Samples were analyzed by both liquid chromatography – selected reaction monitoring (LC-SRM) and liquid chromatography – data dependent acquisition (DDA) tandem mass spectrometry (LC-MS/MS) as previously described46 (link),48 (link). Global analysis was performed on an Orbitrap – Velos coupled with an Eksigent 2D nano-LC, while targeted (LC-SRM) analysis was performed on a Qtrap 5500 coupled with a Dionex Ultimate 3000 UHPLC utilizing optimized conditions described previously. LC-SRM data was directly loaded into Skyline and transition quality, peak shape, and peak boundaries were manually validated. Resulting integrated peak areas were directly exported and protein quantity (pmol/g) was calculated against the known spike of stable isotope labeled peptides. LC-MS/MS data was queried against the SwissProt human database using Mascot (v2.3.1) and directly loaded into Scaffold™ (Proteome Software). Peptide Spectral Matches (PSMs) were directly exported with a 99% confidence in protein identifications and at least 2 unique peptides per protein, resulting in a false discovery rate of 0.54%. Statistical analysis for proteomics data, including principal component analysis, was performed using the MetaboAnalyst (v3.0) software suite49 (link).
2d nanolc
Manufactured by AB Sciex
Sourced in United States, Ireland
The 2D nanoLC is a liquid chromatography system designed for high-resolution separation and analysis of complex samples. It features two-dimensional separation capabilities, enabling improved separation and identification of analytes compared to traditional one-dimensional approaches. The core function of the 2D nanoLC is to provide enhanced chromatographic separation and increased sensitivity for a variety of analytical applications.
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Lab products found in correlation
Picotip emmiters, by New Objective (2 mentions)
Ltq orbitrap xl etd, by Thermo Fisher Scientific (2 mentions)
Ultimate 3000 uhplc, by Thermo Fisher Scientific (1 mentions)
Waters nanoacquity, by Waters Corporation (1 mentions)
Orbitrap elite, by Thermo Fisher Scientific (1 mentions)
Trypsin lys c mix, by Promega (1 mentions)
Ltq orbitrap velos mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Direct ip kit, by Thermo Fisher Scientific (1 mentions)
Anti flag beads, by Merck Group (1 mentions)
Mascot v2, by Matrix Science (1 mentions)
Sequencing grade trypsin, by Promega (1 mentions)
Anti gfp antibody, by Roche (1 mentions)
G a beads, by Santa Cruz Biotechnology (1 mentions)
Mascot, by Matrix Science (1 mentions)
4000 qtrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Multiquant, by Thermo Fisher Scientific (1 mentions)
Endoproteinase lys c, by Roche (1 mentions)
Ltq orbitrap, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Roche (1 mentions)
Ltq orbitrap xl, by Thermo Fisher Scientific (1 mentions)
17 protocols using 2d nanolc
1
Quantitative Proteomics of Biological Samples
2
Gel-free Mass Spectrometry Analysis
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MS sample preparation and analyses were performed by the Stanford University Mass Spectrometry facility. In brief, for gel-free MS analysis, the final elutions from the immunoprecipitations were solubilized and digested using the filter-aided sample preparation (FASP) protocol [70 (link)]. Trypsin/Lys-C Mix (Promega, Madison, WI, USA) was used for protein digestion. Peptides were extracted and dried using a speed-vac prior to reconstitution and analysis. Nano reverse-phase HPLC was performed using either an Eksigent 2D nanoLC (Eksigent, Dublin, CA, USA) or Waters nanoAcquity (Waters, Milford, MA, USA) HPLC system with mobile phase A consisting of 0.1% formic acid in water and mobile phase B consisting of 0.1% formic acid in acetonitrile. A fused silica column self-packed with Duragel C18 (Peeke, Redwood City, CA, USA) matrix was used with a linear gradient from 2% B to 40% B at a flow rate of 600 nL/minute. The nanoHPLC was interfaced with a Bruker/Michrom Advance Captive spray source for nanoESI into either an LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Fremont, CA, USA) or an Orbitrap Elite (Thermo Fisher Scientific) operating in data-dependent acquisition mode to perform MS/MS on the top twelve most intense multiply charged cations.
