Experiments were performed on a Bruker HCT ion trap mass spectrometer in the positive ion mode. Direct infusion electrospray ionization was performed with a 2 μL/min flow rate and a capillary voltage of −4.5 kV. Collision-induced dissociation was used for all MS/MS experiments with a 40 ms activation time and low mass cutoff of 27% of the precursor ion m/z. MS/MS spectra were manually interpreted. The nitrogen drying gas was set to 5 L/min and 300°C. A custom-built differential ion mobility spectrometer was placed on the inlet capillary of the mass spectrometer using the nitrogen drying gas as the ion mobility carrier gas.38 (link) A schematic of the DIMS can be found in the Supporting Information (Figure 1 ). An Agilent 1260 Infinity II HPLC was used for liquid chromatography experiments, with mobile phase A as water (Optima grade) and mobile phase B as methanol (Optima grade), both containing 100 μM lithium acetate and 0.1% formic acid.
Hct ion trap mass spectrometer
Manufactured by Bruker
The HCT ion trap mass spectrometer is a laboratory instrument designed for the analysis of chemical compounds. It utilizes ion trap technology to capture and analyze ions, enabling the identification and quantification of various substances.
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3 protocols using hct ion trap mass spectrometer
1
Mass Spectrometry Analysis of Compounds
2
Soluble Cytosolic Fraction Preparation and STIL Interactome Identification
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For preparing soluble cytosolic fractions of HeLa, U2OS or HEK293T cells, cells were washed by PBS and lysed in ice-cold lysis buffer. The lysates were vortexed for 15 min at 4°C, and insoluble material was removed after centrifugation for 15 min. For STIL immunoprecipitation, soluble cytosolic fractions of HeLa, HEK293T or U2OS cells were incubated with protein A agarose for 1 h at 4°C for preclear and then incubated for 2 h at 4°C with protein A agarose that had been incubated with anti-STIL antibodies or normal rabbit IgG for mock immunoprecipitation. The beads were washed at least three times with lysis buffer and resuspended in SDS-sample buffer before loading onto a SDS–PAGE gel. To identify STIL-interacting proteins, the gel stained with SimplyBlue™ Safe (Invitrogen) was cut into five pieces. The peptides were digested in gel by trypsin and extracted from the gel. The peptides were subsequently desalted and analyzed by a HCT-Ion trap mass spectrometer (Bruker Daltonik GmbH).
For co-immunoprecipitation assays, the lysates of HEK293T cells transfected with the indicated plasmids (pcDNA3-FLAG-RBM14 FL/[N]/[C], pHA-STIL FL/[N]/[C]) for 24 h were lysed in ice-cold lysis buffer and incubated with anti-FLAG M2 agarose beads (Sigma-Aldrich, A2220) for 2 h at 4°C. The beads were then washed three times with ice-cold lysis buffer and resuspended in SDS-sample buffer.
For co-immunoprecipitation assays, the lysates of HEK293T cells transfected with the indicated plasmids (pcDNA3-FLAG-RBM14 FL/[N]/[C], pHA-STIL FL/[N]/[C]) for 24 h were lysed in ice-cold lysis buffer and incubated with anti-FLAG M2 agarose beads (Sigma-Aldrich, A2220) for 2 h at 4°C. The beads were then washed three times with ice-cold lysis buffer and resuspended in SDS-sample buffer.
3
Protein Identification via LC-MS/MS
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The Dionex U3000 Liquid Chromatography System and the Bruker Daltonics HCT Ion Trap Mass Spectrometer were used for protein identification. First, spots of interest were excised from the dried gel using a clean scalpel and rehydrated in UHQ water for 30 minutes. DTT was then added to samples and incubated at 60°C for 20 minutes, S-alkylated with iodoacetamide at 25°C for 10 minutes, and then digested with trypsin for 8 hours at 37°C. Samples were finally dried using a rotary evaporator and then dissolved in 10 μL 0.1% formic acid in water for LC-MS/MS analysis. Next, samples were placed into a 96-well plate and covered with reusable silicone sealing mat, vortexed thoroughly, and centrifuged for 1 minute; the reusable silicone sealing mat was then replaced with sealing film. Samples were then loaded onto the LC system for analysis. Finally, results were analyzed using the Mascot program against a database and identification based on ion scores as a nonprobabilistic basis for ranking possible protein hits.
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