LC-MS/MS was performed with an Easy nLC (Thermo Fisher Scientific, Waltham, MA, USA) coupled to Q Exactive MS (Thermo Finnigan, San Jose, CA, USA) [69 (link)]. Briefly, the iTRAQ-labeled peptides (5 μg) were separated by a Thermo Scientific Easy C18 column (75 μm × 100 mm, 3 μm) with gradient elution from 2% B to 45% B in 120 min (A: 0.1% formic acid in H2O; B: 0.1% formic acid in acetonitrile) at a flow rate of 300 nL/min. All tandem MS were produced following the higher collision energy dissociation (HCD) method. Specifically, MS survey scans were acquired using a data-dependent top 10 method, in which the most abundant precursor ions between 350 and 1500 m/z were dynamically chosen for higher collision energy dissociation (HCD) fragmentation. The resolution was set to 60,000 at 400 m/z, the automatic gain control (AGC) target value was 1 × 106, and the maximum ion accumulation time was 200 ms.
Easy c18 column
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Easy C18 column is a high-performance liquid chromatography (HPLC) column designed for the separation and purification of a wide range of analytes. The column features a C18 stationary phase that provides efficient, reproducible, and robust chromatographic separations.
Automatically generated - may contain errors
Lab products found in correlation
C18 picofrit pepmap, by New Objective (2 mentions)
Easy nanolc 1000, by Thermo Fisher Scientific (2 mentions)
Ltq orbitrap velos, by Thermo Fisher Scientific (2 mentions)
Q exactive ms, by Thermo Fisher Scientific (1 mentions)
Easy nlc, by Thermo Fisher Scientific (1 mentions)
Ltq xl mass spectrometer, by Thermo Fisher Scientific (1 mentions)
μziptipc18 pipette tips, by Merck Group (1 mentions)
4 protocols using easy c18 column
1
Quantitative Proteomics by LC-MS/MS
2
Proteome Profiling by Mass Spectrometry
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Spots from 2D-E were manually excised from gels, triturated, and washed with water. Proteins were in-gel reduced, S-alkylated, and digested with trypsin, as previously reported (D'Ambrosio et al., 2008 (link)). Protein digests were subjected to a desalting/concentration step on μZipTipC18 pipette tips (Millipore Corp., Bedford, MA, USA) and then analyzed by nano-liquid chromatography (nLC)-electrospray ionization (ESI)-linear ion trap (LIT)-tandem (MS/MS) mass spectrometry, using a LTQ XL mass spectrometer (Thermo Fischer Scientific, USA) equipped with a Proxeon nanospray source connected to an Easy-nanoLC (Proxeon, Odense, Denmark). Peptide mixtures were separated on an Easy C18 column (100 × 0.075 mm, 3 μm) (Thermo, USA) using a gradient of acetonitrile containing 0.1% formic acid in aqueous 0.1% formic acid; acetonitrile was ramped from 5 to 35% over 10 min, from 35 to 95% over 2 min, and remained at 95% for 12 min, at a flow rate of 300 nL/min. Spectra were acquired in the range m/z 400–2000. Acquisition was controlled by a data-dependent product ion-scanning procedure over the 3 most abundant ions, enabling dynamic exclusion (repeat count 2; exclusion duration 1 min). The mass isolation window and collision energy were set to m/z 3 and 35%, respectively.
3
Comprehensive nLC-MS/MS proteomic analysis
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The nLC-MS/MS analysis was performed using an Easy Nano LC1000 (Thermo) HPLC coupled with an LTQ Orbitrap Velos (Thermo), where 10 μL of the sample was applied using a 300 nL/min flow rate of Mobile phase A (5% ACN 0.1% formic acid) in a C18 EASY-column (2 cm × 5 μm × 100 μm; 120 Å pore, Thermo) and separated in a C18 PicoFrit PepMap (10 cm × 10 μm × 75 μm; 135 Å pore, New Objective), over 105 min using a linear gradient 2–30% of mobile phase B (100% ACN; 0,1% formic acid). The eluted peptides were ionized using electrospray. The top 20 most intense precursor-ions with charge-state = 2 were fragmented using CID at 35 normalized collision energy and 10 ms activation time. The MS scan range was set between 350 and 1,500 m/z, the MS scan resolution was 60.000, the MS1 ion count was 1 × 10e6, and the MS2 ion count was 3 × 104. The experiments were analyzed in biological triplicates.
4
Differential Proteome Analysis by LC-MS/MS
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Proteins from case and control samples were separated by one-dimensional gel electrophoresis using a 12% gel. Gels were stained using Coomassie brilliant blue and the gel was scanned to identify differentially expressed bands. Bands were excised in the MW range of 10,000–15,000 D corresponding to the “m/z” of discriminant peaks. Bands were in-gel tryptic digested according to Shevchenko and subjected to nanoflow LC-MS/MS analysis. The nLC-MS/MS analysis was performed using an Easy nano LC1000 (Thermo Fisher Scientific) HPLC coupled with an LTQ Orbitrap Velos (Thermo Fisher Scientific). Peptides were loaded on a C18 EASY-column (2 cm × 5 × 100 μm; 120 Å pore; Thermo Fisher Scientific) using a 300 nl/min flow rate of mobile phase A (0.1% formic acid) and separated in a C18 PicoFrit PepMap (10 cm × 10 × 75 μm; 135 Å pore; New Objective), over 105 min using a linear gradient 2–30% of mobile phase B (100% ACN; 0.1% formic acid). The eluted peptides were ionized using electrospray. The top 20 most intense precursor ions with charge-state ≥ 2 were fragmented using collision-induced dissociation at 35 normalized collision energy and 10 ms activation time. The MS scan range was set between “m/z” 350–1,500, the MS scan resolution was 60,000, the MS1 ion count was 1 × 106 and the MS2 ion count was 3 × 104.
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