L3000

A gradient elution (a mixture of mobile phases), composed of mobile phase A [2% acetonitrile, pH 10.0 adjusted with ammonium hydroxide (221228-500ML-A, Sigma)] and B (98% acetonitrile, pH 10.0 adjusted with ammonium hydroxide), was prepared. The lyophilized protein powder obtained was dissolved in solution A and then centrifuged at 12,000 × g for 10 minutes at room temperature. The resulting products were fractionated by a Rigol L3000 HPLC system using a C18 column (Waters BEH C18, 4.6 × 250 mm², 5 μm), and the column oven temperature was set at 45 °C. The detailed elution gradient procedure is shown in Supplementary Table 1. The column eluates were monitored using ultraviolet absorbance at 214 nm and collected into a fresh tube at one-minute intervals. Finally, these were combined into four fractions and dried using a vacuum concentrator. The purified peptides were reconstituted in 0.1% (v/v) FA in water. The peptides of twelve MOGAD samples, ten IND samples and twelve HC samples were pooled and lyophilized before liquid chromatography-mass spectrometry (LC‒MS) analysis.

The sample we tested was plasma. Four spastic CP samples and four control samples were used for proteomic analysis. A 100 μg protein sample was taken from each sample and digested overnight at 37°C. Each sample was processed and labeled with the instruction of iTRAQ Reagent-8 Multiplexing Kit (AB Sciex, UK). The labeled sample was mixed in equal volumes, desalted, and lyophilized. High-performance liquid chromatography was performed with a Rigol L3000 system and a C18 chromatographic column (Waters BEH C18, 5 mm). A Diane NCS3500 system (Thermo Science FicTM) equipped with a trap and an analytical column was used to fractionate the labeled samples, and the precursor ions decomposed by the higher-energy C-trap dissociation (HCD) method were sent to a tandem mass spectrometry Q Exactive HF-X MS (Thermo Fisher, Waltham, MA) for data acquisition and analysis.