3
Nano-LC-MS/MS Peptide Analysis Protocol
4
Interaction Proteomics of Signaling Proteins
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Plasmids encoding Arrb2-Flag, Dvl2-GFP, and hemagglutinin (HA)-tagged Gβ and Gγ were transiently transfected into HEK293T cells. Bait proteins were affinity isolated on anti-Flag beads (Sigma-Aldrich, Munich, Germany) or on anti-GFP beads prepared by coupling an anti-GFP antibody (Acris Antibodies, Herford, Germany) covalently to agarose beads (Direct IP Kit; Thermo Fisher Scientific, Bremen, Germany). Eluates were digested with trypsin and subsequently with Lys-C in solution. After addition of protein digests as quantitative standards, the samples were desalted on C-18 stage tips (Nest Group, Southborough, MA) and analyzed by nanoflow high-performance liquid chromatography–tandem mass spectrometry (2D-NanoLC; Eksigent, Dublin, CA; Orbitrap Velos MS, Thermo Fisher Scientific; Vasilj et al., 2012 (link)). Protein identification was performed with Mascot V2.2 (Matrixscience, London, United Kingdom), and determination of peptide ion signals for label-free quantification was performed with Progenesis LCMS V2.6 (Nonlinear Dynamics, Newcastle on Tyne, United Kingdom); relative quantification was based on the most intense three signals (MI3; Silva et al., 2006 (link); Groessl et al., 2012 (link)). Isoform specificity of peptides was obtained from the proteomicsdb database (www.proteomicsdb.org; Wilhelm et al., 2014 (link)).
5
Affinity Purification and Mass Spectrometry Analysis of Truncated PARD3 Protein
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HEK‐293T cells were stably transfected with pCDH‐SBP‐HIS8‐PARD3‐c.1012dupG. The cells were lysed, and Western blotting was performed to detect the expression. The SBP‐His8‐tagged truncated PARD3 protein was then precipitated by two‐step affinity purification with streptavidin‐agarose and then with nickel resin. The precipitate containing the complex copurified with SBP‐His8‐PARD3‐c.1012dupG was digested with sequencing grade trypsin (Promega). The resulting samples of peptides were analysed by LC–ESI–MS/MS using an Eksigent 2D nanoLC coupled in‐line with an LTQ‐OrbiTrap mass spectrometer. More detailed steps were conducted as we described previously.5
6
GFP-Cdt 2-345 Affinity Purification
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GFP-Cdt 2–345 (Fig. 1 A ) was immunoprecipitated from lysates of transiently transfected U2OS cells by anti-GFP antibodies (Roche) bound to G/A beads (Santa Cruz Biotechnology, Inc.). After three washes and elution from the beads, GFP-Cdt 2–345 and coprecipitated proteins were subsequently digested with trypsin and Lys-C. Samples were desalted on C-18 stage tips (Nest Group) and analyzed by nanoflow HPLC tandem mass spectrometry (2D-NanoLC; Eksigent; Orbitrap Velos mass spectrometry; Thermo Fisher Scientific; Vasilj et al., 2012 (link)). Identification of proteins was performed with Mascot (V2.2; Matrix Science), and isoform specificity of peptides was obtained from the Proteomicsdb database (Wilhelm et al., 2014 (link)).
7
Proteomic Analysis of Sso MCM C-terminus
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The exact molecular weight and the isotope distribution of the Sso MCM C-terminal domain were determined by ESI mass spectrometry using a LTQ Orbitrap XL ETD (Thermo Scientific) coupled to a 2D nanoLC (Eksigent) chromatography system. An amount of 1.5 pmol was loaded on a 75 μm x 10 cm reverse phase column (Nanoseparation) and separated by application of a linear gradient from 20% to 80% buffer B (80% acetonitrile, 0.1% formic acid) against buffer A (5% acetonitrile, 0.1% formic acid).
