Tfa isopropyl alcohol

Trifluoroacetic acid (TFA, Sigma-Aldrich, cat#T6508) was added to the trypsin-digested sample to a final concentration of 1%, and the precipitation of sodium deoxycholate was removed by centrifugation. The resulting supernatant was desalted using in-house-made StageTips that were packed with SDB-RPS (3 M EMPORE, cat#2241) and conditioned with 50 μL of 100% acetonitrile (ACN, Sigma-Aldrich, cat# 34851). After loading the supernatant onto the StageTips, centrifugation was performed at 3000 × g for 5 minutes. The StageTips were washed twice with 50 μL of 1% TFA/isopropyl alcohol (Sigma-Aldrich, cat# I9030) followed by a wash with 50 μL of 0.2% TFA. The peptides were eluted in glass vials (CNW Technologies, cat#A3511040) using 80% ACN/5% NH₄OH and dried at 45 °C using a vacuum centrifuge (Eppendorf, cat#5305). The peptide samples were resolved in 2% ACN/0.1FA for LC-MS analysis. Liquid chromatography was performed on a high-pressure nano-flow chromatography system (Elute UHPLC, Bruker Daltonics). Peptides were separated on a reversed-phase column (40 cm × 75 μm i.d.) at 50 °C packed with 1.8 µm 120 Å C18 material (Welch, Shanghai, China) with a pulled emitter tip. A solution is 0.1% FA in H₂O, and B solution is 0.1% FA in ACN. The gradient time was 60 min, and the total run time was 75 min including washes and equilibration. Peptides were separated with a linear gradient from 0 to 5% B within 5 min, followed by an increase to 30% B within 55 min and further to 35% B within 5 minutes, followed by a washing step at 95% B and re-equilibration. LC was coupled online to a hybrid TIMS quadrupole time-of-flight mass spectrometer (Bruker timsTOF Pro) via a CaptiveSpray nano-electrospray ion source. We performed data-dependent data acquisition in PASEF mode with 10 PASEF scans per topN acquisition cycle. Singly charged precursors were excluded by their position in the m/z-ion mobility plane and precursors that reached a ‘target value’ of 20,000 a.u. were dynamically excluded for 0.4 minutes. We used 100 milliseconds to accumulate and elute ions in the TIMS tunnel. The MS1 m/z-range was acquired from 100 to 1700, and the ion mobility range from 1.5 to 0.7 V cm^-2. For data-independent acquisition, we adopted the isolation scheme of 25 Da × 32 windows to cover 400–1200 mz. DIA files (raw) files were input to DIA-NN (v1.8.1)^{65 (link)} FASTA files downloaded from https://www.uniprot.org (UP000072874)^{66 (link)} were added. “FASTA digest for library-free search” and “Deep learning-based spectra, RTs, and IMs prediction” were enabled. “Generate spectral library” was also enabled. “Protein inference” was set to “gene”. Other parameters were kept at the default settings. Statistical analysis by Perseus software (version 1.6.10.43) were performed as previously reported^{67 (link)}. Parasite protein intensities were imported into Perseus. Protein abundances were normalized with total intensities of all proteins per run and then log2 transformed. The Pearson correlation analysis, hierarchical clustering, and volcano plots were performed with default settings.