B was composed of 98.8% acetonitrile, 1.0% water, and 0.2% formic
acid (v/v). A typical running gradient was 1% B at 0 min →
3% B at 5 min → 30% B at 50 min → 80% B at 59 min →
90% B at 69 min → 1% B at 80 min, with a 10 min equilibration.
Samples were loaded on the trap column at flow rate of 4 μL/min
at 1% solvent B for 10 min. A triple quadruple mass spectrometer was
used, 4000 QTrap from ABSCIEX (Foster City, CA). Transitions for the
native and stable isotope labeled peptides are summarized in Table
10
SWATH-MS Proteomics Protocol
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The proteomics samples were measured on a Sciex TripleTOF 5600 instrument (Sciex, Concord, Canada). The peptides were separated by nano-flow liquid chromatography (NanoLC Ultra 2D, Eksigent) with a flow of 300 nl/min using a NanoSpray III source with a heated interface (Sciex, Concord, Canada). A fused silica PicoTip™ Emitter (inner diameter 75 μm) (New Objective, Woburn, USA) manually packed with 21 cm C18 beads (MAGIC, 3 μm, 200 Å, Michrom BioResources, Auburn, USA) was used to separate about 1 μg of peptides along a linear 120 min gradient from 2% to 35% Buffer B (98% acetonitrile and 0.1% formic acid in H2O) in Buffer A (2% acetonitrile and 0.1% formic acid in H2O). The TripleTOF 5600 was operated in positive ion, high sensitivity SWATH-mode using 64 variable windows between 400 and 1200 m/z (1 m/z overlap). The collision energy was calculated based on a formula for peptides with charge 2+ adding a spread of 15eV. An accumulation time of 250 ms for precursor ions and 50 ms for all fragment ion scans was used that resulted in a total cycle time of about 3.5 s. Samples from the same cell line and biological replicate were injected together in a block design, but randomized within each block. The injection order of the cell line blocks was also randomized.
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