9rkfsg2 ncs 3500r system

Labeled peptides were analyzed by online nano flow liquid chromatography tandem mass spectrometry (MS/MS) using the 9RKFSG2_NCS-3500R system (Thermo Fisher Scientific) connected to the Q_Exactive HF-X system (Thermo Fisher Scientific) via a nanoelectrospray ion source. Briefly, a C18-reversed phase column (75 μm × 25 cm, Thermo Fisher Scientific) was equilibrated with solvent A (A: 2% acetonitrile and 0.1% formic acid) and solvent B (B: 80% acetonitrile and 0.1% formic acid). The peptides were eluted using the following gradient: 0–2 min, 0–3% B; 2–92 min, 5–25% B; 92–102 min, 25–45% B; 102–105 min, 45–100% B; 105–120 min, 100–0% B at a flow rate of 300 μL/min. The Q_Exactive HF-X was operated in the data-dependent acquisition mode to automatically switch between full scan MS and MS/MS acquisition. The survey of full scan MS spectra (m/z 350–1,500) was acquired in the Orbitrap with 70,000 resolutions. The top 20 most intense precursor ions were selected into the collision cell for fragmentation by higher-energy collision dissociation. The MS/MS resolution was set at 35,000 (at m/z 100), with the maximum fill time of 50 ms and a dynamic exclusion of 30 s (Wang et al., 2020a (link),b (link)).

Labeled peptides were analyzed by online nanoflow liquid chromatography tandem mass spectrometry performed on a 9RKFSG2_NCS-3500R system (Thermo, United States) connected to a Q Exactive Plus quadrupole orbitrap mass spectrometer (Thermo, United States) through a nanoelectrospray ion source. The C18 chromatographic column (75 μm × 25 cm, Thermo, United States) as equilibrated with solvent A (A:2% formic acid with 0.1% formic acid) and solvent B (B: 80% ACN with 0.1% formic acid). The peptides were eluted using the gradient (0–4 min, 0–5% B; 4–66 min, 5–23% B; 66–80 min, 23–29% B; 80–89 min,29–38% B; 89–91 min, 38–48% B; 91–92 min, 48–100% B; 92–105 min, 100% B; 105–106 min, 100–0% B) at a flow rate of 300 nL/min. The Q Exactive Plus was operated in the data-dependent acquisition mode to automatically switch between full scan MS and MS/MS acquisition. The survey of full scan MS spectra (m/z 350–1,300) was acquired in the Orbitrap. Then the top 20 most intense precursor ions were selected for secondary fragmentation, and the dynamic exclusion time was 18 s.

Proteomics analyses were performed at Sinotech Genomics Inc. (Shanghai, China) according to a standard procedure. Briefly, bone tissues were ground into powder in liquid nitrogen and suspended in lysis buffer (1% sodium deoxycholate (SDS) and 8 M urea) to extract total protein. Then, the extracted protein was quantified, qualified, and digested. The resulting peptide mixture was labeled using 10-plex tandem mass tag (TMT) reagent (Thermo Fisher, USA). Next, the samples were pooled and fractionated by ACQUITY ultrahigh-performance liquid chromatography (Waters, USA) on an ACQUITY UPLC BEH C18 column (1.7 µm, 2.1 mm × 150 mm, Waters, USA) to increase the proteomic depth. Labeled peptides were analyzed by online nanoflow liquid chromatography with tandem mass spectrometry on a 9RKFSG2_NCS-3500R system (Thermo Fisher, USA) connected to a Q Exactive HF-X (Thermo Fisher, USA) with a nanoelectrospray ion source. Finally, the raw data were analyzed to identify differentially expressed proteins at a false discovery rate (FDR) cutoff of 0.05 (95% confidence interval).

Proteins from cells were extracted with an appropriate amount of protein lysate (8 M urea, 1% SDS), which contains protease inhibitors to inhibit protease activity. The TMT reagent (Thermo Fisher, A44522) was added to 50 μg polypeptide and incubated for 2 h at room temperature.
Labeled peptides were analyzed by online nanoflow liquid chromatography-tandem mass spectrometry (LC/MS). The experiments were performed on a 9RKFSG2_NCS-3500R system (Thermo, USA) connected to Q_Exactive HF-X (Thermo, USA).

The 9RKFSG2_NCS-3500R system (Thermo, Wilmington, DE, USA) was connected to the Q_Exactive HF-X system (Thermo, Wilmington, DE, USA) via an electro-spray ionization for the study. Analysis of labeled peptides by on-line nanoflow liquid chromatography-tandem mass spectrometry. Briefly, a C18 reversed-phase column (75 μm × 25 cm, Thermo, Wilmington, DE, USA) was equilibrated with solvent A (2% formic acid and 0.1% formic acid) and solvent B (80% acetonitrile and 0.1% formic acid). The elution conditions were as follows: 0–2 min, 0–3% B gradient elution; 2–92 min, 5–25% B; 92–102 min, 25–45% B; 102–105 min, 45–100% B; 105–120 min, 100–0% B; flow rate, 300 nL·min⁻¹. The Q_Exactive HF-X operates in Data Dependent Acquisition (DDA) mode and can automatically switch between full scan MS and MS/MS acquisition. In Orbitrap, a full scan mass spectrum was obtained in the m/z 350–1500 range with a resolution of 70,000. The automatic gain control (AGC) target was 3 × 10⁶ and the maximum fill time was 20 ms. The first 20 parent ions were selected to enter the collision cell for high-energy collisional dissociation (HCD) fragmentation. MS/MS resolution was set to 35,000 (m/z 100); automatic gain control (AGC) target was set to 1 × 10⁵; maximum fill time was 50 ms and dynamic rejection time was 30 s. All experiments were performed with three biological replications.