Lc ltq orbitrap

Full length ODIR1 RNA (ODIR1) and ODIR1-antisense RNA (ODIR1-AS) were transcribed in vitro from pcDNA3.1-ODIR1 and pcDNA3.1-ODIR1-AS and labeled with biotin by the Biotin RNA Labeling Mix (Sigma, USA) and T7 RNA polymerase (Sigma, USA). The biotin-labeled RNAs (3 μg) was subjected to heat shock at 90 °C for 2 min and cooled at room temperature (about 25.0 °C) for 20 min, folded with RNA structure buffer, then mixed with cell extract of hUC-MSCs, incubated with Streptavidin Agarose Resin (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 4 °C for 2 h and washed. The retrieved proteins were subjected to SDS-PAGE gel electrophoresis and the protein band of the ODIR1 group was subjected to silver staining, and differential protein bands were identified using high resolution mass spectrometry (LC-LTQ-Orbitrap; Thermo Fisher Scientific, Waltham, MA, USA). The identified proteins were examined using regular western blotting assay. The GO functional annotation was analyzed using the Gene Ontology Consortium (http://www.geneontology.org/).

The OEA − SPC complex was analyzed with H¹NMR (AVANCE III 400 MHz). Free OEA and SPC were used as control. Morphology of the OEA NPs was examined by TEM (Tecnai G2 F20, FEI, USA) at 200 kV. The Size and zeta-potential were determined by Malvern Zetasizer Nano-ZS (Malvern Instruments, Malvern, UK). Three parallel measurements were performed to determine the average values. The drug loading content of OEA in OEA NPs was determined by liquid chromatography-mass spectrometry (LC-LTQ-Orbitrap, Thermo Fisher, USA). The drug loading and encapsulation efficiency was calculated by Equations (1) and (2), respectively.
$$\text{Drug loading content of OEA}\left(\mathrm{\%}\right)=\frac{\left(\text{weight of OEA in NPs}\right)}{\left(\text{weight of NPs}\right)}\times 100\mathrm{\%}$$
$$\text{Entrapment efficiency of OEA}\left(\mathrm{\%}\right)=\frac{\left(\text{weight of OEA in NPs}\right)}{\left(\text{weight of feeding OEA}\right)}\times 100\mathrm{\%}$$

Samples were analyzed using a nano-LC system coupled with a real–hybrid equipment merged into one system (Dual cell liner trap and FT Orbitrap) (nano-LC-LTQ-Orbitrap-MS Thermo Scientific LC-LTQ Orbitrap MS installed at the National Center for Inter-University Research Facilities (NCIRF) at Seoul National University). Split-Free Nano-LC system was equipped with an EASY nLC (Thermo Scientific, US). 4 µL of the concentrated peptide extract was loaded onto a 75 µm × 15 cm column packed with 3 µm Magic C18 particles followed by a 75 min. Linear gradient from 2% to 50% ACN (v/v) in 0.1% formic acid using a 250 nL/min flow rate. The precursor ion mass tolerance was set at ± 0.8Da. Trypsin was designated as the proteolytic enzyme with up to 2 missed cleavages and treated 14–17 h. The mass spectrometer was operated in the data acquisition to automatically switch between MS and MS/MS. Survey full scan MS spectra (from m/z 15 to 4000) were acquired in the Orbitrap mass spectrometer with resolution R = 100,000 max at m/z 4000 (Mass Accuracy: <1 ppm internal calibration). Three LC-MS runs were performed for each digested sample.