Easy nanolc 1000

LC-MS/MS measurements were performed as previously described [18 (link)] on an Orbitrap Q-Exactive plus (Thermo Scientific) mass spectrometer coupled to an Easy nanoLC 1000 (Thermo Scientific). Briefly, high-resolution mass spectrometry data was acquired using the data-dependent acquisition mode. First, a precursor scan covering the mass-to-charge range from 300 to 2000 m/z at 70,000 resolution was performed in profile mode. Subsequently, a series of fragmentation scans of up to 10 most intense precursors at 17,500 resolution was acquired in centroided mode.

The desalted peptides were dried in a speed vacuum and resuspended in buffer A (2% ACN, 0.1% FA), LC-MS/MS analysis was performed using an EASY-nanoLC 1000 nanoflow LC instrument coupled to a high-resolution mass spectrometer (Q Exactive Plus, Thermo Fisher Scientific). A 100 μm (inner diameter) × 2 cm (length) of trap column and a 75 μm (inner diameter) × 12 cm (length) of the analytical column was in-house pulled and packed with C18 particle (DIKMA). Data-dependent acquisition (DDA) was performed in positive ion mode at the flow rate of 300 nL/min. MS spectra were acquired from 350 m/z to 1600 m/z with a resolution of 70,000 at m/z = 200. The automatic gain control (AGC) value was set at 3e6, with a maximum injection time of 20 ms. For MS/MS scans, the top 15 most intense parent ions were selected with a 0.6 m/z isolation window and fragmented with normalized collision energy (NCE) of 30%. The AGC value for MS/MS was set to a target value of 1e5, with a maximum injection time of 100 ms and a resolution of 35,000. Parent ions with a charge state of z = 1 or with unassigned charge states were excluded from fragmentation, and the intensity threshold for selection was set to 2e5.

Tryptic digests of each sample obtained by the FASP method were analyzed by LC/MS/MS using an Easy NanoLC 1000 (Thermo Scientific, Waltham, MA, USA) coupled to an Orbitrap Elite mass spectrometer (Thermo Scientific). Digests were desalted and preconcentrated onto a 2 cm × 100 µm i.d. Pepmap C18 (5 µm particle size) (Thermo Scientific), and then eluted onto and separated using a self-packed PicoFrit (New Objective, Woburn, MA, USA) 75 µm id × 25 cm Magic C18 column (3 µm particle size) with a 60 min linear gradient from 2% mobile phase B to 40% mobile phase B (A = 2% acetonitrile in water, 0.1% formic acid; B = acetonitrile, 0.1% formic acid). An electrospray voltage of 2.8 kV was applied to the PicoFrit column to ionize peptides in the nanoelectrospray ion source of the Obitrap Elite with a heated capillary temperature of 275 °C. The data acquisitions of MS (scan range of m/z 400–2000) and MS/MS (scan range of m/z 140–2000) were collected utilizing the Orbitrap analyzer. A top-five method with higher-energy collisional activation (HCD) for product ion generation in the HCD cell was used with a normalized collision energy setting of 27 V to induce precursor ion fragmentation (+1 charge states were excluded).

The dried samples were dissolved with 20 μl of 0.1% formic acid/water. 2 μl of each sample was analyzed by LC/ESI-MS/MS using a Q-Exactive (Thermo Fisher Scientific, USA) mass spectrometer with an Easy NanoLC-1000 system using data dependent acquisition with dynamic exclusion (DE = 1) settings. The data dependent acquisition settings used was a top 12 higher energy collision induced dissociation (HCD) for the Q-Exactive MS. The Q-Exactive mass spectrometer was used with capillary temperature, 250 °C; spray voltage, 1600 V; and S-lens voltage, 55%. The automatic gain control (AGC) target was 3e6 for Full MS scans and 2e5 for MS/MS scans. Resolving power for Q-Exactive was set at 70,000 for the full MS scan, and 17,500 for the MS/MS scan at m/z 200. LC/ESI-MS/MS analyses were conducted using a C18 column (75 μm × 150 mm). The mobile phases for the reverse phase chromatography were (A) 0.1% HCOOH/water and (B) 0.1% HCOOH in acetonitrile. A four-step, linear gradient was used for the LC separation (2% to 30% B in the first 47 minutes, followed by 80%B in the next 1 minute and holding at 80% B for 12 minutes).

Two μg of digested peptides was analyzed in technical triplicate after 3 h of gradient (5% to 40% B/167 min; 40% to 95% B/5 min; and 95% B/8 min). Easy-nanoLC1000 (Thermo fisher) solvent A consisted of [95% H2O/5% acetonitrile (ACN)/0.1% formic acid] and solvent B of (95% ACN/5% H2O/0.1% formic acid). Trap-column used was Easy column C18, 2 cm × 100 μm i. d. × 5 μm, 120Å and analytical column of 25 cm and internal diameter of 75 μm (3 μm spheres, Reprosil Pur C18). Label-free quantification was performed in an Easy-nLC 1000 (Thermo Scientific) coupled to a QExactive Plus in FullScan-DDA MS2 mode used a dynamic exclusion list of 45 s and spray voltage at 2.70 kV. Full scan was acquired at a resolution of 70000 at m/z 200, with a m/z range of 350-2000, AGC of 1 × 106, and injection time of 50 ms. Selection of the 15 most intense ions for HCD fragmentation used a normalized collision energy of 30, precursor isolation window of m/z 1.2 and 0.5 offset, a resolution of 17 500 at m/z 200, AGC at 5 × 10⁵, and injection time of 100 ms (14 (link)).

Hemolymph from 25 to 30 cold-anesthetized female mosquitoes was collected directly into 1 x Laemmli buffer 48 hr post-blood feeding by clipping the proboscis and gently pressing their abdomen. Samples were precipitated and digested with trypsin, and 1/5 of the digestion product was analyzed on a Q Exactive Plus Mass Spectrometer coupled to an Easy-nanoLC1000 (Thermo). The acquired data was searched against the Anopheles UniProt database plus the Sc2A10 protein sequence using Mascot and the total number of spectra corresponding to ApoLpI, ApoLpII, Sc2A10 and a selection of additional hemolymph proteins that served for normalization were counted.

Proteins from case and control samples were separated by one-dimensional gel electrophoresis using a 12% gel. Gels were stained using Coomassie brilliant blue and the gel was scanned to identify differentially expressed bands. Bands were excised in the MW range of 10,000–15,000 D corresponding to the “m/z” of discriminant peaks. Bands were in-gel tryptic digested according to Shevchenko and subjected to nanoflow LC-MS/MS analysis. The nLC-MS/MS analysis was performed using an Easy nano LC1000 (Thermo Fisher Scientific) HPLC coupled with an LTQ Orbitrap Velos (Thermo Fisher Scientific). Peptides were loaded on a C18 EASY-column (2 cm × 5 × 100 μm; 120 Å pore; Thermo Fisher Scientific) using a 300 nl/min flow rate of mobile phase A (0.1% formic acid) and separated in a C18 PicoFrit PepMap (10 cm × 10 × 75 μm; 135 Å pore; New Objective), over 105 min using a linear gradient 2–30% of mobile phase B (100% ACN; 0.1% formic acid). The eluted peptides were ionized using electrospray. The top 20 most intense precursor ions with charge-state ≥ 2 were fragmented using collision-induced dissociation at 35 normalized collision energy and 10 ms activation time. The MS scan range was set between “m/z” 350–1,500, the MS scan resolution was 60,000, the MS1 ion count was 1 × 10⁶ and the MS2 ion count was 3 × 10⁴.