Vacuum concentrator

Rat metabolic cages (Beijing Jiayuan Xingye Science and Technology Co., Ltd.), frozen high-speed centrifuge (Thermo Fisher Scientific, United States), vacuum concentrator (Thermo Fisher Scientific, United States), DK-S22 electric thermostatic water bath (Shanghai Jinghong Experimental Equipment Co., Ltd.), full-wavelength multifunctional enzyme labeling instrument (BMG Labtech, Germany), oscillator (Thermo Fisher Scientific, United States), TS100 constant temperature mixer (Hangzhou Ruicheng Instruments Co. BMG Labtech), electronic balance (METTLER TOLEDO, Switzerland), −80°C ultralow-temperature freezing refrigerator (Thermo Fisher Scientific, United States), EASY-nLC1200 Ultra High Performance Liquid Chromatography system (Thermo Fisher Scientific, United States), and Orbitrap Fusion Lumos Tribird Mass Spectrometer (Thermo Fisher Scientific, United States) were used.

DNA was synthesized by Genscript (Nanjing, Jiangsu Province, China). Biotinylated TFRE primers (Forward primer: 5'-CATTCAGGCTGCGCAACTGTTG-3', Reverse primer: 5'-GTGAGTTAGCTCACTCATTAGG-3') were synthesized by Sigma. Dynabeads (M-280 streptavidin) were purchased from Invitrogen. Approximately 2–3 pmol of biotinylated DNA was pre-immobilized on Dynabeads and then mixed with nuclear extracts (NEs) from the tissues. The mixtures were incubated for 2 h at 4 °C. The supernatant was discarded, and the Dynabeads were washed twice with NETN solution (100 mM NaCl, 20 mM Tris-HCl, 0.5 mM ethylenediaminetetraacetic acid and 0.5% (vol/vol) Nonidet P-40) and then twice with phosphate-buffered saline. The TFRE pull-down beads were resuspended with 20 μL of SDS loading buffer and boiled for 5 min at 95 °C. The samples were then loaded on 10 cm 10% SDS-polyacrylamide gel electrophoresis gels and run to 1/3 of the length. The gel was stained with coomassie brilliant blue and then destained in 5% ethanol/10% acetic acid solution. Six bands were excised according to the molecular weight ranges and then subjected to in-gel trypsin digestion. 0.1% formic acid was used to stop digestion and 50% acetonitrile was used to extract peptides. Peptide solution was dried in a vacuum concentrator (Thermo Scientific) and then analyzed by LC-MS/MS.

L. longipalpis sand flies were collected in Mali (39 (link), 40 (link)) and reared at the Laboratory of Malaria and Vector Research (LMVR), NIAID. Salivary glands were dissected from 50, 5–7-day old colonized female L. longipalpis sand flies. The salivary glands were pooled, aliquoted, and used as needed to perform in vitro experiments. Salivary gland sonicate (SGS) was prepared by ultrasonication, followed by centrifugation at 10,000 g for 3 min at 4°C. Supernatants were collected and dried using a vacuum concentrator (Thermo, Asheville, NC). SGS were shipped to the Gonçalo Moniz Institute (IGM-FIOCRUZ, Salvador, Bahia-Brazil) and rehydrated with ultra-pure water (KD-Medical, Columbia, MD) immediately before use.

In total, 200 µL of the sample from each beverage was transferred to a Nanosep 3 kDa (Pall Corp, New York, NY, USA) size-exclusion spin column to separate the small molecules from the proteins and molecules with higher molecular weight. Samples were centrifuged with 12,800× g at 4 °C, 3 times for 10 min, and the flow-through was completely dried in a vacuum concentrator (ThermoScientific, San Jose, CA, USA).

Serum samples (50 μl) were thawed and deactivated for COVID-19 in 150 μl of acetone:methanol (50:50, v/v) for 60 min at room temperature. Control samples were treated in exactly the same manner. To precipitate proteins, the samples were incubated for 2 hours at −20°C, which was followed by centrifugation at 15,000g at 4°C for 15 min. The resulting supernatant was removed and evaporated to dryness for 12 hours with a vacuum concentrator (Thermo Fisher Scientific). The dry extracts were then reconstituted in 100 μl of acetonitrile:H₂O (1:1, v/v), sonicated for 10 min, and centrifuged at 15,000g at 4°C for 15 min to remove insoluble debris. The supernatants were transferred to ultraperformance liquid chromatography (UPLC) autosampler vials (Thermo Fisher Scientific). A pooled QC sample was prepared by mixing 5 μl of extracted solution from each sample into a similar UPLC vial. All the vials were then capped and stored at −80°C before being subjected to UPLC–mass spectrometry (MS) analysis.

Protein spots showing significant differences between the studied groups were excised from the gel and subjected to in-gel trypsin digestion. The spots were destained using a 1:1 ratio of 25 mM ammonium bicarbonate (pH 8.5) and 50% acetonitrile followed by digestion with 100 ng stabilized MS grade trypsin (ABSciex, Framingham, MA, USA) at 37 °C overnight. The reaction was stopped by the addition of concentrated formic acid. The tryptic peptides were extracted from the gel pieces, dried in a vacuum concentrator (Thermo Scientific, Waltham, MA, USA), and kept at −20 °C until mass spectrometric (MS) analysis.