Q exactive series mass spectrometer

Mobile phase A (100% water and 0.1% formic acid) and B solution (80% acetonitrile and 0.1% formic acid) were prepared. The lyophilized powder was dissolved in 10 μl of solution A, centrifuged at 15,000 rpm for 20 min at 4°C, and 1 μg of the supernatant was injected into a homemade C18 Nano-Trap column (2 cm × 75 μm, 3 μm). Peptides were separated in a homemade analytical column (15 cm × 150 μm, 1.9 μm) using linear gradient elution, as listed in Supplementary Table S1. The isolated peptides were analyzed by the Q Exactive series mass spectrometer (Thermo Fisher), with ion source of Nanospray Flex™ (ESI), spray voltage of 2.3 kV, and ion transport capillary temperature of 320°C. Full scan ranges from m/z 350 to 1,500 with resolution of 60,000 (at m/z 200), an automatic gain control (AGC) target value was 3 × 10 6, and a maximum ion injection time was 20 ms. The top 20 (Huang et al., 2009 (link)) precursors of the highest abundant in the full scan were selected and fragmented by higher-energy collisional dissociation (HCD) and analyzed in MS/MS, where resolution was 15,000 (at m/z 200), the automatic gain control (AGC) target value was 5 × 10⁴, the maximum ion injection time was 45 ms, a normalized collision energy was set as 27%, and intensity threshold was 2.2 × 10⁴. The dynamic exclusion parameter was 20 s. The raw data of M.S. detection were named as “raw.”

Biotin-labelled control (antisense) and circCRIM1 (sense) probes (Supplementary Table S1) were synthesized by TSINGKE. RNA pull-down assays were conducted as described previously [18 (link)]. The bound proteins in the pull-down samples were detected using Western blotting or mass spectrometry. Silver staining was performed according to the manufacturer’s instructions of the PAGE Gel Silver Staining Kit (Solarbio, Beijing, China). Mass spectrometry analysis was accomplished by Novogene (Tianjin, China) using an EASY-nLCTM 1200 UHPLC system (Thermo Fisher Scientific) coupled with a Q ExactiveTM series mass spectrometer (Thermo Fisher Scientific). Furthermore, protein identification and quantification were performed by Proteome Discoverer software (version 2.2, ThermoFisher Scientific).

The isolated neutrophils were transferred to a 1.5 ml centrifuge tube, and subsequently lysed with lysis buffer containing 8 mol/L urea, 100 mmol/L triethylammonium bicarbonate at pH 8.5. Following that, the sample was ultrasonicated for 5 min on ice. The lysate was centrifuged at 12,000 g for 15 min at 4 ℃, and the supernatant was subsequently reduced with 10 mmol/L dl-dithiothreitol for 1 h at 56 ℃. This was followed by alkylation using sufficient iodoacetamide for 1 h at room temperature in the dark. Each protein sample underwent labeling with tandem mass tag and subsequent desalting and lyophilization. The sample was fractionated on a Rigol L3000 HPLC system (RIGOL TECHNOLOGIES CO., LTD, China) using a C18 column (Waters BEH C18, 4.6 mm × 250 mm, 5 μm) with the column chamber maintained at a temperature of 45 °C. For the construction of transition libraries, we conducted shotgun proteomic analyses using an EASY-nLCTM 1200 UHPLC system (Thermo Fisher, USA) and a Q ExactiveTM series mass spectrometer (Thermo Fisher, USA) in the data-dependent acquisition mode. The statistical analysis of protein quantitation results was performed using t-test. Proteins showing significant different were considered differentially expressed if they exhibited fold changes greater than 1.5 and a P-value less than 0.05 [29 (link)].

FLAG-NKAP Co-IP product and m⁶A Co-IP product were loaded on a 10% SDS- PAGE gel and stained with Coomassie brilliant blue. About 100 μL with 100 mM TEAB, 1 ug trypsin, and 1/300 CaCl₂ were added into the gel, proteins were digested overnight at 37 °C. After gel digestion, the separated peptides were analyzed by Q Exactive^TM series mass spectrometer (Thermo Fisher), with ion source of Nanospray Flex™ (ESI), spray voltage of 2.1 kV, and ion transport capillary temperature of 320 °C. The resulting spectra were searched by the search engines: Proteome Discoverer 2.2 (PD 2.2, Thermo). Gene Ontology (GO) functional analysis was conducted using the InterProScan program against the non-redundant protein database (including Pfam, PRINTS, ProDom, SMART, ProSite, PANTHER), and the databases of COG (Clusters of Orthologous Groups) and KEGG (Kyoto Encyclopedia of Genes and Genomes) were used to analyze the protein family and pathway.