Easy nlc 1000 uplc system

The tryptic peptides were dissolved in 0.1% formic acid (solvent A), directly loaded onto a home-made reversed-phase analytical column (15 cm length, 75 μm internal diameter). The gradient was comprised of an increasing from 6 to 23% solvent B (0.1% formic acid in 98% acetonitrile) over 26 min, 23% to 35% in 8 min, climbing to 80% in 3 min, and then holding at 80% for the last 3 min. The experiment was processed at a constant flow rate of 400 nL/min on an EASY-nLC 1,000 UPLC system (Thermo, Shanghai, China).
The peptides were subjected to NSI source followed by MS/MS in Q ExactiveTM Plus (Thermo, Shanghai, China) coupled online to UPLC. The electrospray voltage applied was set at 2.0 kV. The m/z scan range was set from 350 to 1,800 for full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using NCE setting as 28 and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans with 15.0 s dynamic exclusion. Automatic gain control was set at 5E4. Fixed first mass was set as 100 m/z^{31 (link)}. The MS proteomics data have been set to the ProteomeXchange Consortium by PRIDE partner repository program under identifier PXD016423.

The dissolution of tryptic peptides was performed in formic acid (0.1%, solvent A), followed by direct loading on a self-made separating column (reverse phase, 15-cm length, 75-μm i.d.). The gradient included an increase of 5–20% solvent B (0.1% formic acid in 98% ACN) within 24 min, an increase of 20–32% within 8 min, and an increase to 80% within 4 min and a holding at 80% for 4 min. All processes were conducted at a fixed flow rate (700 nL/min) on an EASY-nLC 1000 UPLC system (Thermo Fisher Scientific). The peptides were charged to the NSI source, followed by MS/MS in Orbitrap Fusion^TM (Thermo Fisher Scientific) mounted to the UPLC online, under an electrospray voltage of 2.0 kV. The m/z scan range was 350–1,550 for a full scan, and intact peptides were detected at a resolution of 60,000. The 28 peptides were chosen for MS/MS with NCE setting, and the detection of fragments was conducted at a resolution of 30,000. The variation of the data-dependent process was conducted between one MS scan and 20 MS/MS scans with a dynamic exclusion of 15.0 s. The automatic gain control was set as 5E4, and the constant first mass was 100 m/z.

Three parallel analyses for each fraction were performed. Peptides were dissolved in 0.1% formic acid (FA), directly loaded onto a reversed-phase pre-column (Acclaim PepMap 100, Thermo Scientific). Peptide separation was performed using a reversed-phase analytical column (Acclaim PepMap RSLC, Thermo Scientific). The gradient was comprised of an increase from 6% to 23% solvent B (0.1% FA in 98% acetonitrile) for 24 min, 23% to 35% for 8 min and climbing to 80% in 4 min then holding at 80% for the last 4 min, all at a constant flow rate of 280 nl/min on an EASY-nLC 1000 UPLC system, the resulting peptides were analyzed by Q Exactive^TM Plus hybrid Quadrupole-Orbitrap mass spectrometer (ThermoFisher Scientific).

Proteins were extracted from the same batch of L. donovani samples used for the genomic DNA isolation. In brief, the purified promastigotes were incubated in lysis buffer (8 M urea and 1% protease inhibitor cocktail) at 4 °C for 3 min, sonicated on ice for three times using a high intensity ultrasonic processor (Scientz, Ningbo, China). Sonicated samples were centrifuged at 12,000× g at 4°C for 10 min to remove debris. Supernatant was collected, and the protein concentration was measured using a BCA Protein Assay Kit (Thermo Fisher Scientific).
For each of the L. donovani samples (HCZ isolate, DD8 and 9044 strains), proteins were subjected to an in-solution reduction, alkylation and digestion approach. In brief, 300 µg protein aliquots (n = 3) was reduced with 5 mM dithiothreitol at 56 °C for 30 min, alkylated with 11 mM iodoacetamide at room temperature in darkness for 15 min, digested with trypsin (1:50 trypsin-to-protein) at 37 °C overnight, and further digested with trypsin (1:100 trypsin-to-protein) at 37 °C for 4 h. The processed samples were dissolved in 1.0% (v/v) formic acid, and then subjected to liquid chromatography–mass spectrometry/mass spectrometry (LC-MS/MS) analysis using a Q ExactiveTM Plus Orbitrap mass spectrometer (Thermo Fisher Scientific) coupled online to the EASY-nLC 1000 UPLC system.

The tryptic peptides were separated by an EASY-nLC 1000 UPLC system and then subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q Exactive TM Plus (Thermo) coupled online to the UPLC. The resulting MS/MS data were processed using Maxquant search engine (v.1.5.2.8). Tandem mass spectra were searched against SwissProt Human database concatenated with reverse decoy database. Trypsin/P was specified as cleavage enzyme allowing up to two missing cleavages. The mass tolerance for precursor ions was set as 20 ppm in First search and 5 ppm in Main search, and the mass tolerance for fragment ions was set as 0.02 Da. Carbamidomethyl on Cys was specified as fixed modification, and oxidation on Met was specified as variable modifications. False positive rate (FDR) was adjusted to <1% and minimum score for peptides was set >40.