An Acquity M-class nanoLC system (Waters, Milford, MA, USA) was used, loading 5 µL of the sample (1 mg) at a rate of 15 mL/min for 3 min onto a nanoEase Symmetry C18 trapping column (180 mm × 20 mm). It was then washed onto a PicoFrit column (75 mm ID × 250 mm; New Objective, Woburn, MA, USA) packed with Magic C18AQ resin (Michrom Bioresources, Auburn, CA, USA). The column was then eluted of peptides into the Q Exactive Plus mass spectrometer (Thermofisher Scientific, NSW, Australia) using the following program: 5%–30% MS buffer B (98% acetonitrile + 0.2% formic acid) for 90 min, 30%–80% MS buffer B for 3 min, 80% MS buffer B for 2 min, 80%–5% for 3 min. The peptides that were eluted were ionised at 2000 V. A data-dependent MS/MS (dd-MS2) experiment was performed with a 350–1500 Da survey scan performed at a resolution of 70,000 m/z for peptides of charge state 2+ or higher with an automatic gain control (AGC) target of 3 × 106 and a 50 ms maximum injection time. The top 12 peptides were selectively fragmented in the higher-energy collisional dissociation (HCD) cell using a 1.4 m/z isolation window, an AGC target of 1 × 105 and a 100 ms maximum injection time. The fragments were scanned in the Orbitrap analyser at a resolution of 17,500 and the product ion fragment masses were measured over a 120–2000 Da mass range. The mass of the precursor peptide was then excluded for 30 s.
Magic c18aq resin
Manufactured by Bruker
Sourced in United States
Magic C18AQ resin is a high-performance reversed-phase chromatography material developed by Bruker. It is designed for the separation and purification of a variety of organic compounds. The resin features a proprietary silica-based matrix and C18 bonded phase that provide efficient and selective retention of analytes.
Automatically generated - may contain errors
Lab products found in correlation
Picofrit column, by New Objective (4 mentions)
Acquity m class nanolc system, by Waters Corporation (3 mentions)
Q exactive plus mass spectrometer, by Thermo Fisher Scientific (3 mentions)
Nanoease symmetry c18 trapping column, by Waters Corporation (2 mentions)
Nanoease symmetry c18 trapping column, by New Objective (1 mentions)
Orbitrap analyzer, by Thermo Fisher Scientific (1 mentions)
Ltq orbitrap elite, by Thermo Fisher Scientific (1 mentions)
Tempo nanolc system, by AB Sciex (1 mentions)
As 1 autosampler, by AB Sciex (1 mentions)
Qstar elite hybrid quadrupole time of flight mass spectrometer, by AB Sciex (1 mentions)
Picotip emitter, by New Objective (1 mentions)
Analyst tf 1, by AB Sciex (1 mentions)
Tripletof 5600 mass spectrometer, by AB Sciex (1 mentions)
Nanoelectrospray ion source, by New Objective (1 mentions)
Agilent 1100 series binary hplc pump, by Agilent Technologies (1 mentions)
Ltq ft icr ms, by Thermo Fisher Scientific (1 mentions)
7 protocols using magic c18aq resin
1
Nanoflow LC-MS/MS Proteomics Pipeline
2
Nanoflow LC-MS/MS Proteomics Workflow
3
Comprehensive Mass Spectrometry Analysis of Alpha-Crystallin
4
Peptide Separation and Identification via Nano-LC-MS
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Using an Eksigent AS-1 autosampler connected to a Tempo nanoLC system (Eksigent, Dublin, CA, USA) as previously described [13 (link)], 10 µL of each combined extract sample was loaded at 20 µL/min with MS buffer A (2% Acetonitrile + 0.2% Formic Acid) onto a C8 trap column (Michrom Bioresources, Auburn, CA, USA). After washing the trap for three min, the peptides were washed off the trap at 300 nL/min onto a PicoFrit column (75 µm ID × 150 mm; New Objective, Woburn, MA, USA) packed with Magic C18AQ resin (Michrom Bioresources, Auburn, CA, USA), then eluted from the column and into the source of a QSTAR Elite hybrid quadrupole-time-of-flight mass spectrometer (AB Sciex, Eksigent, Dublin, CA, USA) using the following program [14 (link),15 (link)]: 5%–30% MS buffer B (98% acetonitrile + 0.2% formic acid) over 60 min, 30%–80% MS buffer B over 3 min, 80% MS buffer B for 2 min, 80%–85% for 3 min. The eluting peptides were ionised at 2300 V. An Intelligent Data Acquisition (IDA) experiment was performed, with a mass range of 350–1500 Da continuously scanned for peptides of charge state 2+–5+ with an intensity of more than 30 counts/s. The selected peptides were fragmented and the product ion fragment masses measured over a mass range of 100–1500 Da. The mass of the precursor peptide was then excluded for 120 s.
