Acclaim pepmap c18 reversed phase column

The desalted peptide mixture was delivered in duplicate onto a Acclaim PePmap C₁₈-reversed phase column (75 μm × 2 cm, 3 μm, Thermo Scientific, California, USA) and separated with reversed phase C₁₈ column (75 μm × 10 cm, 5 μm, Agela Technologies, Wilmington, Delaware, USA) mounted in a nano-HPLC system (SHIMADZU, Kyoto, Japan). Peptides were eluted using a gradient of 5–80% (v/v) acetonitrile in 0.1% formic acid (FA) over 45 min at a flow rate of 300 nL/min combined with a Q Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA).
The eluates were directly entered Q-Exactive MS, setting in positive ion mode and data-dependent manner with full MS scan from 350 to 2000 m/z, full scan resolution at 70,000, MS/MS scan resolution at 17,500 with minimum signal threshold 1E + 5, isolation width at 2 Da. To evaluate the performance of this mass spectrometry on the iTRAQ labeled samples, two MS/MS acquisition modes, higher collision energy dissociation (HCD) was employed. And to optimize the MS/MS acquisition efficiency of HCD, normalized collision energy (NCE) was systemically examined 28, stepped 20%. Analysis was carried out with 3 technical replications.

Desalted peptide mixture of the supernatants was loaded onto an Acclaim PePmap C18-reversed phase column (75 μm×2 cm, 3 μm, thermo scientific) and separated with a reversed-phase C18 column (75 μm×10 cm, 5 μm, Agela Technologies) mounted on a Dionex ultimate 3000 nano LC system. Peptides were eluted using a gradient of 5–80% (v/v) acetonitrile in 0.1% formic acid over 45 min at a flow rate of 300 nL/min combined with a Q Exactive mass spectrometer (Thermo Fisher Scientific, MA, USA). The eluates were directly entered Q-Exactive MS (Thermo Fisher Scientific, Waltham, MA, USA), setting in positive ion mode and data-dependent manner with full MS scan from 350 to 2000 m/z, full scan resolution at 70,000. Only proteins with scores corresponding to p < 0.05 and with at least two independent peptides were considered. Visualization of protein interaction networks was performed with the String database using a high confidence interaction score (0.7) and experiments, databases, gene fusion, and neighborhood as active interaction sources. GO enrichment analysis was performed using the open access platform Term Finder. Venn diagrams were generated using online tools (http://bioinformatics.psb.ugent.be/webtools/Venn/).

The peptides were dissolved in 50 μL mobile phase A (0.1% formic acid) and loaded onto an Acclaim PePmap C18-reversed phase column (75 μm × 2 cm, 3 μm, 100 Ǻ Thermo Scientific). The peptides were isolated by reversed phase C18 column (75 μm × 10 cm, 5 μm, 300 Ǻ, Agela Technologies, New York, NY, USA), which was mounted on a Dionex ultimate 3000 nano LC system. Peptide elution was performed with the following gradient: 0–6 min 5% buffer B; 6–6.5 min 10% buffer B; 6.5–45 min 10–24% buffer B; 45–51 min 24–40% buffer B, 51–54 min 40–80% buffer B; 54–59 min 80% buffer B; 59–59.9 min 5% buffer B; 59.9–65 min 5% buffer B. This method was used in combination with a Q Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) at a flow rate of 300 nL/min. The eluates went directly to Q-Exactive MS, set to cation mode, and relied on data with full MS scanning at 350–2000 m/z, 70,000 full scanning resolution, and MS/MS scanning resolution at 17,500. For MS/MS scans, the minimum signal threshold was 1 × 10⁵, and the isolation width was 2 Da. To evaluate the performance of this mass spectrometry on iTRAQ-labeled samples, two MS/MS acquisition modes and higher collision energy dissociation (HCD) were used. To optimize the MS/MS acquisition efficiency of HCD, the normalized collision energy (NCE) was systemically checked 28 times, stepped 20%.