Orbitrap lumos mass spectrometer

The enriched glycopeptides from MCI and normal controls were dissolved in 0.1% Formic Acid (FA) and subject to targeted quantitative analysis on the Orbitrap Lumos Mass Spectrometer (Thermo). The NanoLC system and the analysis column were the same as described above. The analytical gradient was 60 min long from 5 to 42% at a flow rate of 300 nL/min. Data acquisition was achieved in PRM mode which only performs MS/MS for the targeted precursors on the preloaded mass list. MS conditions were as described above with minor modifications on the MS1 scan range (700–1800 m/z) and the MS2 scan range (130–2000 m/z).

Tryptic peptides were separated by reverse phase chromatography on a Dionex Ultimate 3000 RSLCnano UHPLC system (Thermo Scientific) with an Acclaim C18 PepMap RSLC column using a 3–42% acetonitrile gradient over 60 minutes. Peptides were eluted directly into a Thermo Orbitrap Lumos mass spectrometer by nano-electrospray. Data-dependent acquisition (DDA) was applied, with precursor ion scans (MS1) collected by FTMS at 120,000 resolution and HCD fragmentation scans (MS2) collected in parallel by ITMS with 3-second cycle times. Monoisotopic precursor selection and charge-state screening were enabled, with ions > +1 charge selected. Dynamic exclusion was applied to selected ions +/− 10 ppm for 30 seconds. Raw mass spectrometry data have been deposited on MassIVE (MSV000092793).

Peptides were analysed in an Ultimate 3000 RSLCnano system (Thermo Scientific) coupled to an Orbitrap Lumos mass spectrometer (Thermo Scientific). Peptides were injected onto a 50 cm long C18 Pepmap EasySpray column with an internal diameter of 75 μm and 2 μm particles (Thermo Scientific) at a flow rate of 300 nl per min. Peptides were separated at the same flow rate with a gradient of 1–27% Acetonitrile 0.1% formic acid over 30 min followed by 27–45% acetonitrile 0.1% formic acid over an additional 10 min. The emitter eluted the peptides directly into the MS source at a spray voltage of 1800 V. The mass spectrometer was operated in data dependent mode. An MS survey scan was acquired from m/z 350–1200 at a nominal resolution of 120,000 (defined at m/z 200) with a target value of 5e5 ions and a maximum fill time of 50 ms. The most intense multiply charged ions were selected for HCD MS2 analysis at a normalised collision energy of 29% followed by dynamic exclusion for 15 s with as many precursors selected per cycle as possible and a maximum time of 1.2 s.

Drug affinity responsive target stability (DARTS) assay was completed to identify the target of ISP I in vitro. For this assay, we used the protocol published by Lomenick et al. [19 (link)]. Briefly, LN229 cells were lysed with M-PER (Pierce) and supplemented with protease and phosphatase inhibitors. After centrifugation at 14, 000 rpm for 15 min, lysates were diluted to the same final volume and protein concentration with M-PER and proposed in TNC buffer [50 mM Tris·HCl (pH 8.0), 50 mM NaCl, 10 mM CaCl₂]. All steps were performed on ice or at 4 °C to prevent premature protein degradation. After incubation, the protein sample was incubated with ISP I (40 μM) or DMSO (control) at room temperature for one hour. Each sample was subsequently proteolyzed with 2 μL 1:100 Pronase at room temperature for 32 min. To stop proteolysis, 3 μL cold 20 × Protease inhibitor was added to each sample, mixed, and placed on ice. The digested peptides were filtered through Vivacon 500 10 K spin column, precipitated using acetone, reduced with TCEP, alkylated with NEM, and digested with trypsin. Digests were desalted and used for LC–MS/MS data acquisition on an Orbitrap Lumos mass spectrometer (Thermo Fisher Scientific) coupled with an UltiMate 3000 RSLC-nano HPLC (Thermo Fisher Scientific) in data-dependent acquisition (DDA) mode.