Ltq ft icr ms

All ESI-MS experiments were conducted using a LTQ/FT-ICR MS (Thermo Scientific) equipped with an external ESI source. Analyte solutions were infused into the ESI source at 3 μL min⁻¹ flow rates (500 μL gas tight syringe, Hamilton 1700 Series) and a voltage of 3.5–5.0 kV was applied between the ESI capillary and the capillary entrance to the MS to form ions. LTQ-MS ion optic parameters were optimised to obtain stable and intense protein ion signal and to maximise the abundance of the highest protein ion charge states. For comparing the supercharging effects of different additives, the same ion optic parameters were used for all samples, and the mass spectra were collected using the LTQ-MS. Protein solutions were prepared from aqueous stock solutions (100 μM) that were stored at 4 °C for a maximum of three days. ESI solutions consisted of a constant solvent matrix (∼80/19/1% v/v methanol/water/acetic acid), 1–6% of the solution additive of interest, and 5–10 μM of a peptide or protein, specifically 10 μM cytochrome c, 10 μM [Val [5 (link)]]-Angiotensin II acetate, 5 μM carbonic anhydrase II, and 5 μM bovine serum albumin. For ESI-MS, the synthesised additives were isolated and purified, without the separation of any diastereomers or enantiomers. For assignment of the diastereomers of S1-S8, please refer to the Supplementary Information and Fig. S3.

EFHD2-mediated protein changes were identified by mass spectrometric analysis (MS). Total proteins of H1299 and F4 cells were extracted using RIPA lysis and extraction buffer (Thermo Fisher) and quantified using the Bio-Rad Protein Assay kit by the measurement of absorbance at 595 nm. Total protein (20 μg) of each sample was separated using 10% SDS-PAGE and divided into eight gel fractions. After finely cutting (<1 mm³), gel pieces were subjected to in-gel digestion to produce tryptic peptides, followed our previously described method [25 ]. The linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer (LTQ-FTICR MS, Thermo Fisher) was used for survey scan analysis (range: m/z 320–2000) with a mass resolution of 100,000 at m/z 400. Top ten most abundant multiply charged ions were sequentially isolated for tandem mass analysis using LTQ. Protein identification and label-free quantification were performed using the MaxQuant and MaxLFQ software [26 (link)], and the identification threshold was set to P < 0.01.