5
Proteomic Analysis Using Nanoflow LC-MS/MS
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An Acquity M-class nanoLC system (Waters, USA) was used, loading 5 µL of the sample (1 mg) at a rate of 15 mL/min for 3 min onto a nanoEase Symmetry C18 trapping column (180 mm × 20 mm). It was then washed onto a PicoFrit column (75 mm ID × 250 mm; New Objective, Woburn, MA, USA) packed with Magic C18AQ resin (Michrom Bioresources, Auburn, CA, USA). The column was then eluted of peptides into the Q Exactive Plus mass spectrometer (Thermofisher Scientific, NSW, Australia) using the following program: 5%–30% MS buffer B (98% Acetonitrile +0.2% Formic Acid) over 90 min, 30%–80% MS buffer B over 3 min, 80% MS buffer B for 2 min, 80%–5% for 3 min. The peptides that were eluted were ionised at 2000 V. A data dependant MS/MS (dd-MS2) experiment was performed, with a 350–1500 Da survey scan was performed at a resolution of 70,000 m/z for peptides of charge state 2+ or higher with an Automatic Gain Control (AGC) target of 3 × 106 and a 50 ms maximum injection time. The top 12 peptides were selectively fragmented in the Higher-energy collisional dissociation (HCD) cell using a 1.4 m/z isolation window, an AGC target of 1 × 105 and a 100 ms maximum injection time. The fragments were scanned in the Orbitrap analyser at a resolution of 17,500 and the product ion fragment masses were measured over a 120–2000 Da mass range. The mass of the precursor peptide was then excluded for 30 s.
6
SWATH-MS and DDA Proteomic Analysis
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The MS data acquisition (SWATH‐MS and DDA mode) was performed on TripleTOF 5600 mass spectrometer equipped with a NanoSpray III source and operated by Analyst TF 1.5.1 software (AB Sciex). The samples were injected onto a C18 nanocolumn packed in‐house directly in a fused silica PicoTip emitter (New Objective, Woburn, MA, USA) with 3‐μm 200 Å Magic C18 AQ resin (Michrom BioResources, Auburn, CA, USA) and reverse phase peptide separation was performed on a NanoLC‐Ultra 2D Plus system (Eksigent–AB Sciex, Dublin, CA, USA). The total acquired data were analyzed using a pipeline configured on the Euler‐Portal platform at ETH Zurich.
7
Label-free Quantification of Proteins by LC-MS/MS
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Mass spectrometer analysis was performed using the linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer (LTQ-FTICR MS; Thermo Fisher) equipped with a nano-electrospray ion source (New Objective) and a nano-HPLC system. Nano-HPLC separation used a reverse nano-column (75 μm I.D. × 200 mm) packaged with Magic C18AQ resin (particle size 5 μm, pore size 200 A°; Michrom Bioresources) and an Agilent 1100 series binary HPLC pump (Agilent Technologies). The analytic program was set at a linear gradient from 10% to 50% ACN with a 60 min running cycle. The survey scan of MS analysis (m/z 320–2,000) was performed in LTQ-FTICR MS with a mass resolution of 100,000 at m/z 400. Top ten most abundant multiply charged ions were sequentially isolated for MS/MS by LTQ. The resulting data were applied to the MaxQuant software [23 (link)] for protein identification. Accurate label-free quantification was performed using the MaxLFQ program by normalization and maximal peptide ratio extraction methods [24 (link)]. The significance threshold for the identification was set to P < .01.
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