8
Nano-LC-MS/MS Peptide Analysis Protocol
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Peptide samples were loaded directly onto an in house packed reverse phase column using 5 µm, 200Å particles (magic C18, Michrom) and PicoTip Emmiters (New Objective) with an autosampler / nanoLC setup (2D nanoLC, Eksigent) at a flow rate of 1 µl/min. After loading the column was washed for 5 min at 1 µl/min at 99 %A (water with 0.2 % FA) 1 %B (acetonitrile with 0.2 % FA) followed by elution with a linear gradient from 1 % B to 35 % B at 400 nl/min in 60 min. Peptides eluting from the column were ionized in the positive ion mode and the 6 most abundant ions were fragmented in the PQD-mode30 (link) to allow for the detection of low mass range reporter ions. Briefly, the LTQ-Orbitrap was run in positive ion mode. Full scans were carried out with a scan range of 395 to 1200 m/z. Normalized collision energy of 35 was used to activate both the reporter ions and parent ions for fragmentation. Scans were carried out with an activation time of 30 ms. The isolation window was set to 1.0 m/z.
9
Proteomic Analysis of Lipid Droplets
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Briefly, protein samples were run using a Eksigent 2D nanoLC with buffer A consisting of 0.1% formic acid in water and buffer B consisting of 0.1% formic acid in acetonitrile. A fused silica column, self packed with Ultro120 3 µm C18Q from Peeke Scientific was used with a linear gradient from 5% B to 40% B over 60 min at a flow rate of 750 nl/min. The mass spectrometer was a LTQ-Orbitrap Velos that is set in data dependent acquisition mode to perform MS/MS in HCD mode. The RAW files were searched with a Sorcerer (SageN) processor using the ipi Rat (EMBL) database with a precursor mass tolerance of 50 ppm and later filtered to <25 ppm. At least two peptides for protein assignment are used so as to decrease the false positive discovery rate and add significant values to the quantitative measurements by isobaric tags. Samples were run in duplicate and results were averaged. Using Scaffold Q+ function, protein levels in CE-enriched LDs were expressed as a ratio over TAG-enriched LDs. The data were exported to Excel for further evaluation.
10
Mass Spectrometry Serum Sample Preparation
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Serum samples were prepared for mass spectrometry as described [23] (link). Samples were lyophilized and redissolved in 2% ACN containing 0.1% formic acid, and peaked with 50 fmol of peptide mixture of βgalactosidase, as a relative internal standard peptide for LC-MS/MS analysis as described [24] (link).
MRM experiments were performed on 4000 QTRAP mass spectrometer (Applied Biosystems) interfaced with a 2-D nanoLC (Eksigent) was used to perform LC-MS/MS analysis. MRM data on the 4000 QTRAP mass spectrometer were acquired with NanoSpray II source. The optimal acquisition parameters were as follows: ion spray voltage (2300 V), curtain gas (30 p.s.i.), nebulizer gas (16 p.s.i.), interface heater temperature (150 ℃), declustering potential (100). The resolution parameters of the rst and the third quadrupoles were set as "unit". In the MRM runs, the scan time was maintained at 50ms for each transition, and the pause between transition scans was set to 5ms. Result les (wiff and wiff.scan) were imported into peak area integration software, MultiQuant (Applied Biosystems, version 1.1) to extract the peak areas of transitions and to normalize using the peak area of internal standard peptide for the βgalactosidase peptide (VDEDQPFPAVPK, IDPNAWVER, GDFQFNISR) to adjust for variations between runs, as described.
MRM experiments were performed on 4000 QTRAP mass spectrometer (Applied Biosystems) interfaced with a 2-D nanoLC (Eksigent) was used to perform LC-MS/MS analysis. MRM data on the 4000 QTRAP mass spectrometer were acquired with NanoSpray II source. The optimal acquisition parameters were as follows: ion spray voltage (2300 V), curtain gas (30 p.s.i.), nebulizer gas (16 p.s.i.), interface heater temperature (150 ℃), declustering potential (100). The resolution parameters of the rst and the third quadrupoles were set as "unit". In the MRM runs, the scan time was maintained at 50ms for each transition, and the pause between transition scans was set to 5ms. Result les (wiff and wiff.scan) were imported into peak area integration software, MultiQuant (Applied Biosystems, version 1.1) to extract the peak areas of transitions and to normalize using the peak area of internal standard peptide for the βgalactosidase peptide (VDEDQPFPAVPK, IDPNAWVER, GDFQFNISR) to adjust for variations between runs, as described.
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